EP2313419A1 - Novel compounds and methods for their production - Google Patents
Novel compounds and methods for their productionInfo
- Publication number
- EP2313419A1 EP2313419A1 EP09785241A EP09785241A EP2313419A1 EP 2313419 A1 EP2313419 A1 EP 2313419A1 EP 09785241 A EP09785241 A EP 09785241A EP 09785241 A EP09785241 A EP 09785241A EP 2313419 A1 EP2313419 A1 EP 2313419A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- alkyl
- acyl
- strain
- compound according
- avermectin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
- C07D498/18—Bridged systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
Definitions
- the present invention relates to novel FK506 and FK520 analogues generated by feeding non-natural starter units to bacterial strains and to use of such compounds in therapy.
- FK506 tacrolimus/fujimycin/Prograf
- FK520 ascomycin or immunomycin
- Figure 1 are lipophilic macrolides produced by a variety of actinomycetes, including Streptomyces tsukubuaensis No. 9993 (Hatanaka et al., 1989), Streptomyces sp, MA8858, Streptomyces sp.
- FK525 also known as FR-900525 (Hatanaka H, et al., 1989)
- FK523 also known as FR-900523
- antascomicins Fehr, T., et al., 1996.
- a number of semisynthetic derivatives of these molecules have also been shown to be of utility, including pimecrolimus (SDZ ASM 981 , Elidel), which is a derivative of FK520 (Meingassner et al., 1997).
- PKS polyketide synthases
- the avermectin PKS naturally incorporates branched chain keto acids (Ikeda et al., 1999), but has been found to additionally incorporate a variety of other non- natural carboxylic acids when fed exogenously to fermentation broths (Dutton et al., 1991 ).
- This capability is encoded on the PKS by the loading module, which consists of a section of the PKS including an Acyltransferase domain (AT) and an Acyl Carrier Protein domain (ACP) (Ikeda et al., 1999).
- PKS with loading modules with similar specificities to the avermectin PKS include nemadectin (Carter et al., 1988) and milbemycin (Takiguchi et al., 1980; Okazaki et al., 1983). Methods for elucidating the sequence of PKS gene clusters have been published previously, examples include using homologous probes to screen cosmid libraries (e.g. Oliynyk et al., 2003, Fang et al., 2007, Choi et al., 2007) and full genome sequencing is another approach.
- FK506, FK520 and close analogues suppress the immune system by inhibiting signal transduction pathways required for T-cell activation and growth.
- they have been shown to inhibit Ca 2+ -dependent T-cell proliferation, via initial formation of a complex with an FK-binding protein (FKBP), which binds to and blocks calcineurin (CaN).
- FKBP FK-binding protein
- This FK506-FKBP-CaN complex inhibits the activation of nuclear factor of activated t-cells (NF-AT), preventing its entrance into the nucleus, and subsequent activation of the promoter of lnterleukin-2 (IL-2), which initiates IL-2 production.
- NF-AT nuclear factor of activated t-cells
- IL-2 lnterleukin-2
- FK506 can interfere with the action of calcineurin on substrates other than NFAT, including IKB, Na-K- ATPase and nitric oxide synthase, which may lead to some of the side-effects (Kapturczak et al., 2004).
- TGF- ⁇ transforming growth factor beta
- FK506 and FK520 also have antifungal activity (Heitman et al., 1991 , Brizuela et al., 1991 ).
- USES: FK506 in particular, is an important immunosuppressant used to aid prevention of organ rejection after transplantation.
- it is used intravenously and orally for the prevention of organ rejection after allogeneic liver or kidney transplantation and in bone marrow transplantation. It has been shown to have potential utility in a wide variety of autoimmune, inflammatory and respiratory disorders, including Crohn's disease, Behcet syndrome, uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, nephritic syndrome, aplastic anaemia, biliary cirrhosis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease.
- COPD chronic obstructive pulmonary disease
- FK506 FK506, which include renal dysfunction, gastrointestinal effects, neurological effects, hyperthrichosis and gingival hyperplasia.
- Pimecrolimus and FK506 are both used in topical formulations, such as ointments and creams, as treatments for a variety of skin conditions, in particular atopic dermatitis (Nghiem et al., 2002).
- Cytochrome P450 3A4 (Cyp3A4) and Cyp3A5 are the most important contributors to FK506 metabolism while the P-glycoprotein pump (MDR-1 ) modulates its bioavailability (Roy et al., 2006). The complexity of FK506 dosing is therefore enhanced by significant drug-drug interactions (Kapturczak et al., 2004).
- the mechanism of toxicity of FK506 and FK520 has been related to the mechanism of action of immunosuppression (F. Dumont et al., 1992). This strong link between the mechanism of action and the toxicity has presented significant challenges to improving the therapeutic index through chemical modification. Segregation of efficacy and toxicity of new analogues may be possible by altering distribution or metabolism (NH Signal et al., 1991 ). By limiting the exposure of the compound to organs that are sensitive to such inhibition, such as the kidney, systemic toxicity can be avoided. Additionally, topical administration of the calcineurin inhibitor at the site of administration (such as skin, lungs, gut, eye etc.) can be maximized. One way this can be achieved is by using a 'soft drug' approach, which involves designing compounds to have limited systemic exposure such as through increased metabolism, higher blood/plasma protein binding, poor absorption or bioavailability.
- variable metabolism of FK506 leads to some of the toxicity, due to variable levels of systemic exposure, which led to the need for constant drug monitoring (Armstrong and Oellerich, 2001 ). Therefore, analogues of FK506 with reduced or less variable metabolism could be useful in reducing toxicity, and reducing the need for constant monitoring of drug levels.
- FK506 is also poorly bioavailable (Tamura et al., 2003), which leads to variable systemic exposure when dosed orally, and the frequent need for intravenous dosing. Therefore, analogues with improved oral bioavailability would be very useful, to reduce systemic toxicity through incorrect dosing, and improve the ease of oral dosing. Therefore, there remains a need to identify novel FK506 and FK520 analogues, which may have utility in the maintenance of immunosuppression, both for organ transplantation, and for the treatment of inflammatory conditions, and for the treatment of fungal infections.
- novel FK506 and FK520 analogues which have improved pharmaceutical properties compared with the currently available FK506 and FK520 analogues; these properties may be useful for therapies requiring good systemic bioavailability, including, but not limited to oral therapies to maintain immunosuppression, which in particular are expected to show improvements in respect of one or more of the following properties: increased metabolic stability, increased bioavailability, increased oral bioavailability, reduced efflux via membrane transporters and low plasma protein binding;
- the novel FK506 and FK520 analogues may also be useful for therapies requiring local availability but with poor systemic availability, including, but not limited to topically administered therapies for inflammatory disorders such as atopic dermatitis, asthma and inflammatory bowel diseases, which in particular are expected to show improvements in respect of one or more of the following properties: decreased metabolic stability, decreased bioavailability, decreased oral bioavailability, increased efflux via membrane transporters and high plasma protein binding;
- non-natural starter units are fed to strains normally producing FK506 or FK520 or analogues thereof, said strains optionally having been mutated to have targeted inactivation or deletion of one or more genes responsible for post-PKS modification, and/or targeted inactivation or deletion of one or more precursor supply genes, including fkbO and homologues thereof.
- the natural loading module from the FK506 or FK520 polyketide synthase is replaced by the loading module from the avermectin or an avermectin-like polyketide synthase, and optionally non-natural starter units are fed to these strains, said strains optionally having been mutated to have targeted inactivation or deletion of one or more genes responsible for post-PKS modification, and/or targeted inactivation or deletion of one or more precursor supply genes, including bkd genes (Ward et al., 1999) and homologues thereof.
- unnatural starter acids have been found to be incorporated at different levels into the FK506 and FK520 systems when compared to the rapamycin system.
- pyrans are very well incorporated, leading to high yields of FK506 and FK520 analogues.
- X a represents bond or CH 2 ;
- Ri is selected from the group consisting of
- R 10 , Rn and R 12 are independently selected from H, F, Cl, OR 13 , SR 13 and NHR 13 and R 13 is selected from H, CrC 4 alkyl and Ci-C 4 acyl; and
- A is selected from O, S and NR 13a
- R 13a represents H, CrC 4 alkyl or Ci-C 4 acyl
- R 14 and R 15 are independently selected from H, CrC 4 alkyl, OR 16 , SR 16 and NHR 16
- R 16 represents CrC 4 alkyl or Ci-C 4 acyl
- X b represents a bond or CH 2
- R 17 represents H and when X b is CH 2 then R 17 represents H, F, Cl, OH, SH, Ci-C 4 alkyl
- OR i6a , SRi6a or NHR 163 and Ri 6a represents Ci-C 4 alkyl or Ci-C 4 acyl
- Ri 6a represents Ci-C 4 alkyl or Ci-C 4 acyl
- Ri3b represents H, Ci-C 4 alkyl, or Ci-C 4 acyl
- Ri 4a represents Ci-C 4 alkyl
- Ri6 represents Ci-C 4 alkyl or Ci-C 4 acyl
- Ri 8 represents H, F, Cl, OH, SH, Ci-C 4 alkyl
- R i6a represents Ci-C 4 alkyl or Ci-C 4 acyl
- X c and X d independently represent bond or CH 2 provided that X c and X d do not both represent CH 2 and when X c is bond then Ri 7a represents H and when X c is CH 2 then Ri 7a represents H, F, Cl, OH, SH, Ci-C 4 alkyl, ORi 6b , SRi 6b or NHRi 6b , Ri 6b represents CrC 4 alkyl
- Ri 9b and R 20 independently represent H, F, Cl, OH, SH, CrC 4 alkyl, ORi 6d , SRi 6d or NHRi 6d , and Ri 6d represents Ci-C 4 alkyl or CrC 4 acyl;
- Ri 9c represents H, F, Cl, OH, SH, C r C 4 alkyl, ORi 6e , SRi 6e or NHRi 6e , and R i6e represents CrC 4 alkyl or CrC 4 acyl;
- R 19d and R 2 Oa independently represent H, F, Cl, OH, SH, d-C 4 alkyl, ORi 6f , SRi6f or NHR 16 f and Ri 6 f represents CrC 4 alkyl or d-C 4 acyl ;
- R 18a , Ri 9e and R 2Ob independently represent H, F, Cl, OH, SH, d-C 4 alkyl, ORi6g, SRi6g or NHR 16g and Ri 6g represents CrC 4 alkyl or d-C 4 acyl;
- R 2 i, R 22 and R 23 independently represent H, F, Cl, OR 26 , SR 26 , CrC 4 alkyl or CN
- R 26 represents H, d-C 4 alkyl or d-C 4 acyl
- R 24 and R 25 independently represent H, F, Cl, OH, SH or CrC 4 alkyl, provided that at least one of R 2 i, R 22 and R 23 is not H or d-C 4 alkyl
- R 2 i a , R22a and R 2 3a are independently selected from H, F, Cl, OR 26a , SR 26a , C1- C4 alkyl and CN
- R 26a represents H, d-C 4 alkyl or d-C 4 acyl
- R 24a and R 25a independently represent H, F, Cl, OH, SH or d-C 4 alkyl and provided that at least one of R 2 i a , R 22a and R 23a does not represent H or d-C 4 alky
- the invention embraces all stereoisomers of the compounds defined by structure (I) as shown above.
- the present invention provides FK506 or FK520 analogues such as compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.
- analogue means one analogue or more than one analogue.
- analogue(s) refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).
- FK506 and FK520 analogues refer to compounds related to FK506, FK520 and similar compounds in structure. Such compounds are 22-membered rings with one lactone and one amide bond. The N of the amide bond forms a 2-carboxyl piperidine or a 2-carboxyl pyrrolidine. This carboxyl group forms the lactone group, with an oxygen that is allylic to a double bond that is exo to the main 22-membered ring.
- Such compounds include, without limitation, FK520, FK506, antascomicin, FK523, FK525, pimecrolimus and tsukubamycin as well as compounds of formula (I).
- FK506 or FK520 producing strain refers to a strain (natural or recombinant) which is capable of producing one or more FK506 or FK520 analogues when fed appropriately.
- recombinant strain of a FK506 or FK520 producing host refers to a recombinant strain based on a natural FK506 or FK520 producing strain which is capable of producing one or more FK506 or FK520 analogues when fed appropriately.
- FK506 or FK520 cluster means the PKS and associated enzymes responsible for production of FK506 or FK520 analogues.
- modifying gene(s) includes the genes required for post- polyketide synthase modifications of the polyketide, for example but without limitation cytochrome P-450 monooxygenases, ferredoxins and SAM-dependent O-methyltransferases.
- these modifying genes include fkbD and fkbM, but a person of skill in the art will appreciate that PKS systems related to FK520 (for example but without limitation: FK506, antascomicin, FK523, FK525 and tsukubamycin) will have homologues of at least a subset of these genes, some of which are discussed further below.
- precursor supply gene(s) includes the genes required for the supply of the natural or non-natural precursors, the genes required for the synthesis of any naturally or non-naturally incorporated precursors and the genes required for the incorporation of any naturally or non-naturally incorporated precursors.
- these genes include fkbL, fkbO and fkbP but a person of skill in the art will appreciate that PKS systems related to FK506 and FK520 (for example but without limitation: antascomicin, FK523, FK525 and tsukubamycin) will have homologues of these genes, some of which are discussed further below.
- auxiliary gene(s) includes references to modifying genes, precursor supply genes or both modifying genes and precursor supply genes.
- One example of an auxiliary gene is an oxygenase which may hydroxylate the starter unit.
- basic product refers to the initial product of the polyketide synthase enzyme before the action of any modifying genes.
- non-natural starter unit refers to any compounds which can be incorporated as a starter unit in polyketide synthesis that are not the starter unit usually chosen (i.e. incorporated) by that PKS.
- avermectin-like PKS means the PKS of a bacterium producing a avermectin-like polyketide such as nemadectin or milbemycin which contains a loading domain consisting of AT and ACP domain and which naturally incorporates branched chain starter acids such as those of the present invention.
- C1-4 acyl groups include C2-4 acyl groups such as -COMe and -COEt, especially COMe. CHO is a further example which is less preferred.
- C1-4 alkyl groups include Me, Et, n-Pr, i-Pr, n-Bu, especially Me.
- Physiologically functional derivatives of compounds of formula (I) include physiologically acceptable salts, esters and solvates.
- Pharmaceutically acceptable salts include the non-toxic acid addition salt forms of the compounds of formula (I).
- the pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid.
- Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulphuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.
- butanedioic acid maleic, fumaric, malic (i.e. hydroxyl- butanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.
- salt forms can be converted by treatment with an appropriate base into the free base form.
- Example esters include labile esters which are cleaved in the body, for example carboxylic acid esters formed with hydroxyl groups.
- Example solvates include hydrates.
- X a represents a bond. In another embodiment X a represents CH 2 . Preferably X a represents CH 2 .
- X b represents bond.
- X c represents bond
- X d represents bond
- R 1 represents [A]. In another embodiment R 1 represents [B]. In another embodiment R 1 represents [C]. In another embodiment R 1 represents [D]. In another embodiment R 1 represents [E]. In another embodiment R 1 represents [F]. In another embodiment R 1 represents [G]. In another embodiment R 1 represents [H]. In another embodiment R 1 represents [J]. In another embodiment R 1 represents [K].
- Ri 0 represents OH.
- Rn represent H.
- Ri 2 represents H.
- Ri 3 , Ri 3a and Ri 3b represent H.
- A represents O or S.
- Ri 4 represents H.
- Ri 4a represents H.
- Ri 5 represents H.
- Ri 6 represents H.
- Ri 6a represents H.
- Ri 6 b represents H.
- Ri 6c represents H.
- Ri 6 d represents
- Ri 6e represents H.
- Ri 6f represents H.
- Ri 6g represents H.
- Ri 7 represents H.
- Ri 7a represents H.
- Ri 8 represents H.
- Ri 8a represents H.
- Ri 9 represents H.
- Ri 9a represents H.
- Ri 9b represents H.
- Ri 9c represents H.
- Ri 9e represents OH.
- R 20 represents H.
- R 20 b represents OH.
- R 2 i represents H or F. More preferably R 2 i represents F.
- R 2 i a represents H or F. More preferably R 2 i a represents F.
- R 22 represents H or F. More preferably R 22 represents F.
- R 22a represents H or F. More preferably R 22a represents F.
- R 23 represents H or F. More preferably R 23 represents F.
- R 23a represents H or F. More preferably R 23a represents F.
- R 24 represents OH.
- R 24a represents OH.
- R 25 represents H
- R 25a represents H.
- R 26 and R 26a represent H.
- Ri represents G
- R 20 b represents OH
- Ri 9e and Ri 8a represent H
- R 2 represents CH 2 CH 3
- R 3 0
- X a represents CH 2 , as shown by the following s sttrruuccttuurree:
- R 1 represents [J]
- R 22 represents OH
- R 2 i, R 23 , R 24 and R 25 represent H
- R 2 represents CH 2 CH 3
- R 3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [B]
- A represents S
- R 17 , R 1S , and R 14 represent H
- X b represents bond
- R 2 represents CH 2 CH 3
- R 3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [B]
- A represents O
- Ri 7 , Ri 5 , and Ri 4 represent H
- X b represents bond
- R 2 represents CH 2 CH 3
- R 3 0
- X a represents CH 2 , as shown by the following structure, also known as Pyran FK520 or 28-des(3-c/s-methoxy-4- frans-hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520:
- R 1 represents [E]
- Ri 9c represents H
- R 2 represents CH 2 CH 3
- R 3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [D]
- R 20 and Rigb represent H
- R 2 represents CH 2 CH 3
- R 3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [A]
- R 12 represents OH
- R 11 and R 10 represent H
- R 2 represents CH 2 CH 3
- R 3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents G
- R 2 Ob represents OH
- R 19e and Ri 8a represent H
- R3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [J]
- R 22 represents OH
- R 21 , R 23 , R 24 and R 25 represent H
- R 3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [B]
- A represents S
- Ri 7 , Ri 5 , and Ri 4 represent H
- X b represents bond
- R3 0
- X a represents CH 2 , as shown by the following structure:
- Ri represents [B]
- A represents O
- Ri 7 , Ri 5 , and R 14 represent H
- X b represents bond
- X a represents CH 2 , as shown by the following structure, also known as Pyran FK520 or 28-des(3-c/s-methoxy-4- frans-hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520:
- R 1 represents [E]
- Ri 9c represents H
- R3 0
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [D]
- R 20 and R 19b represent H
- X a represents CH 2 , as shown by the following structure:
- R 1 represents [A]
- R 12 represents OH
- R 11 and R 1 O represent H
- R 3 0
- X a represents CH 2 , as shown by the following structure:
- Compounds of formula (I) may be produced by feeding an appropriate non-natural starter unit to an FK506 or FK520 producing strain, culturing the strain and optionally isolating the compounds thereafter.
- the strain is mutated to have targeted inactivation or deletion of one or more genes that contribute to the biosynthesis or regulation of precursor supply.
- the gene(s) that contribute to the biosynthesis or regulation of precursor supply may contribute to the biosynthesis or regulation of the a starter unit (starter acid) e.g. 4,5 dihydroxycyclohex-1-ene carboxylic acid, such as fkbO (Motamedi and Shafiee, 1998, Wu et al., 2000).
- the strain has the natural loading module of the FK506 or FK520 PKS replaced with that of the avermectin PKS or an avermectin-like PKS (such as the nemadectin or milbemycin PKS), and is then fed appropriate starter units, the strain cultured and the compounds optionally isolated.
- other genes may be inactivated, such as the gene(s) that contribute to the biosynthesis or regulation of precursor supply which may contribute to the biosynthesis or regulation of branched chain keto acid, such as bkd (Ward et al., 1999).
- the strain may be mutated to inactivate or delete one or more genes responsible for biosynthesis of pipecolic acid, such as fkbL (Wu et al., 2000). Pipecolic is naturally incorporated into the chain as a final step prior to ring closure. This particular modification increases the yield of prolyl derivatives of formula (I) when proline is fed to the strain.
- the strain may be mutated to have targeted inactivation or deletion of one or more genes responsible for post-PKS modification, for example the gene responsible for oxidation at the C-9 position, fkbJ (Motamedi and Shafiee, 1998, Wu et al., 2000). This particular modification increases the yield of C-9 desketo derivatives of formula (I).
- avermectin loading module may be subject, after their isolation, to synthetic alteration using processes known to a skilled person e.g. alkylation of hydroxyl and amino groups and the like. It should be understood by one skilled in the art that it may be possible to use alternative junctions to join the avermectin loading module to the rapamycin PKS. Examples of this include taking just the Acyltransferase (AT) and Acyl Carrier Protein (ACP) domains from the avermectin or avermectin-like PKS and joining to the FK506/FK520 PKS before the fist ketosynthase (KS) domain.
- AT Acyltransferase
- ACP Acyl Carrier Protein
- the junction may be made between the KS from the avermectin or avermectin-like PKS and the first extender AT from the FK506/FK520 PKS. Analogous possibilities are discussed in WO 98/01546. Additionally, homologues of the avermectin loading module may be used, such as those found in the milbemycin PKS or nemadectin PKS.
- a precursor supply gene such as fkbO or an fkbO homologue or the bkd genes or homologues thereof may be manipulated by targeted inactivation or deletion or modified by other means such as exposing cells to UV radiation and selection of the phenotype indicating that starter unit biosynthesis or branched chain alpha keto acid biosynthesis has been disrupted.
- the optional targeting of the post-PKS genes may occur via a variety of mechanisms, e.g. by integration, targeted deletion of a region of the FK506 or FK520 cluster including all or some of the post-PKS genes optionally followed by insertion of gene(s) or other methods of rendering the post-PKS genes or their encoded enzymes non-functional e.g. chemical inhibition, site-directed mutagenesis or mutagenesis of the cell for example by the use of UV radiation.
- WO2004/007709 provides methods for the alteration of a gene system which comprises a core portion responsible for the production of a basic product, and a multiplicity of modifying genes responsible for effecting relatively small modifications to the basic product - e.g. effecting oxidation, reduction, alkylation, dealkylation, acylation or cyclisation of the basic product, and a multiplicity of precursor supply genes which are involved in the production of particular precursor compounds.
- the basic product may be a modular polyketide and the modifying genes may be concerned with modifications of a polyketide chain (such as oxidation at the 9 position), and the precursor supply genes may be involved in the production and/or incorporation of natural or non-natural precursors (e.g. pipecolate and/or 4,5 dihydroxycyclohex-1-ene carboxylic acid).
- the core portion may not function properly or even at all in the absence of a precursor supply gene (unless a natural or unnatural precursor compound is supplied or is otherwise available). Therefore, the deletion or inactivation of a precursor supply gene provides a system where it is possible to incorporate non-natural starter units with no competition from the natural starter unit.
- the present invention provides a method for the incorporation of non-natural starter acids into FK506 and FK520 analogues, said method comprising feeding said starter units to a strain which contains a gene system in which the precursor supply gene has been deleted or inactivated, or the natural FK506/520 loading module has been replaced with the avermectin loading module, or a homologue thereof.
- Suitable gene systems include, but are not limited to, antascomicin, FK520 (Wu et al., 2000; U.S. 6,150,513; AF235504), FK506 (Motamedi et al., 1996; Motamedi et al., 1997; Motamedi and Shafiee, 1998; AF082100,
- the precursor supply gene which is deleted or inactivated is preferably fkbO or a homologue of fkbO.
- the gene system is preferably the FK506 or FK520 cluster.
- the precursor supply gene deleted or inactivated is more preferably fkbO.
- the precursor gene which is deleted or inactivated is involved in branched chain keto acid formation, such as one of the bkd genes, when the loading module is replaced with the avermectin loading module.
- fkbM and/or fkbl are deleted or inactivated in addition to fkbO.
- the present invention provides a method of producing compounds of formula (I) comprising:
- the precursor for which one or more starter genes have been deleted or inactivated may be a starter unit (e.g. starter acid). Additionally or instead the precursor for which one or more starter genes have been deleted or inactivated may be pipecolic acid. Optionally one or more genes responsible for post-PKS modification are also deleted or inactivated.
- a starter unit e.g. starter acid
- the precursor for which one or more starter genes have been deleted or inactivated may be pipecolic acid.
- one or more genes responsible for post-PKS modification are also deleted or inactivated.
- the natural KS1 module is replaced by the KS1 module of the avermectin or an avermectin-like PKS in addition to the loading module being so replaced as described above.
- the natural KS1 module is replaced by the KS1 module of the avermectin or an avermectin-like PKS in addition to the loading module being so replaced as described above.
- the recombinant strain is generated using the methods described in WO2004/007709 and in the examples below.
- the host strain is a selected from the group consisting of Streptomyces tsukubaensis No. 9993 (Ferm BP-927), Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822), Streptomyces sp. AA6554, Streptomyces hygroscopicus var. ascomyceticus MA 6475 ATCC 14891 , Streptomyces hygroscopicus var. ascomyceticus MA 6678 ATCC 55087, Streptomyces hygroscopicus var. ascomyceticus MA 6674, Streptomyces hygroscopicus var.
- the host strain is selected from the group consisting of: S. hygroscopicus var. ascomyceticus ATCC 14891 , Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) or Streptomyces tsukubaensis No. 9993 (Ferm BP-927).
- S. hygroscopicus var. ascomyceticus ATCC 14891 Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) or Streptomyces tsukubaensis No. 9993 (Ferm BP-927).
- DSM 40822 Streptomyces tsukubaensis No. 9993
- auxiliary genes may be deleted or inactivated in the host strain.
- one or more of the deleted or inactivated genes of the host strain may be reintroduced by complementation (e.g. at an attachment site, on
- polyketide gene clusters may be expressed in heterologous hosts (Pfeifer et al., 2001 ). Accordingly, the present invention includes the transfer of the FK506 or FK520 biosynthetic gene cluster with or without resistance and regulatory genes, either complete, engineered, containing mutations or containing deletions, for complementation in heterologous hosts. Methods and vectors for the transfer as defined above of such large pieces of DNA are well known in the art (Rawlings, 2001 ; Staunton and Weissman, 2001 ) or are provided herein in the methods disclosed.
- the present invention provides a method of producing compounds of formula (I) comprising: (a) generating a recombinant strain of a non-FK506 or FK520 producing host (i.e. a heterologous host) that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene is absent or non-functional or the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; and (b) feeding a non-natural starter unit to said recombinant strain;
- a non-FK506 or FK520 producing host i.e. a heterologous host
- a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene is absent or non-functional or the natural loading module has been replaced by the loading module from the avermectin
- a non-natural starter unit to a recombinant strain of a non-FK506 or FK520 producing host (i.e. a heterologous host) that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene is absent or non-functional or the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; (b) culturing said strain; and
- a preferred heterologous host cell strain is a prokaryote, more preferably an actinomycete or Escherichia coli, still more preferably include, but are not limited to S. hygroscopicus, S. hygroscopicus sp. , S. hygroscopicus var.
- PKS polyketide synthase
- FK506 or FK520 analogue or other polyketide analogue a modified FK506 or FK520 analogue or other polyketide analogue.
- PKS in a homologous or heterologous could be a hybrid PKS in which one or more domains have been removed, replaced or inserted, such replacements or insertions coming from other heterologous (or homologous) PKS clusters.
- actinomycetes contain multiple biosynthetic gene clusters for different secondary metabolites, including polyketides and non-ribosomally synthesised peptides. Specifically, it has been demonstrated that strains of S. hygroscopicus produce a variety of polyketides and non-ribosomally synthesised peptides in addition to FK506, FK520, FK523, meridamycin, FK525, antascomicin or tsukubamycin.
- biosynthetic gene clusters represent a competing requirement for biosynthetic precursors and an additional metabolic demand on the host strain.
- elaiophylin elaiophylin, bialaphos, hygromycin, augustmycin, endomycin (A, B), glebomycin, hygroscopin, ossamycin and nigericin.
- additional biosynthetic gene clusters represent a competing requirement for biosynthetic precursors and an additional metabolic demand on the host strain.
- the starter unit is selected from the following carboxylic acids or carboxylic acid derivatives: wherein R 10 , Rn and Ri 2 are independently selected from H, F, Cl, ORi 3 , SRi 3 and NHRi 3 and Ri 3 is selected from H, d-C 4 alkyl and Ci-C 4 acyl; or
- A is selected from O, S and NRi 3a
- Ri 3a represents H, Ci-C 4 alkyl or Ci-C 4 acyl
- Ri 4 and Ri 5 are independently selected from H, Ci-C 4 alkyl
- Ri6 represents CrC 4 alkyl or Ci-C 4 acyl
- X b represents a bond or CH 2
- Ri 7 represents H and when X b is CH 2 then
- R 17 represents H, F, Cl, OH, SH
- ORi 63 , SRi 6a or NHRi 63 and Ri 6a represents Ci-C 4 alkyl or Ci-C 4 acyl; or
- Ri 3 b represents H, Ci-C 4 alkyl, or Ci-C 4 acyl
- Ri 4a represents Ci-C 4 alkyl
- Ri6 represents Ci-C 4 alkyl or Ci-C 4 acyl
- Ri 8 represents H, F, Cl, OH, SH, Ci-C 4 alkyl
- R i6a represents Ci-C 4 alkyl or Ci-C 4 acyl
- X c and X d independently represent bond or CH 2 provided that X c and X d do not both represent CH 2 and when X c is bond then Ri 7a represents H and when X c is CH 2 then Ri 7a represents H, F, Cl, OH, SH, Ci-C 4 alkyl, ORi 6b , SRi 6b or NHRi 6b , Ri 6b represents Cr
- Ri 9b and R 2 o independently represent H, F, Cl, OH, SH, d-C 4 alkyl, ORi 6 d, SRi6d or NHRi6d, and Ri 6 d represents Ci-C 4 alkyl or d-C 4 acyl; or
- Ri 9c represents H, F, Cl, OH, SH, C r C 4 alkyl, ORi 6e , SR 16e or NHRi 6e , and R i6 e represents d-C 4 alkyl or d-C 4 acyl; or
- R 19d and R 2 Oa independently represent H, F, Cl, OH, SH, d-C 4 alkyl, ORi 6f ,
- SRi6f or NHR 16 f and Ri 6 f represents d-C 4 alkyl or d-C 4 acyl ; or wherein R 18a , Ri9e and R 2 Ob independently represent H, F, Cl, OH, SH, d-C 4 alkyl, ORi6g, SRi6g or NHR 16g and Ri 6g represents CrC 4 alkyl or d-C 4 acyl; or
- R 2 i, R 22 and R 2 3 independently represent H, F, Cl, OR 2 6, SR 2 6, CrC 4 alkyl or CN, R 2 6 represents H, d-C 4 alkyl or d-C 4 acyl, R 24 and R 25 independently represent H, F, Cl, OH, SH or CrC 4 alkyl, provided that at least one of R 2 i, R 22 and R 23 is not H or d-C 4 alkyl; or
- R 21a , R 22a and R 23a are independently selected from H, F, Cl, OR 26 , SR 26 , C1- C4 alkyl and CN, R 26a represents H, d-C 4 alkyl or d-C 4 acyl, R 24a and R 25a independently represent H, F, Cl, OH, SH or d-C 4 alkyl and provided that at least one of R 2 i a , R 22a and R 23a does not represent H or d-C 4 alkyl.
- Starter units are suitably provided as the free carboxylic acid, but derivatives that may be employed include salts and esters.
- the aforementioned starter unit substances are either known or may be prepared by a skilled person using conventional methods.
- Standard methods known to those of skill in the art may be used to culture the host or recombinant strain in order to produce compounds of formula (I). Such methods include, without limitation, those described in the examples below; additional methods may also be found in Reynolds and Demain, 1997 and references therein.
- Compounds of formula (I) are useful as pharmaceuticals for example, but without limitation, having potential utility as immunosuppressants, antifungal agents, anticancer agents, neuroregenerative agents, or agents for the treatment of psoriasis, rheumatoid arthritis, fibrosis and other hyperproliferative diseases.
- the invention provides for the use of a compound of formula (I) as disclosed herein, in the preparation of a medicament for the prophylaxis and/or treatment of organ rejection after transplantation, autoimmune diseases, inflammatory disorders, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders.
- the invention provides for a method of treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders comprising administering a compound of formula (I) to a subject in need thereof.
- the compounds of formula (I) disclosed herein may be used in the preparation of a medicament for the prevention of organ allograft rejection.
- the compounds of formula (I) are used in the preparation of a medicament for the treatment of autoimmune diseases or inflammatory disorders.
- the compounds of this invention are useful for inducing immunosuppression and therefore relate to methods of therapeutically or prophylactically inducing a suppression of a human's or an animal's immune system for the treatment or prevention of rejection of transplanted organs or tissue, the treatment of autoimmune, inflammatory, proliferative and hyperproliferative diseases (examples include but are not inclusively limited to autoimmune diseases, diabetes type I, acute or chronic rejection of an organ or tissue transplant, asthma, tumours or hyperprolific disorders, psoriasis, eczema, rheumatoid arthritis, fibrosis, allergies and food related allergies).
- Such assays are well known to those of skill in the art, for example but without limitation: Immunosuppressant activity - Warner, LM.,et al., 1992, Kahan et al. (1991 ) & Kahan & Camardo, 2001 ); Allografts - Fishbein, T.M., et al., 2002, Kirchner et al. 2000; Autoimmune / Inflammatory / Asthma - Carlson, R.P. et al., 1993, Powell, N. et al., 2001 ; Diabetes I - Rabinovitch, A. et al., 2002; Psoriasis - Reitamo, S.
- the ability of the compounds of this invention to induce immunosuppression may be demonstrated in standard tests used for this purpose.
- the compounds of this invention are useful in relation to antifibrotic, neuroregenerative and anti-angiogenic mechanisms, one skilled in the art would be able by routine experimentation to determine the ability of these compounds to prevent angiogenesis (e.g. Guba, M.,et al., 2002, ).
- the compounds of formula (I) are also, or in particular, expected to be useful as a therapeutic or prophylactic agents for one or more of the following conditions: rejection reactions after transplantation of organs or tissues (for example heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, intervertebral disc, trachea, myoblast and cartilage); graft-versus-host reactions following bone marrow transplantation; autoimmune diseases (for example rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes); infections caused by pathogenic microorganisms, in particular fungal infections; inflammatory or hyperproliferative skin diseases or cutaneous manifestations of immunologically-mediated diseases (for example psoriasis, atopic dermatitis, contact dermatitis, eczematoid dermatitis, py
- the compounds of formula (I) may also be useful as a therapeutic or prophylactic agents for Bronchiolitis Obliterans Syndrome (BOS).
- BOS Bronchiolitis Obliterans Syndrome
- the aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including topically (for example by inhalation, vaginally, intranasally, or by eye or ear drop), enterally (for example orally or rectally) or parenterally (for example by intravenous, intracavernosal, subcutaneous, intramuscular, intracardiac or intraperitoneal injection) or via a medical device (for example via a stent).
- the treatment may consist of a single dose or a plurality of doses over a period of time.
- a compound of the invention Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more physiologically acceptable diluents or carriers.
- the diluents or carrier(s) must be "physiologically acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. In some cases, the diluent or carrier will be water or saline which will be sterile and pyrogen free.
- the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformLy and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
- Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
- Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
- Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
- the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
- a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
- Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
- Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
- the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
- Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
- the active ingredient may also be presented as a bolus, electuary or paste.
- Aerosol formulations suitable for administering via inhalation can also be made using methods known in the art. Examples of this include administration of the compounds of the invention by inhalation in the form of a powder (e.g. micronized) or in the form of atomized solutions or suspensions.
- the aerosol formulation may be placed in a suitable pressurized propellant, and may be used with additional equipment such as nebulizer or inhaler.
- compositions are preferably applied as a topical ointment or cream.
- the active agent may be employed with either a paraffinic or a water-miscible ointment base.
- the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
- a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. 5,399,163; U.S. 5,383,851 ; U.S. 5,312,335; U.S. 5,064,413; U.S.
- implants and modules useful in the present invention include : US 4,487,603, which discloses an implantable micro- infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi- chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.
- the compounds can be administered as the sole active agent, or in combination with other pharmaceutical agents, such as other agents that stimulate or inhibit cell proliferation of immune responses.
- agents include e.g. cyclosporine, rapamycin, FK506, leflunomide, butenamides, corticosteroids, Doxorubicin, and the like.
- each active ingredient can be administered either in accordance with its usual dosage range, or at a lower dose level.
- the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
- Pharmaceutical compositions of the invention may optionally contain further active ingredients.
- strains wherein the strain is a FK506 or FK520 producing strain which is Streptomyces hygroscopicus subsp hygroscopicus or Streptomyces tsukubaensis;
- Figure 1 Diagram describing the methodology used for generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
- Figure 2 Diagram describing the methodology used for generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
- Figure 3 Diagram describing the methodology used for generation of a Streptomyces tsukubaensis strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
- FK506, FK520 or the Pyran FK520 as a single 1 mg/kg Lv. dose (filled diamonds) or 10mg/kg p.o. dose (open diamonds), with concentration measured in whole blood (mean of triplicates in ng/mL) compared to time after dosing (in hours).
- Vector pKC1139B01 was obtained by inserting a linker into pKC1 139 (Bierman et al., 1992).
- the 674bp BgIW PvuW fragment of pKC1 139 was replaced by the annealing product of oligos B01 and B02 to give the plasmid pKC1 139B01
- Escherichia coli DH 10B (GibcoBRL) was grown in 2xTY medium as described by
- E. coli VCS257 was used for transfection of in vitro packaged cosmids. According to the instructions of
- E. coli transformants were selected for with 100 mg/L ampicillin or 50 mg/L apramycin.
- the avermectin producer Streptomyces avermitilis (DSM41443 / ) was grown on TSB at 28 ° for genomic DNA isolation.
- the FK506 producer Streptomyces tsukubaensis no. 9993 (FERM BP-927) (International Patent Organism Depositary, Tsukuba, Japan) and its derivatives were maintained on medium 1 agar plates or ISP4, ISP3,or ISP2 (see below) at 28 0 C.
- FK520 was carried out by fermentation of Streptomyces hygroscopicus subsr. hygroscopicus (DSM 40822), also termed BIOT-4081. A single spore isolate of this strain, terme BIOT-4168, was also used. Streptomyces tsukubaensis no. 9993 (FERM BP-927), also termei
- BIOT-31 19 was used for producing FK506.
- BIOT-420f A single spore isolate of this strain, termed BIOT-420f was also used.
- Polypropylene Centrifuge tubes (cat nc 227261 , purchased from Greiner Bio-One Ltd, Stonehouse, Gloucestershire, UK) with foam plugs or in Erlenmeyer flasks as described below.
- the culture tubes were incubated at 28°C, 300 rpm (2. cm throw) for 48 h. From the seed culture 0.5 ml. was transferred into 7 ml. production maximr PYDG or PYDG+MES in 50-mL Centrifuge tubes with foam plugs. Cultivation was carried out for days at 28°C and 300 rpm (2.5 cm throw). If necessary a selected starter unit was fed to thi production medium 24 hours post inoculation. Enough feed was dissolved in 0.05 ml. to 0.1 m methanol and added to the culture to give final concentration of 2.12 mM of the feed compound.
- Vegetativ cultures were prepared by removing 4-10 agar plugs (6 mm in diameter) from thn MAM, ISP4, ISP3 or ISP2 plate and inoculating into 50-250 ml. medium NGY in 250 ml. or 2000 m Erlenmeyer flasks with foam plugs. The seed flasks were incubated at 28°C, 200-250 rpm (5 or 2. cm throw) for 48 h. From the seed culture 2-10% v/v was transferred into 50 or 250 ml. productio medium PYDG (or PYDG + MES) in 250 ml. or 2000 ml.
- PYDG or PYDG + MES
- the pH may be maintained using acid or base addition on demand.
- the selected feed starting unit for biosynthesis of target compound
- thi production medium 12 - 24 hours post inoculation Enough feed was dissolved in 3 ml. to 5 m methanol and added to the culture to give final concentration of 2 mM of the feed compound. Th amount of methanol does not exceed 1 % of the total volume. Fermentation was continued fc further five days post-feeding Media Recipes
- Medium 1 Modified A-medium (MAM) component Source g/L
- Oatmeal is cooked/steamed in the water for 20 mins, strained through a muslin and more water added to replace lost volume.
- ISP Trace Elements Solution is added and pH adjusted to 7.2 with NaOH.
- Agar is added before autoclaving at 121 0 C, 15 minutes.
- the medium is adjusted to pH 7.0, with NaOH and then sterilised by autoclaving 121 0 C, 15 minutes.
- the medium is adjusted to pH 7.0 with NaOH, and then sterilised by autoclaving 121 0 C, 15 minutes.
- a paste is made using a little cold water and the starch. This is brought up to a volume of 500 ml_. All other ingredients are then added, and the pH of the media is adjusted to pH 7.0-7.4. Sterilise by autoclaving 121 0 C, 15 minutes.
- DNA manipulation and sequencing DNA manipulations were carried out as described in Sambrook et al. (1989).. DNA sequencing was performed as described previously (Gaisser et al., 2000). Genome sequencing was carried out using 454 technology (Margulies et al., 2005) at Cogenics and the University of Cambridge.
- STE buffer supplemented with 2 mg/mL lysozyme were added and the resuspension incubated for 30 min at 37 0 C.
- 20 DL of RNaseA (10 mg/mL) were added and the mixture incubated for another 30 min at 37 0 C. 4.8 mL EDTA (0.1 M final concentration) were added to stop the reaction.
- 1.4 ml. 20% SDS were added.
- the lysate was incubated on ice for 5 min, then extracted with one volume of phenol/chloroform/isoamylalcohol (25:24:1 ) and centrifuged at 2300 g and 4 0 C for at least 15 min up to 1 h.
- Extractions were repeated until no more protein was visible at the interface, followed by a final chloroform/isoamylalcohol (49: 1 ) extraction.
- the upper phase was precipitated with 1/10 vol. 5 M NaCI and 1 vol. cold isopropanol. After a few min, the DNA was spooled out with a glass rod and washed in ice cold 70% EtOH. After brief drying, the recovered DNA was dissolved in 0.5 - 1 ml. TE 10:1.
- the proteinase K method (Kieser et al., 2000) was also applied successfully to recover genomic DNA from S. tsukubaensis.
- a cosmid library of genomic DNA of S. tsukubaensis was constructed. High molecular weight DNA from several genomic DNA preps was partially digested with BfuC ⁇ , an isoschizomer of Sau3A, to a mean size of 30 - 60 kb, ligated to Supercos-1 , packaged into A phage using Gigapack® I I I XL Packaging Extract (Stratagene) and transfected into Escherichia coli VCS257. The titre was 6.7 x 10 5 cfu / ⁇ g vector. DNA of 10 cosmids was isolated and digested with EcoRI to check the insert size which was 40 kb on average.
- 2000 clones were grown in 96-well microtitre plates (150 ⁇ l_ LB AmplOO Kan50 per well) at 37 0 C and frozen at -80 0 C after mixing wells with 50 ⁇ L LB/glycerol 1 :1.
- a DIG labeled fkbO probe was used to detect cosmids containing this region of the FK506 biosynthetic cluster.
- the probe was prepared by PCR using DIG labeled dNTP mix (Roche). It comprises 410 bp of 3'-terminal fkbO sequence. Sequence information for primer design had been obtained by 454 sequencing of BIOT-31 19 - see Fig. 1 and SEQ ID 1. Primer sequences were:
- UES2for (Seq ID 2) 5'-CACTCCTTCGATCTCCACGAGCAGGTCGCCACGGGC-S' and UES2rev (Seq ID 3) 5'-ACCCTGCCGTCCTCACGGCACACCACTACCCCACGG-S'. Annealing temperatures between 66 and 71 0 C and extension for 20 sec at 68 0 C proved to be successful.
- DNA was crosslinked by exposing membranes to UV (302nm) for 5 min. Membranes were kept between two sheets of filter paper soaked with 2xSSC at 4 0 C or used immediately for hybridization. Hybridization was carried out using standard hybridization buffer and DIG labeled fkbO probe (see above) at a hybridization temperature of 68 0 C. Stringent washes were performed at 68 0 C. The nonradioactive DIG Nucleic Acid Detection kit from Roche was used to identify 5 positive clones on 4 library plates. The procedure followed the instructions of the DIG Application Manual for Filter Hybridization (Roche).
- Fresh spores were harvested in water from plates of Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) and were heat-shocked at 50 0 C for 10 minutes. These were then mixed with the E. coli donor strain, which had been washed twice with 2xTY, in a ratio of 1 :3 streptomycete to E. coli, and plated on R6 medium, incubating at 28°C. After -20 hr, the plates were overlaid with water containing 2 mg of apramycin sulphate and 1 mg of nalidixic acid and incubation continued at 28°C.
- Escherichia coli ET12567 harbouring the plasmid pUZ8002 (Macneil et al., 1992, Paget et al., 1999) was transformed with pAES10 by electroporation to generate the E. coli donor strain for conjugation.
- This strain was used to transform Streptomyces tsukubaensis no. 9993 (FERM BP-927) by spore conjugation (Kieser et a 1.2000).
- Fresh spores were harvested in water from plates of Streptomyces tsukubaensis no. 9993 (FERM BP-927) and were heat-shocked at 50 0 C for 10 minutes. These were then mixed with the E. coli donor strain, which had been washed twice with 2xTY, in a ratio of 3:1 Streptomycete to £. coli, and the mixture shaken at 37°C, 200 rpm, 1 " throw for 3 hr. The conjugation mixture was then plated on R6 medium, incubating at 37°C. After -20 hr, the plates were overlaid with water containing 2 mg of apramycin sulphate and 1 mg of nalidixic acid and incubation continued at 37°C. After 4-6 days, apramycin resistant colonies were seen, indicating integration of the pAESI O plasmid into the genome.
- Culture broth (0.9 ml.) is added to an Eppendorf tube (2.0 ml.) and ethyl acetate (0.9 mL) added.
- the broth is mixed with the solvent for 30 minutes on a shaking platform (vibrax) at 400 rpm.
- the phases are then separated by centrifugation (10 minutes, 13,200 rpm).
- An aliquot of the organic layer (0.1 mL) is then transferred to either a clean glass LC-vial or a vial containing 0.005 mg of pimecrolimus (as an internal standard for quantification).
- the solvent is removed in vacuo and then residue re-dissolved in methanol (1 mL) by gentle agitation on a shaking platform (5 minutes).
- the HPLC system comprised an Agilent HP1100 equipped with a Hyperclone ODS2,
- LC samples that have been spiked with 0.005 mg/mL pimecrolimus were analysed on the same instrument and with the same chromatographic conditions.
- MS was conducted in multiple reaction monitoring mode (MRM mode) in order to quantify the amount of FK analog in the sample. Details of the quantification are:
- the amount of analyte present is then calculated by dividing the integral for the analyte transition (as detailed above) with that for the internal standard, pimecrolmius. This ratio is then compared with a standard calibration curve for FK520 or FK506 up to 100 ng on column with 50 ng on column pimecrolimus.
- NMR spectra 1 H, 13 C, DQF-COSY, TOCSY, HMQC, HMBC, NOESY
- 500 MHz for proton derived spectra, pro rata for other nuclei
- Chemical shifts are described in parts per million (ppm) and are referenced to solvent signal e.g. CHCI3 at ⁇ 7.26 ( 1 H) and CHCI 3 at ⁇ c 77.0 ( 13 C). J values are given in Hertz (Hz).
- NFAT-bla Jurkat cells were thawed and resuspended in Assay Media (OPTI-MEM, 0.5% dialyzed FBS, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 100 U/mL/100 ⁇ g/mL Pen/Strep) to a concentration of 781 ,250 cells/mL.
- 4 ⁇ L of a 10X serial dilution of the test compounds were added to appropriate wells of a TC-Treated assay plate.
- 32 ⁇ L of cell suspension was added to the wells and pre- incubated at 37°C/5% CO 2 in a humidified incubator with test compounds for 30 minutes.
- Anti CD4:CD8 activator at the pre-determined EC80 concentration was added to wells containing the test compounds. The plate was incubated for 5 hours at 37°C/5% CO 2 in a humidified incubator. 8 ⁇ L of 1 ⁇ M Substrate Loading Solution was added to each well and the plate was incubated for 2 hours at room temperature. The plate was read on a fluorescence plate reader.
- Water solubility may be tested as follows: A 10 mM stock solution of the sanglifehrin analogue is prepared in 100% DMSO at room temperature. Triplicate 0.01 mL aliquots are made up to 0.5 mL with either 0.1 M PBS, pH 7.3 solution or 100% DMSO in amber vials. The resulting 0.2 mM solutions are shaken, at room temperature on an IKA® vibrax VXR shaker for 6 h, followed by transfer of the resulting solutions or suspensions into 2 mL Eppendorf tubes and centrifugation for 30 min at 13200 rpm. Aliquots of the supernatant fluid are then analysed by the LCMS method as described above. Assessment of cell permeability
- Test compound was dissolved in DMSO and then diluted further in buffer to produce a final 1 ⁇ M dosing concentration. The fluorescence marker lucifer yellow was also included to monitor membrane integrity. Test compound was then applied to the apical surface of Caco-2 cell monolayers and compound permeation into the basolateral compartment is measured. This is performed in the reverse direction (basolateral to apical) to investigate active transport. LC-MS/MS was used to quantify levels of both the test and standard control compounds (such as Propanolol and Acebutolol).
- In vivo assays may also be used to measure the bioavailability of a compound.
- a compound is administered to a test animal (e.g. mouse) both intravenously (i.v.) and orally ⁇ p.o.) and blood samples are taken at regular intervals to examine how the plasma (or whole blood) concentration of the drug varies over time.
- the time course of plasma (or whole blood) concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models.
- Example 1 Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
- a 5' extension was designed in oligo SG167 to introduce the restriction site to aid cloning of the amplified fragment ( Figure 1 ) and SG168 included the restriction site present in the genomic sequence.
- This 2034 bp fragment was cloned into pUC19 that had been linearised with Sma ⁇ , resulting in plasmid pAESBI and the insert verified by sequencing.
- pAESABI The -1.7 kb Bgl ⁇ /Xho ⁇ fragment from pAESA5 and -2.0 kb Xho ⁇ /EcoR ⁇ fragment from pAESBI were cloned into the -5.9 kb Sg/ll/EcoRI fragment of pKC1 139 (Bierman et al., 1992) to make pAESABI .
- pAESABI therefore contained the upstream and downstream regions such that the double crossover event would result in the desired small deletion, introducing a frameshift in the fkbO gene.
- Escherichia coli ET12567 harbouring the plasmid pUZ8002, was transformed with pAESABI by electroporation to generate the E. coli donor strain for conjugation.
- This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) by spore conjugation (as described in General Methods). Exconjugants were plated on R6 agar and incubated at 28°C.
- pAESABI is able to self-replicate in Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) at 28°C.
- Transformants were subcultured onto MAM plates with apramycin (0.050 mg/mL) at 28°C to ensure the pAESABI plasmid with resistance marker was present. Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured again on to MAM plates with apramycin at 37°C to induce the plasmid to integrate, as the plasmid cannot self-replicate at 37°C. The transformants were then subcultured for four subsequent rounds at 37°C on MAM plates with no antibiotic. The transformants from the fourth subculture on antibiotic free plates were plated for spore harvest on ISP3 medium at 28°C. Serial dilutions were made from the filtered collected spores and were plated on MAM plates to achieve single colonies.
- FK production medium See Media Recipes
- 50 ml. falcon tube at 28°C, 300 rpm with a 1 inch throw.
- each falcon tube was fed with 0.050 ml. 0.32 M 4-trans- hydroxycyclohexane carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation was continued as before.
- the cultures were sampled after 6 days growth and analysed by LC-MS, using the methods described above.
- Example 2 Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
- 2.1 Cloning of DNA homologous to the upstream flanking region of fkbO disruption.
- Oligos SG165 SEQ ID NO: 1
- SG166 SEQ ID NO: 2
- aesFK520A SEQ ID NO: 10
- DSM 40822 standard PCR reaction
- genomic DNA Kieser et a/.2000
- a 5' extension was designed in each oligo to include the restriction sites present in the genomic sequence to aid cloning of the amplified fragment ( Figure 2).
- This 1727 bp fragment was cloned into pUC19 that had been linearised with Sma ⁇ , resulting in plasmid pAESA5 and the insert verified by sequencing.
- a 5' extension was designed in oligo SG169 to introduce the restriction site to aid cloning of the amplified fragment (Figure 2) and SG168 included the restriction site present in the genomic sequence.
- This 1460 bp fragment was cloned into pUC19 that had been linearised with Sma ⁇ , resulting in plasmid pAESC ⁇ and the insert verified by sequencing.
- pAESAC4 therefore contained the upstream and downstream regions such that the double crossover event would result in the desired large deletion of the fkbO gene.
- FK production medium See Media Recipes
- 50 ml. falcon tube at 28°C, 300 rpm with a 1 inch throw.
- each falcon tube was fed with 0.050 ml. 0.32 M 4-frans-hydroxy cyclohexane carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation was continued as before.
- the cultures were sampled after 6 days growth and analysed by LC-MS using the methods described above.
- Example 3 Generation of a Streptomyces tsukubaensis strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift. 3.1 Cloning of DNA homologous to the upstream flanking region of fkbO disruption.
- Oligos AES43 (SEQ ID NO: 6) and AES40 (SEQ ID NO: 7) were used to amplify a 2057 bp region of DNA, termed aesFK506D1 1 (SEQ ID NO: 13) from Streptomyces tsukubaensis no. 9993 (FERM BP-927) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et al., 2000) as the template and hot start KOD DNA polymerase.
- a 5' extension was designed in each oligo to introduce the restriction sites to aid cloning of the amplified fragment (Figure 3). This 2057 bp fragment was cloned into pUC19 that had been linearised with Sma ⁇ , resulting in plasmid pAES ⁇ and the insert verified by sequencing.
- Oligos AES41 SEQ ID NO: 8 and AES42 (SEQ ID NO: 9) were used to amplify a 1985 bp region of DNA, termed aesFK506E3 (SEQ ID NO: 14) from Streptomyces tsukubaensis no. 9993 (FERM BP-927) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et al., 2000) as the template and hot start KOD DNA polymerase.
- a 5' extension was designed in each oligo to introduce the restriction sites to aid cloning of the amplified fragment ( Figure 3).
- This 1985 bp fragment was cloned into pUC19 that had been linearised with Sma ⁇ , resulting in plasmid pAES9 and the insert verified by sequencing.
- pAES10 therefore contained the upstream and downstream regions such that the double crossover event would result in the desired small deletion, introducing a frameshift in the fkbO gene.
- Escherichia coli ET12567 harbouring the plasmid pUZ8002, was transformed with pAES10 by electroporation to generate the E. coli donor strain for conjugation.
- This strain was used to transform Streptomyces tsukubaensis no. 9993 (FERM BP-927) by spore conjugation (as described in General Methods).
- Exconjugants were plated on R6 agar and incubated at 37°C.
- pAESIO is not able to self-replicate in Streptomyces tsukubaensis no. 9993 (FERM BP-927) at 37°C and must integrate into the genome.
- Transformants were subcultured onto MAM plates with apramycin (0.050 mg/mL) at 37°C two to three times, to ensure that the pAESI O plasmid with resistance marker had integrated. Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured for two subsequent rounds at 37°C on MAM plates with no antibiotic and then a final time at 28°C. The transformants from the fourth subculture on antibiotic free plates were plated for spore harvest on ISP4 medium at 28°C. Serial dilutions were made from the filtered collected spores and plated on ISP4 plates at 28°C to achieve single colonies.
- Example 4 Array feeding to BIOT-4131 with various starter units
- Spore stocks of BIOT-4131 were prepared after growth on MAM, ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0 C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 mL medium NGY in 50 mL Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h.
- the solvent was removed under reduced pressure to impregnate the silica with the crude extract.
- the impregnated silica was dry packed above silica (1 10 g) in a column with a 5 cm diameter.
- the column was conditioned with and eluted with ethyl acetate / hexane (1 :1 , 1 litre), ethyl acetate (1 litre). After a 250 ml. pre-fraction, 50 ml. fractions were collected and the target compound identified in fractions 2 - 10. The fractions were combined and taken to dryness (500 mg).
- This material was impregnated onto silica (4 g) and this material was dry- packed onto silica (20 cm x 2.5 cm diameter) and conditioned with ethyl acetate / hexane (1 :2, 300 ml.) and eluted with ethyl actate / hexane (1 :1 , 300 ml.) , ethyl acetate / hexane (2:1 , 300 ml.) and finally ethyl actate. After a prefraction (150 ml.) 15 ml. fractions were collected and the target compound was identified in fractions 13 to 20. These were combined and taken to dryness under reduced pressure (133 mg).
- Example 6 Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the loading module of the FK520 PKS (comprising DHCHCA CoA ligase-like domain, enoyl CoA reductase and ACP) is replaced by the loading module of the avermectin PKS (comprising AT and ACP) from Streptomyces avermitilis.
- the loading module of the FK520 PKS comprising DHCHCA CoA ligase-like domain, enoyl CoA reductase and ACP
- PCR1 1 F and PCR1 1 R were used to amplify a 2.19 kb region of DNA from Streptomyces hygroscopicus subsp. hygroscopicus (DSM40822) using genomic DNA template (Kieser et al., 2000) and the KOD Polymerase kit from Novagen. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 65 and 70 0 C and extension for 1 min at 70 0 C were used. A 5' extension was designed for each oligo to introduce restriction sites (Spel, ⁇ / ⁇ tel) to aid cloning of the amplified fragment.
- the Nde ⁇ site of PCR1 1 R comprises the loading module start codon (ATG), and the 3 non-coding bases upstream are mutated to CAT (replacing TCC).
- the 2.19 kb PCR fragment was ligated with Sma ⁇ digested and SAP-treated pUC19, resulting in plasmid pUC19 PCR1 1.
- the insert sequence was verified by sequencing.
- PCR11 F (Seq ID 18) CGACTAGTGCAGCGCGAGCGTGTTGACGAACATGCCGATCAGG
- a 5' extension was designed for each oligo to introduce restriction sites ⁇ Nhe ⁇ , Xba ⁇ ) to aid cloning of the amplified fragment.
- the 2.20 kb PCR fragment was ligated with Sma ⁇ digested and SAP-treated pUC19.
- a construct was selected with the insert orientated such that the Nhe ⁇ site of the PCR fragment would be close to the Nde ⁇ site of pUC19. This was relevant for the subsequent cloning procedure and the resulting plasmid was named pUC19 PCR13F.
- the insert sequence was verified by sequencing.
- PCR13F2 comprises the 5' end of KS1 and the choice of Nhe ⁇ introduces a mutation resulting in the KS1 N-terminal sequence of 'DPLA' instead of the original FK520 KS1 sequence 'DPVA'.
- PCR13F2 (Seq ID 20)
- Oligos PCR12F and PCR12R were used to amplify a 1.46 kb region of DNA from Streptomyces avermitilis (DSM41443 / ) using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were supplemented with 1 0% DMSO . An nealing temperatures between 65 and 70 0 C and extension for 1 min at 70 0 C were used. A 5' extension was designed for each oligo to introduce restriction sites ( ⁇ /c/el, Apa ⁇ ) to aid cloning of the amplified fragment.
- PCR12F comprises the 5' end of ave loading module and the choice of ⁇ /c/el introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS.
- PCR12R comprises the 5' end of KS1 and as mentioned for PCR13F2 the choice of Nhe ⁇ introduces a mutation resulting in the KS1 N-terminal sequence of 'DPLA' instead of 'DPVA' in the FK520 cluster and 'DPIA' in the avermectin cluster.
- PCR12F (Seq ID 22)
- Escherichia coli ET12567 (pUZ8002) was transformed with pUS4. This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar, incubated at 37 0 C and overlaid with nalidixic acid (25 mg/L) and apramycin (50 mg/L) the next day. pUS4 is not able to self-replicate in Streptomyces hygroscopicus subsp. hygroscopicus at 37 0 C and is forced to integrate into the genome by recombination.
- transformants were subcultured on MAM plates containing apramycin (50 mg/L) at 37 0 C to ensure the pUS4 plasmid with resistance marker was present.
- Subculturing for two subsequent rounds at 37 0 C on MAM plates without apramycin was carried out to allow secondary recombination. This event would either cause the loss of the plasmid via the second region of homology, not the one by which it had originally integrated resulting in the desired gene replacement; or the loss of the plasmid via the same region of homology as the original integration resulting in a wildtype revertant. Unexpected recombination events could occur as well. This required single spore isolation of subcultured patches.
- the transformants were subcultured on MAM plates without apramycin at 28°C and subsequently incubated at 28 0 C on ISP3 plates for spore harvest. Serial dilutions were made from the collected spores and plated on MAM plates to achieve single colonies.
- FK production medium (see Media Recipes) in a 50 ml. falcon tube and incubated at 28 0 C, 300 rpm, 2.5 cm throw. The cultures were harvested after 6 days growth and analysed by LC-MS, using the methods described above. 61 out of 93 apramycin sensitive strains tested had undergone the desired recombination event. These strains produced FK520 derivates arising from the use of isobutyrate and 2-methylbutyrate starter units, most of them producing FK520 as well.
- Example 7 Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the loading module and KS1 of the FK520 PKS are replaced by the loading module and KS1 of the avermectin PKS from Streptomyces avermitilis.
- Oligos PCR23F and PCR23R were used to amplify a 2.15 kb region of DNA from Streptomyces hygroscopicus subsp. hygroscopicus using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 65 and 70 0 C and extension for 1 min at 70 0 C proved to be most successful. A 5' extension was designed for each oligo to introduce restriction sites (Apa ⁇ , Xba ⁇ ) to aid cloning of the amplified fragment. The 2.15 kb PCR fragment was ligated with Sma ⁇ digested and SAP-treated pUC19.
- PCR23F (Seq ID 24) TTGGGCCCTCGCGTGTGGAGTCGGGTGGTGATGGGTTG
- Oligos PCR12F and PCR22R were used to amplify a 2.75 kb region of DNA from Streptomyces avermitilis using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 65 and 70 0 C and extension for 1.5 min at 70 0 C proved to be most successful. A 5' extension was designed for each oligo to introduce restriction sites ( ⁇ / ⁇ tel, Apa ⁇ ) to aid cloning of the amplified fragment.
- PCR12F comprises the 5' end of ave loading module and the choice of Nde ⁇ introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS.
- PCR22R was designed such that the sequence of the amplified fragment ends slightly downstream of the KS1 gene where Apa ⁇ links to the FK520 sequence.
- the Spe ⁇ Nde ⁇ fragment of pUC19PCR1 1 was ligated with Spe ⁇ Nde ⁇ cut pKC1139B01 resulting in pUS1. Since the pUC19PCR22 insert contains 2 internal Apa ⁇ sites, a third single- cutting restriction enzyme (C/al) was chosen to aid cloning. C/al is affected by dam methylation.
- the dam " dcm " strain E. coli JM1 10 was transformed with pUC19PCR22 and the isolated plasmid digested with Nde ⁇ C/al and C/al Apa ⁇ .
- a 3-part ligation was performed with the 1.76 kb Nde ⁇ C/al fragment, the 0.98 kb C/al Apa ⁇ fragment and pUC19PCR23F cut with Nde ⁇ Apa ⁇ resulting in plasmid pUS3.
- the Nde ⁇ Xba ⁇ fragment of pUS3 was then cloned into pUS1 digested with Nde ⁇ Xba ⁇ , the final plasmid being pUS5.
- Escherichia coli ET12567 (pUZ8002) was transformed with pUS5 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar, incubated at 37°C and overlaid with nalidixic acid (25 mg/L) and apramycin (50 mg/L) the next day. pUS5 is not able to self- replicate in Streptomyces hygroscopicus subsp.
- hygroscopicus at 37 0 C and is forced to integrate into the genome by recombination.
- transformants Six days later, transformants were subcultured on MAM plates with apramycin (50 mg/L) at 37 0 C to ensure the pUS5 plasmid with resistance marker was present. Subculturing for two subsequent rounds at 37 0 C on MAM plates without apramycin was carried out to allow secondary recombination. This event would either cause the loss of the plasmid via the second region of homology, not the one by which it had originally integrated resulting in the desired gene replacement; or the loss of the plasmid via the same region of homology as the original integration resulting in a wildtype revertant. Unexpected recombination events could occur as well.
- FK production medium (see Media Recipes) in a 50 ml. falcon tube and incubated at 28 0 C, 300 rpm, 2.5 cm throw. The cultures were harvested after 6 days growth and analysed by LC-MS, using the methods described above. 2 out of 93 apramycin sensitive strains tested had undergone the desired recombination event. These strains produced FK520 derivates arising from the use of isobutyrate and 2-methylbutyrate starter units (FK520 background). Feeding experiments were undertaken with the two strains to further confirm the integration of the ave loading module sequence. After inoculation of FK production medium (see above), tubes were incubated at 28 0 C, 300 rpm, 2.5 cm throw.
- each falcon tube was fed with 50 ⁇ l_ 0.32 M cyclobutyl carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation was continued for five days.
- Culture extracts were analysed by LC-MS, using the methods described above. Both isolates designated “#1-7” and “#1-8” incorporated cyclobutyl carboxylic acid as a starter unit. Isolate #1-7 was designated as "BIOT-4230".
- Example 8 Generation of a Streptomyces tsukubaensis strain in which the loading module of the FK506 PKS (comprising DHCHCA CoA ligase-like domain, enoyl CoA reductase and ACP) is replaced by the loading module of the avermectin PKS (comprising AT and ACP) from Streptomyces avermitilis.
- the loading module of the FK506 PKS comprising DHCHCA CoA ligase-like domain, enoyl CoA reductase and ACP
- the avermectin PKS comprising AT and ACP
- FK506 cluster including fkbO and fkbP, but neither of them covering fkbB sufficiently.
- the 5 positive cosmids obtained via hybridization of the BIOT-31 19 cosmid library with an FK506 fkbO probe were end-sequenced.
- the alignment of end sequences with the FK520 cluster sequence showed that two of the cosmids contained fkbO, P, B completely.
- Oligos UES4_For and UES4_Rev were used to amplify a 2.27 kb region of DNA from Streptomyces tsukubaensis no. 9993 using cosmid 3G9 template and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures of 66 and 71 0 C and extension for 1 .5 min at 68 0 C were used. A 5' extension was designed for each oligo to introduce restriction sites (Spel, ⁇ / ⁇ tel) to aid cloning of the amplified fragment.
- the Nde ⁇ site of UES4_For comprises the loading module start codon (ATG), and the 3 non-coding bases upstream are mutated to CAT (replacing TCC).
- the 2.27 kb PCR fragment was ligated with Sma ⁇ digested and SAP-treated pUC19, resulting in plasmid 'pUC19 fkbP'O 506 left'. The insert sequence was
- Oligos UES7_For and UES7_Rev were used to amplify a 2.29 kb region of DNA from Streptomyces tsukubaensis no. 9993 using 3G9 cosmid and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 66 and 71 0 C and extension for 1 .5min at 68 0 C proved to be successful. A 5' extension was designed for UES7_Rev to introduce an Xba ⁇ site.
- UES7_For comprises the internal Pvu ⁇ site at the 5' end of ave KS1 followed by the internal Pvu ⁇ site of FK506 KS1 which was mutated to 'CCATCG'.
- Oligos PCR12F and UES5_Rev were used to amplify a 1.46 kb region of DNA from Streptomyces avermitilis using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 66 and 71 0 C and extension for 1 .5 min at 68 0 C were used. A 5' extension was designed for PCR12F to introduce an Nde ⁇ site, whereas UES5_Rev comprises the internal Pvu ⁇ site at the 5'-end of ave KS1.
- PCR12F comprises the 5' end of ave loading module and the choice of Nde ⁇ introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS.
- UES5_Rev comprises the 5' end of ave KS1 and introduces a mutation resulting in the KS1 N-terminal sequence of 'EPIA' instead of 'DPIA' in the avermectin cluster and as 'EPIA in the FK506 cluster (see 3.5).
- Escherichia coli ET12567 (pUZ8002) was transformed with pUS14 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces tsukubaensis by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar and incubated at 37°C. pUS14 is not able to self- replicate in Streptomyces tsukubaensis at 37 0 C and must integrate into the genome.
- Transformants were subcultured onto MAM plates with apramycin (100 mg/L) and nalidixic acid (50 mg/L) at 37 0 C and subcultured again onto MAM plates with apramycin (50 mg/L) and nalidixic acid (25 mg/L) at 37 0 C, and once more onto MAM plates containing apramycin (50 mg/L), to ensure that the pUS14 plasmid with resistance marker had integrated.
- Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured for three subsequent rounds at 37 0 C on MAM plates with no antibiotic and a final time at 28 0 C. The transformants from the last subculture on antibiotic free plates were plated for spore harvest on ISP4 medium at 28 0 C. Serial dilutions were made from the filtered collected spores and plated on MAM plates to achieve single colonies.
- Example 9 Generation of a Streptomyces tsukubaensis strain in which the loading module and KS1of the FK506 PKS are replaced by the loading module and KS1 of the avermectin PKS from Streptomyces avermitilis.
- Oligos UES10_For and UES10_Rev were used to amplify a 2.23 kb region of DNA from Streptomyces tsukubaensis using cosmid 3G9 template and KOD polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 66 and 71 0 C and extension for 1 .5 min at 68 0 C were used. A 5' extension was designed for each oligo to introduce restriction sites (Sfo ⁇ , Xba ⁇ ) to aid cloning of the amplified fragment.
- the 2.23 kb PCR fragment was ligated with Sma ⁇ digested and SAP-treated pUC19 resulting in plasmid 'pUC19 fkbB" 506 right'.
- the insert sequence was verified by sequencing.
- the choice of Sfo ⁇ as restriction site immediately downstream of fkbB KS1 introduces a mutation (GTG to GCG) with the translated code changing from VPEVS' to 'APEVS' (see 4.4).
- Oligos PCR12F and UES9_Rev were used to amplify a 2.75 kb region of DNA from Streptomyces avermitilis (DSM41443J using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were su pplemented with 1 0% DMSO. Anneal ing temperatures between 66 and 71 0 C and extension for 1 .5 min at 68 0 C proved to be most successful. A 5' extension was designed for each oligo to introduce restriction sites ( ⁇ /c/el, Afe ⁇ ) to aid cloning of the amplified fragment.
- PCR12F comprises the 5' end of ave loading module and the choice of ⁇ /c/el introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS.
- Escherichia coli ET12567 (pUZ8002) was transformed with pUS16 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces tsukubaensis by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar and incubated at 37°C. pUS16 is not able to self- replicate in Streptomyces tsukubaensis at 37 0 C and must integrate into the genome.
- Transformants were subcultured onto MAM plates with apramycin (100 mg/L) and nalidixic acid (50 mg/L) at 37 0 C and subcultured again onto MAM plates with apramycin (50 mg/L) and nalidixic acid (25 mg/L) at 37 0 C, and once more onto MAM plates containing apramycin (50 mg/L), to ensure that the pUS16 plasmid with resistance marker had integrated.
- Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured for three subsequent rounds at 37 0 C on MAM plates with no antibiotic and a final time at 28 0 C. The transformants from the last subculture on antibiotic free plates were plated for spore harvest on ISP4 medium at 28 0 C. Serial dilutions were made from the filtered collected spores and plated on MAM plates to achieve single colonies.
- PYDG+MES medium See Media Recipes
- Example 10 Array feeding to BIOT-4254 with various starter units
- Spore stocks of BIOT-4254 were prepared after growth on MAM , ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0 C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 ml. medium NGY in 50 ml. Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h. From the seed culture 0.5 ml.
- Example 11 Biological data - In vitro evaluation of potency with a NFAT gene reporter.
- IC50 values were additionally generated for FK506 and FK520 as controls.
- Example 12 Biological data - In vitro evaluation of cell permeability and efflux.
- both FK506 and FK520 show high levels of efflux, possibly due to the action of a transporter, such as Pgp.
- a transporter such as Pgp.
- 28-des(3-c/s-methoxy-4- frans-hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520 showed much higher apical to basolateral transport and lower basolateral to apical transport, suggesting that it is more permeable and less of a substrate for efflux, predicting improved and more consistent oral bioavailability.
- Example 13 Biological data - In vivo evaluation of PK.
- the oral bioavailability (F%) of the Pyran FK520 is higher than that for both FK506 and FK520, whilst the half life (T1/2) is shorter.
- Example 14 Array feeding to BIOT-4225 with various starter units
- Spore stocks of BIOT-4225 were prepared after growth on MAM, ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0 C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 ml. medium NGY in 50 ml. Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h. From the seed culture 0.5 ml.
- Example 15 Array feeding to BIOT-4276 with various starter units
- Spore stocks of BIOT-4276 were prepared after growth on MAM , ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0 C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 ml. medium NGY in 50 ml. Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h. From the seed culture 0.5 ml.
- Streptomyces hygroscopicus analysis of the enzymatic domains in the modular polyketide synthase. Gene 169: 9-16.
- Antascomicinc A, B, C, D and E Novel FKBP12 binding compounds from a
- Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Its effect on cellular, growth factor and cytokine response in injured vessels.
- FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in
- Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia. Proceedings of the National Academy of Sciences of the United States of America 91 :3191-3195. MacNeil, DJ. , Gewain, K.M., Ruby, C. L., Dezeny, G., Gibbons, P. H., and MacNeil, T. (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111 : 61-68. Maes, B. D. and Vanrenterghem, Y.F.Ch. (2004) Cyclosporine: Advantages Versus
- Streptomyces hygroscopicus analysis of genes flanking the polyketide synthase. Gene 169: 1-7.
- A mycophenolate mofetil, and sirolimus (rapamycin) inhibit allergen-induced proliferation and IL-5 production by PBMCs from atopic asthmatic patients.
- the biosynthetic gene cluster for the polyketide immunosuppressant rapamycin is the biosynthetic gene cluster for the polyketide immunosuppressant rapamycin.
- Streptomyces hygroscopicus var. ascomyceticus contains genes for biosynthesis of unusual polyketide extender units. Gene 251 : 81-90.
- Hsp56 component of steroid receptor complexes binds to immobilized FK506 and shows homology to FKBP-12 and FKBP-13. Journal of
- Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis. Gastroenterology. 117(5): 1198-204.
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Abstract
Compounds of formula (I) their use as medicaments and related aspects.
Description
NOVEL COMPOUNDS AND METHODS FOR THEIR PRODUCTION
Field of the Invention
The present invention relates to novel FK506 and FK520 analogues generated by feeding non-natural starter units to bacterial strains and to use of such compounds in therapy.
Background of the invention
FK506 (tacrolimus/fujimycin/Prograf) (Schreiber and Crabtree, 1992) and FK520 (ascomycin or immunomycin) (Wu et al., 2000) (Figure 1 ) are lipophilic macrolides produced by a variety of actinomycetes, including Streptomyces tsukubuaensis No. 9993 (Hatanaka et al., 1989), Streptomyces sp, MA8858, Streptomyces sp. MA6548, Streplomyces kanamyceticus KCC S-G433 and Streptomyces clavuHgerus CKD11 19 (Kim arid Park, 2008) which have been shown to produce FK506 (Muramatsu et al., 2005), and Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822), which is equivalent to Streptomyces hygroscopicus var ascomyceϋcus (ATCC 14891 ) and Streptomyces tubercidicus (Kόnya et aL 2008) producing FK520 (Garrity et aL 1993). Other closely related macrolides include FK525 also known as FR-900525 (Hatanaka H, et al., 1989), FK523 also known as FR-900523 (Hatanaka, H., et al., 1988) and antascomicins (Fehr, T., et al., 1996). A number of semisynthetic derivatives of these molecules have also been shown to be of utility, including pimecrolimus (SDZ ASM 981 , Elidel), which is a derivative of FK520 (Meingassner et al., 1997).
FK506, 1 : R1= -CH=CH2, R2 = trans-OH FK520, 2 : R1= -CH3, R2 = trans-OH FK523, 3 : R1= -H, R2 = trans-OH Pimecrolimus, 4 : R1= -CH3, R2 = c/s-CI
BIOSYNTHESIS: FK506 and FK520 are synthesised by type I polyketide synthases (PKS). This biosynthesis involves a shikimate derived starter unit, followed by 10 extensions utilising malonyl, or substituted malonyl derivative, thioesters, and addition of a lysine derived pipecolate group. The structure is then finally completed by O-methylation at C-31 and oxidation at C-9 (Motamedi et al., 1996). FK520 and FK506 differ at the C-21 position. FK520 has a C-21 ethyl substituent, whereas FK506 has a C-21 allyl substituent.
Meanwhile, the avermectin PKS naturally incorporates branched chain keto acids (Ikeda et al., 1999), but has been found to additionally incorporate a variety of other non- natural carboxylic acids when fed exogenously to fermentation broths (Dutton et al., 1991 ). This capability is encoded on the PKS by the loading module, which consists of a section of the PKS including an Acyltransferase domain (AT) and an Acyl Carrier Protein domain (ACP) (Ikeda et al., 1999).
It has been shown previously, that if the loading module, and optionally the first ketosynthase domain (KS), of the avermectin PKS replaces the natural loading module of another PKS, such as erythromycin, a broad variety of starter acids can be incorporated into the product of this hybrid PKS (Marsden et al., 1998, WO 98/01546).
Other examples of PKS with loading modules with similar specificities to the avermectin PKS include nemadectin (Carter et al., 1988) and milbemycin (Takiguchi et al., 1980; Okazaki et al., 1983). Methods for elucidating the sequence of PKS gene clusters have been published previously, examples include using homologous probes to screen cosmid libraries (e.g. Oliynyk et al., 2003, Fang et al., 2007, Choi et al., 2007) and full genome sequencing is another approach.
MECHANISM OF ACTION: FK506, FK520 and close analogues suppress the immune system by inhibiting signal transduction pathways required for T-cell activation and growth. In particular, they have been shown to inhibit Ca2+-dependent T-cell proliferation, via initial formation of a complex with an FK-binding protein (FKBP), which binds to and blocks calcineurin (CaN). This FK506-FKBP-CaN complex inhibits the activation of nuclear factor of activated t-cells (NF-AT), preventing its entrance into the nucleus, and subsequent activation of the promoter of lnterleukin-2 (IL-2), which initiates IL-2 production. Additionally, FK506 can interfere with the action of calcineurin on substrates other than NFAT, including IKB, Na-K- ATPase and nitric oxide synthase, which may lead to some of the side-effects (Kapturczak et al., 2004).
Treatment with FK506 also seems to be associated with up-regulation of transforming growth factor beta (TGF-β). This cytokine not only has immunosuppressive properties, but may be associated with the development of allograft fibrosis, which can lead to serious complications after long term treatment with these agents (Kapurtzak et al., 2004).
FK506 and FK520 also have antifungal activity (Heitman et al., 1991 , Brizuela et al., 1991 ).
USES: FK506, in particular, is an important immunosuppressant used to aid prevention of organ rejection after transplantation. For example, it is used intravenously and orally for the prevention of organ rejection after allogeneic liver or kidney transplantation and in bone marrow transplantation. It has been shown to have potential utility in a wide variety of autoimmune, inflammatory and respiratory disorders, including Crohn's disease, Behcet
syndrome, uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, nephritic syndrome, aplastic anaemia, biliary cirrhosis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease.
Treatment of many of these disorders is currently limited to patients with severe disease that are either refractory or hypersensitive to standard treatments. This limitation is due to the side effects of administration of FK506, which include renal dysfunction, gastrointestinal effects, neurological effects, hyperthrichosis and gingival hyperplasia.
Chronic treatment with FK506 requires strict therapeutic monitoring due to its narrow therapeutic index and great inter-individual variability which lead to dangers of over (or under) dosing (Roy et al., 2006).
Pimecrolimus and FK506 are both used in topical formulations, such as ointments and creams, as treatments for a variety of skin conditions, in particular atopic dermatitis (Nghiem et al., 2002).
Cytochrome P450 3A4 (Cyp3A4) and Cyp3A5 are the most important contributors to FK506 metabolism while the P-glycoprotein pump (MDR-1 ) modulates its bioavailability (Roy et al., 2006). The complexity of FK506 dosing is therefore enhanced by significant drug-drug interactions (Kapturczak et al., 2004).
The mechanism of toxicity of FK506 and FK520 has been related to the mechanism of action of immunosuppression (F. Dumont et al., 1992). This strong link between the mechanism of action and the toxicity has presented significant challenges to improving the therapeutic index through chemical modification. Segregation of efficacy and toxicity of new analogues may be possible by altering distribution or metabolism (NH Signal et al., 1991 ). By limiting the exposure of the compound to organs that are sensitive to such inhibition, such as the kidney, systemic toxicity can be avoided. Additionally, topical administration of the calcineurin inhibitor at the site of administration (such as skin, lungs, gut, eye etc.) can be maximized. One way this can be achieved is by using a 'soft drug' approach, which involves designing compounds to have limited systemic exposure such as through increased metabolism, higher blood/plasma protein binding, poor absorption or bioavailability.
It has also been suggested that the variable metabolism of FK506 leads to some of the toxicity, due to variable levels of systemic exposure, which led to the need for constant drug monitoring (Armstrong and Oellerich, 2001 ). Therefore, analogues of FK506 with reduced or less variable metabolism could be useful in reducing toxicity, and reducing the need for constant monitoring of drug levels.
FK506 is also poorly bioavailable (Tamura et al., 2003), which leads to variable systemic exposure when dosed orally, and the frequent need for intravenous dosing. Therefore, analogues with improved oral bioavailability would be very useful, to reduce systemic toxicity through incorrect dosing, and improve the ease of oral dosing.
Therefore, there remains a need to identify novel FK506 and FK520 analogues, which may have utility in the maintenance of immunosuppression, both for organ transplantation, and for the treatment of inflammatory conditions, and for the treatment of fungal infections. The present invention discloses novel FK506 and FK520 analogues which have improved pharmaceutical properties compared with the currently available FK506 and FK520 analogues; these properties may be useful for therapies requiring good systemic bioavailability, including, but not limited to oral therapies to maintain immunosuppression, which in particular are expected to show improvements in respect of one or more of the following properties: increased metabolic stability, increased bioavailability, increased oral bioavailability, reduced efflux via membrane transporters and low plasma protein binding; The novel FK506 and FK520 analogues may also be useful for therapies requiring local availability but with poor systemic availability, including, but not limited to topically administered therapies for inflammatory disorders such as atopic dermatitis, asthma and inflammatory bowel diseases, which in particular are expected to show improvements in respect of one or more of the following properties: decreased metabolic stability, decreased bioavailability, decreased oral bioavailability, increased efflux via membrane transporters and high plasma protein binding; Other properties the molecules might be expected to have that are of general use, include improved formulation ability, improved potency, increased FKBP binding, improved toxicological profile, reduced nephrotoxicity and neurotoxicity, improved crystallinity, improved water solubility or improved lipophilicity.
Summary of the Invention
In the present invention, non-natural starter units are fed to strains normally producing FK506 or FK520 or analogues thereof, said strains optionally having been mutated to have targeted inactivation or deletion of one or more genes responsible for post-PKS modification, and/or targeted inactivation or deletion of one or more precursor supply genes, including fkbO and homologues thereof.
Alternatively, the natural loading module from the FK506 or FK520 polyketide synthase is replaced by the loading module from the avermectin or an avermectin-like polyketide synthase, and optionally non-natural starter units are fed to these strains, said strains optionally having been mutated to have targeted inactivation or deletion of one or more genes responsible for post-PKS modification, and/or targeted inactivation or deletion of one or more precursor supply genes, including bkd genes (Ward et al., 1999) and homologues thereof. Surprisingly, unnatural starter acids have been found to be incorporated at different levels into the FK506 and FK520 systems when compared to the rapamycin system. In particular, pyrans are very well incorporated, leading to high yields of FK506 and FK520 analogues.
Thus in one aspect of the invention there is provided a compound of formula (I)
wherein
Xa represents bond or CH2;
R2 represents -CH3, -CH2CH3, Or -CH2CH=CH2;
R3 represents H2 or =0;
Ri is selected from the group consisting of
wherein R10, Rn and R12 are independently selected from H, F, Cl, OR13, SR13 and NHR13 and R13 is selected from H, CrC4alkyl and Ci-C4acyl; and
wherein A is selected from O, S and NR13a, R13a represents H, CrC4alkyl or Ci-C4acyl, R14 and R15 are independently selected from H, CrC4alkyl, OR16, SR16 and NHR16, R16 represents CrC4alkyl or Ci-C4acyl, Xb represents a bond or CH2, when Xb is bond then R17
represents H and when Xb is CH2 then R17 represents H, F, Cl, OH, SH, Ci-C4 alkyl, ORi6a, SRi6a or NHR163 and Ri6a represents Ci-C4alkyl or Ci-C4 acyl; and
wherein A represents O, S, or NR13b, Ri3b represents H, Ci-C4alkyl, or Ci-C4acyl, Ri4a represents Ci-C4alkyl, ORi6, SRi6 or NHRi6, Ri6 represents Ci-C4alkyl or Ci-C4acyl, Ri8 represents H, F, Cl, OH, SH, Ci-C4 alkyl, ORi63, SRi63 or NHRi63, Ri6a represents Ci-C4 alkyl or Ci-C4acyl, Xc and Xd independently represent bond or CH2 provided that Xc and Xd do not both represent CH2 and when Xc is bond then Ri7a represents H and when Xc is CH2 then Ri7a represents H, F, Cl, OH, SH, Ci-C4alkyl, ORi6b, SRi6b or NHRi6b, Ri6b represents CrC4 alkyl or CrC4 acyl and when Xd is bond Ri9 represents H and wherein when Xd represents CH2 then Rig represents H, F, Cl, OH, SH, CrC4 alkyl, ORi6c, SRi6c or NHRi6c, and Ri6c represents d- C4alkyl or Ci-C4acyl; and
wherein Ri9b and R20 independently represent H, F, Cl, OH, SH, CrC4 alkyl, ORi6d, SRi6d or NHRi6d, and Ri6d represents Ci-C4alkyl or CrC4 acyl; and
wherein Ri9c represents H, F, Cl, OH, SH, CrC4alkyl, ORi6e, SRi6e or NHRi6e, and Ri6e represents CrC4 alkyl or CrC4 acyl;
and
wherein R19d and R2Oa independently represent H, F, Cl, OH, SH, d-C4 alkyl, ORi6f, SRi6f or NHR16f and Ri6f represents CrC4 alkyl or d-C4 acyl ; and
wherein R18a, Ri9e and R2Ob independently represent H, F, Cl, OH, SH, d-C4 alkyl, ORi6g, SRi6g or NHR16g and Ri6g represents CrC4 alkyl or d-C4 acyl; and
wherein R2i, R22 and R23 independently represent H, F, Cl, OR26, SR26, CrC4 alkyl or CN, R26 represents H, d-C4 alkyl or d-C4 acyl, R24 and R25 independently represent H, F, Cl, OH, SH or CrC4 alkyl, provided that at least one of R2i, R22 and R23 is not H or d-C4 alkyl; and
wherein R2ia, R22a and R23a are independently selected from H, F, Cl, OR26a, SR26a, C1- C4 alkyl and CN, R26a represents H, d-C4 alkyl or d-C4 acyl, R24a and R25a independently represent H, F, Cl, OH, SH or d-C4 alkyl and provided that at least one of R2ia, R22a and R23a does not represent H or d-C4 alkyl; and physiologically functional derivatives thereof.
The above structure shows a representative tautomer and the invention embraces all tautomers of the compounds of formula (I) for example keto compounds where enol compounds are illustrated and vice versa.
The invention embraces all stereoisomers of the compounds defined by structure (I) as shown above.
In a further aspect, the present invention provides FK506 or FK520 analogues such as compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.
Definitions:
The articles "a" and "an" are used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example "an analogue" means one analogue or more than one analogue.
As used herein the term "analogue(s)" refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).
As used herein the terms "FK506 and FK520 analogues" / "FK506 or FK520 analogues" refer to compounds related to FK506, FK520 and similar compounds in structure. Such compounds are 22-membered rings with one lactone and one amide bond. The N of the amide bond forms a 2-carboxyl piperidine or a 2-carboxyl pyrrolidine. This carboxyl group forms the lactone group, with an oxygen that is allylic to a double bond that is exo to the main 22-membered ring. Such compounds include, without limitation, FK520, FK506, antascomicin, FK523, FK525, pimecrolimus and tsukubamycin as well as compounds of formula (I).
As used herein the term "FK506 or FK520 producing strain" refers to a strain (natural or recombinant) which is capable of producing one or more FK506 or FK520 analogues when fed appropriately.
As used herein the term "recombinant strain of a FK506 or FK520 producing host" refers to a recombinant strain based on a natural FK506 or FK520 producing strain which is capable of producing one or more FK506 or FK520 analogues when fed appropriately. As used herein the term "FK506 or FK520 cluster" means the PKS and associated enzymes responsible for production of FK506 or FK520 analogues.
As used herein the term "modifying gene(s)" includes the genes required for post- polyketide synthase modifications of the polyketide, for example but without limitation cytochrome P-450 monooxygenases, ferredoxins and SAM-dependent O-methyltransferases. In the FK520 system these modifying genes include fkbD and fkbM, but a person of skill in the art will appreciate that PKS systems related to FK520 (for example but without limitation: FK506, antascomicin, FK523, FK525 and tsukubamycin) will have homologues of at least a subset of these genes, some of which are discussed further below.
As used herein the term "precursor supply gene(s)" includes the genes required for the supply of the natural or non-natural precursors, the genes required for the synthesis of any naturally or non-naturally incorporated precursors and the genes required for the incorporation of any naturally or non-naturally incorporated precursors. For example but without limitation in the FK506 and FK520 system these genes include fkbL, fkbO and fkbP but a person of skill in the art will appreciate that PKS systems related to FK506 and FK520 (for example but without limitation: antascomicin, FK523, FK525 and tsukubamycin) will have homologues of these genes, some of which are discussed further below. Additionally it may be useful to inactivate homologues of the bkd genes, which are involved in the generation of branched chain keto- acids, such as might be expected to incorporate preferentially into avermectin type loading modules, such as those described in Ward et al. 1999 (e.g. Wei et al., 2006).
As used herein, the term "auxiliary gene(s)" includes references to modifying genes, precursor supply genes or both modifying genes and precursor supply genes. One example of an auxiliary gene is an oxygenase which may hydroxylate the starter unit. As used herein the term "basic product" refers to the initial product of the polyketide synthase enzyme before the action of any modifying genes.
As used herein, the term "precursor" includes the natural starter units (i.e. 4,5- dihydroxycyclohex-1-ene carboxylic acid), non-natural starter units (e.g non-cyclic or heterocyclic starter units), and naturally incorporated amino acids (i.e. pipecolic acid) and non- naturally incorporated amino acids
As used herein the term "non-natural starter unit" refers to any compounds which can be incorporated as a starter unit in polyketide synthesis that are not the starter unit usually chosen (i.e. incorporated) by that PKS.
The term "avermectin-like PKS" means the PKS of a bacterium producing a avermectin-like polyketide such as nemadectin or milbemycin which contains a loading domain consisting of AT and ACP domain and which naturally incorporates branched chain starter acids such as those of the present invention.
Examples of C1-4 acyl groups include C2-4 acyl groups such as -COMe and -COEt, especially COMe. CHO is a further example which is less preferred. Examples of C1-4 alkyl groups include Me, Et, n-Pr, i-Pr, n-Bu, especially Me.
Detailed Description of the Invention
Physiologically functional derivatives of compounds of formula (I) include physiologically acceptable salts, esters and solvates. Pharmaceutically acceptable salts include the non-toxic acid addition salt forms of the compounds of formula (I). The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulphuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxyl- butanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form. Example esters include labile esters which are cleaved in the body, for example carboxylic acid esters formed with hydroxyl groups. Example solvates include hydrates.
In one embodiment Xa represents a bond. In another embodiment Xa represents CH2. Preferably Xa represents CH2.
Preferably Xb represents bond. Preferably Xc represents bond
Preferably Xd represents bond.
In one embodiment R1 represents [A]. In another embodiment R1 represents [B]. In another embodiment R1 represents [C]. In another embodiment R1 represents [D]. In another embodiment R1 represents [E]. In another embodiment R1 represents [F]. In another embodiment R1 represents [G]. In another embodiment R1 represents [H]. In another embodiment R1 represents [J]. In another embodiment R1 represents [K].
Preferably R2 represents -CH3, -CH2CH3, Or -CH2CH=CH2. More preferably R2
represents -CH2CH3, Or -CH2CH=CH2. In one embodiment, R2 represents -CH2CH3. In another embodiment R2 represents -CH2CH=CH2.
Preferably R3 represents =0 (keto).
Preferably Ri0 represents OH. Preferably Rn represent H. Preferably Ri2 represents H.
Preferably Ri3 , Ri3a and Ri3b represent H.
Preferably A represents O or S.
Preferably Ri4 represents H. Preferably Ri4a represents H.
Preferably Ri5 represents H. Preferably Ri6 represents H. Preferably Ri6a represents H.
Preferably Ri6b represents H. Preferably Ri6c represents H. Preferably Ri6d represents
H. Preferably Ri6e represents H. Preferably Ri6f represents H.
Preferably Ri6g represents H.
Preferably Ri7 represents H. Preferably Ri7a represents H. Preferably Ri8 represents H.
Preferably Ri8a represents H.
Preferably Ri9 represents H.
Preferably Ri9a represents H. Preferably Ri9b represents H. Preferably Ri9c represents H. Preferably Ri9e represents OH.
Preferably R20 represents H.
Preferably R20b represents OH.
Preferably R2i represents H or F. More preferably R2i represents F.
Preferably R2ia represents H or F. More preferably R2ia represents F. Preferably R22 represents H or F. More preferably R22 represents F.
Preferably R22a represents H or F. More preferably R22a represents F.
Preferably R23 represents H or F. More preferably R23 represents F.
Preferably R23a represents H or F. More preferably R23a represents F.
Preferably R24 represents OH. Preferably R24a represents OH.
Preferably R25 represents H
Preferably R25a represents H.
Preferably R26 and R26a represent H.
In one embodiment, Ri represents G, R20b represents OH, Ri9e and Ri8a represent H, R2 represents CH2CH3, R3 represents =0, Xa represents CH2, as shown by the following s sttrruuccttuurree:
In another embodiment, R1 represents [J], R22 represents OH, R2i, R23, R24 and R25 represent H, R2 represents CH2CH3, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [B], A represents S, R17, R1S, and R14 represent H, Xb represents bond, R2 represents CH2CH3, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [B], A represents O, Ri7, Ri5, and Ri4 represent H, Xb represents bond, R2 represents CH2CH3, R3 represents =0, Xa represents CH2, as shown by the following structure, also known as Pyran FK520 or 28-des(3-c/s-methoxy-4- frans-hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520:
In another embodiment, R1 represents [E], Ri9c represents H, R2 represents CH2CH3, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [D], R20 and Rigb represent H, R2 represents CH2CH3, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [A], R12 represents OH, R11 and R10 represent H, R2 represents CH2CH3, R3 represents =0, Xa represents CH2, as shown by the following structure:
In one embodiment, R1 represents G, R2Ob represents OH, R19e and Ri8a represent H, R2 represents -CH2CH=CH2, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [J], R22 represents OH, R21, R23, R24 and R25 represent H, R2 represents -CH2CH=CH2, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [B], A represents S, Ri7, Ri5, and Ri4 represent H, Xb represents bond, R2 represents -CH2CH=CH2, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, Ri represents [B], A represents O, Ri7, Ri5, and R14 represent H, Xb represents bond, R2 represents -CH2CH=CH2, R3 represents =0, Xa represents CH2, as shown by the following structure, also known as Pyran FK520 or 28-des(3-c/s-methoxy-4- frans-hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520:
In another embodiment, R1 represents [E], Ri9c represents H, R2 represents - CH2CH=CH2, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [D], R20 and R19b represent H, R2 represents CH2CH=CH2, R3 represents =0, Xa represents CH2, as shown by the following structure:
In another embodiment, R1 represents [A], R12 represents OH, R11 and R1O represent H, R2 represents -CH2CH=CH2, R3 represents =0, Xa represents CH2, as shown by the following structure:
Compounds of formula (I) may be produced by feeding an appropriate non-natural starter unit to an FK506 or FK520 producing strain, culturing the strain and optionally isolating the compounds thereafter. In alternative embodiment the strain is mutated to have targeted inactivation or deletion of one or more genes that contribute to the biosynthesis or regulation of precursor supply. For example the gene(s) that contribute to the biosynthesis or regulation of precursor supply may contribute to the biosynthesis or regulation of the a starter unit (starter acid) e.g. 4,5 dihydroxycyclohex-1-ene carboxylic acid, such as fkbO (Motamedi and Shafiee, 1998, Wu et al., 2000). By removal of the means of generation of the natural starter unit, incorporation of non-natural starter acids is facilitated due to lack of competition with the natural substrate. In another embodiment, the strain has the natural loading module of the FK506 or FK520 PKS replaced with that of the avermectin PKS or an avermectin-like PKS
(such as the nemadectin or milbemycin PKS), and is then fed appropriate starter units, the strain cultured and the compounds optionally isolated. Additionally, other genes may be inactivated, such as the gene(s) that contribute to the biosynthesis or regulation of precursor supply which may contribute to the biosynthesis or regulation of branched chain keto acid, such as bkd (Ward et al., 1999). By removal of the means of generation of branched chain keto acids, incorporation of other non-natural starter acids is facilitated due to lack of competition with the preferential substrate of the avermectin load. In an alternative embodiment the strain may be mutated to inactivate or delete one or more genes responsible for biosynthesis of pipecolic acid, such as fkbL (Wu et al., 2000). Pipecolic is naturally incorporated into the chain as a final step prior to ring closure. This particular modification increases the yield of prolyl derivatives of formula (I) when proline is fed to the strain. Additionally or instead in an alternative embodiment the strain may be mutated to have targeted inactivation or deletion of one or more genes responsible for post-PKS modification, for example the gene responsible for oxidation at the C-9 position, fkbJ (Motamedi and Shafiee, 1998, Wu et al., 2000). This particular modification increases the yield of C-9 desketo derivatives of formula (I).
If appropriate or necessary compounds so produced may be subject, after their isolation, to synthetic alteration using processes known to a skilled person e.g. alkylation of hydroxyl and amino groups and the like. It should be understood by one skilled in the art that it may be possible to use alternative junctions to join the avermectin loading module to the rapamycin PKS. Examples of this include taking just the Acyltransferase (AT) and Acyl Carrier Protein (ACP) domains from the avermectin or avermectin-like PKS and joining to the FK506/FK520 PKS before the fist ketosynthase (KS) domain. Alternatively, the junction may be made between the KS from the avermectin or avermectin-like PKS and the first extender AT from the FK506/FK520 PKS. Analogous possibilities are discussed in WO 98/01546. Additionally, homologues of the avermectin loading module may be used, such as those found in the milbemycin PKS or nemadectin PKS.
In one method of generating a suitable strain, a precursor supply gene such as fkbO or an fkbO homologue or the bkd genes or homologues thereof may be manipulated by targeted inactivation or deletion or modified by other means such as exposing cells to UV radiation and selection of the phenotype indicating that starter unit biosynthesis or branched chain alpha keto acid biosynthesis has been disrupted. The optional targeting of the post-PKS genes may occur via a variety of mechanisms, e.g. by integration, targeted deletion of a region of the FK506 or FK520 cluster including all or some of the post-PKS genes optionally followed by insertion of gene(s) or other methods of rendering the post-PKS genes or their encoded enzymes non-functional e.g. chemical inhibition, site-directed mutagenesis or mutagenesis of
the cell for example by the use of UV radiation.
WO2004/007709 provides methods for the alteration of a gene system which comprises a core portion responsible for the production of a basic product, and a multiplicity of modifying genes responsible for effecting relatively small modifications to the basic product - e.g. effecting oxidation, reduction, alkylation, dealkylation, acylation or cyclisation of the basic product, and a multiplicity of precursor supply genes which are involved in the production of particular precursor compounds. Thus the basic product may be a modular polyketide and the modifying genes may be concerned with modifications of a polyketide chain (such as oxidation at the 9 position), and the precursor supply genes may be involved in the production and/or incorporation of natural or non-natural precursors (e.g. pipecolate and/or 4,5 dihydroxycyclohex-1-ene carboxylic acid).
The methods of WO2004/007709 may hence be suitable for implementation of the present invention.
The core portion may not function properly or even at all in the absence of a precursor supply gene (unless a natural or unnatural precursor compound is supplied or is otherwise available). Therefore, the deletion or inactivation of a precursor supply gene provides a system where it is possible to incorporate non-natural starter units with no competition from the natural starter unit.
Therefore, the present invention provides a method for the incorporation of non-natural starter acids into FK506 and FK520 analogues, said method comprising feeding said starter units to a strain which contains a gene system in which the precursor supply gene has been deleted or inactivated, or the natural FK506/520 loading module has been replaced with the avermectin loading module, or a homologue thereof. Suitable gene systems include, but are not limited to, antascomicin, FK520 (Wu et al., 2000; U.S. 6,150,513; AF235504), FK506 (Motamedi et al., 1996; Motamedi et al., 1997; Motamedi and Shafiee, 1998; AF082100,
Y10438), FK523, FK525 and tsukubamycin biosynthetic clusters. The precursor supply gene which is deleted or inactivated is preferably fkbO or a homologue of fkbO. The gene system is preferably the FK506 or FK520 cluster. The precursor supply gene deleted or inactivated is more preferably fkbO. Optionally, the precursor gene which is deleted or inactivated is involved in branched chain keto acid formation, such as one of the bkd genes, when the loading module is replaced with the avermectin loading module. Optionally fkbM and/or fkbl are deleted or inactivated in addition to fkbO. Optionally fkbL, or an analogue of rapL is also deleted to allow more efficient incorporation of pipecolate analogues (Motamedi and Shafiee, 1998, Khaw et al., 1998) Therefore in one aspect the present invention provides a method of producing compounds of formula (I) comprising:
(a) generating a recombinant strain of a FK506 or FK520 producing host that contains
a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene has been deleted or inactivated, or in which the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; and (b) feeding a non-natural starter unit to said recombinant strain;
(c) culturing said strain; and
(d) optionally isolating compounds of formula (I).
In step (a) the precursor for which one or more starter genes have been deleted or inactivated may be a starter unit (e.g. starter acid). Additionally or instead the precursor for which one or more starter genes have been deleted or inactivated may be pipecolic acid. Optionally one or more genes responsible for post-PKS modification are also deleted or inactivated.
Optionally the natural KS1 module is replaced by the KS1 module of the avermectin or an avermectin-like PKS in addition to the loading module being so replaced as described above.
There is also provided a method of producing compounds of formula (I) comprising:
(a) feeding a non-natural starter unit to a recombinant strain of a FK506 or FK520 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene has been deleted or inactivated, or in which the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS;
(b) culturing said strain; and
(c) optionally isolating compounds of formula (I).
Optionally the natural KS1 module is replaced by the KS1 module of the avermectin or an avermectin-like PKS in addition to the loading module being so replaced as described above.
In a preferred embodiment the recombinant strain is generated using the methods described in WO2004/007709 and in the examples below.
In one embodiment, the host strain is a selected from the group consisting of Streptomyces tsukubaensis No. 9993 (Ferm BP-927), Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822), Streptomyces sp. AA6554, Streptomyces hygroscopicus var. ascomyceticus MA 6475 ATCC 14891 , Streptomyces hygroscopicus var. ascomyceticus MA 6678 ATCC 55087, Streptomyces hygroscopicus var. ascomyceticus MA 6674, Streptomyces hygroscopicus var. ascomyceticus ATCC 55276, Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891 , Streptomyces kanamyceticυs KCC S-0433, Sireptornyces clavuhgerus CKDI 1 19 (Kim and Park, 2008), Streptomyces hygroscopicus subsp. yakushimaensis, Streptomyces sp. DSM 7348, Micromonospora n.sp. A92-306401 DSM 8429
Streptomyces sp. MA 6548 and Streptomyces sp. MA 6858 ATCC 55098. In a preferred embodiment the host strain is selected from the group consisting of: S. hygroscopicus var. ascomyceticus ATCC 14891 , Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) or Streptomyces tsukubaensis No. 9993 (Ferm BP-927). If desired or necessary one or more auxiliary genes may be deleted or inactivated in the host strain. If desired or necessary one or more of the deleted or inactivated genes of the host strain may be reintroduced by complementation (e.g. at an attachment site, on a self- replicating plasmid or by insertion into a homologous region of the chromosome).
If desired or necessary, further chemical steps, known to one skilled in the art may be used to generate the final compound (for example see March, Wiley Interscience).
It is well known to those skilled in the art that polyketide gene clusters may be expressed in heterologous hosts (Pfeifer et al., 2001 ). Accordingly, the present invention includes the transfer of the FK506 or FK520 biosynthetic gene cluster with or without resistance and regulatory genes, either complete, engineered, containing mutations or containing deletions, for complementation in heterologous hosts. Methods and vectors for the transfer as defined above of such large pieces of DNA are well known in the art (Rawlings, 2001 ; Staunton and Weissman, 2001 ) or are provided herein in the methods disclosed.
Therefore in another aspect the present invention provides a method of producing compounds of formula (I) comprising: (a) generating a recombinant strain of a non-FK506 or FK520 producing host (i.e. a heterologous host) that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene is absent or non-functional or the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; and (b) feeding a non-natural starter unit to said recombinant strain;
(c) culturing said strain; and
(d) optionally isolating compounds of formula (I).
There is also provided a method of producing compounds of formula (I) comprising:
(a) feeding a non-natural starter unit to a recombinant strain of a non-FK506 or FK520 producing host (i.e. a heterologous host) that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene is absent or non-functional or the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; (b) culturing said strain; and
(c) optionally isolating compounds of formula (I).
In this context a preferred heterologous host cell strain is a prokaryote, more preferably an actinomycete or Escherichia coli, still more preferably include, but are not limited to S. hygroscopicus, S. hygroscopicus sp. , S. hygroscopicus var. ascomyceticus, Streptomyces tsukubaensis, Streptomyces coelicolor, Streptomyces lividans, Saccharopolyspora erythraea, Streptomyces fradiae, Streptomyces avermitilis, Streptomyces cinnamonensis, Streptomyces rimosus, Streptomyces albus, Streptomyces griseofuscus, Streptomyces longisporoflavus, Streptomyces venezuelae, Micromonospora griseorubida, Amycolatopsis mediterranei or Actinoplanes sp. N902-109.
Those skilled in the art will appreciate that the methods of the present invention could be applied to recombinant host strains in which the polyketide synthase (PKS) has been altered by genetic engineering to express a modified FK506 or FK520 analogue or other polyketide analogue. For example the PKS in a homologous or heterologous could be a hybrid PKS in which one or more domains have been removed, replaced or inserted, such replacements or insertions coming from other heterologous (or homologous) PKS clusters. The prior art describes several methods for the production of novel polyketides by the deletion or inactivation of individual domains (WO93/13663, WO97/92358), construction of hybrid polyketide synthases (WO98/01546, WO00/00618, WO00/01827) or alteration of domain specificity by site-directed mutagenesis (WO02/14482).
It is well known that many actinomycetes contain multiple biosynthetic gene clusters for different secondary metabolites, including polyketides and non-ribosomally synthesised peptides. Specifically, it has been demonstrated that strains of S. hygroscopicus produce a variety of polyketides and non-ribosomally synthesised peptides in addition to FK506, FK520, FK523, meridamycin, FK525, antascomicin or tsukubamycin. These include, but are not limited to, elaiophylin, bialaphos, hygromycin, augustmycin, endomycin (A, B), glebomycin, hygroscopin, ossamycin and nigericin. These additional biosynthetic gene clusters represent a competing requirement for biosynthetic precursors and an additional metabolic demand on the host strain. In order to enhance production of the desired rapamycin, or other polyketide, analogues, it may therefore be advantageous to delete or inactivate any other biosynthetic gene clusters present in the host strain. Methods for the deletion or inactivation of biosynthetic gene clusters are well known in the art.
Suitably, the starter unit is selected from the following carboxylic acids or carboxylic acid derivatives:
wherein R10, Rn and Ri2 are independently selected from H, F, Cl, ORi3, SRi3 and NHRi3 and Ri3 is selected from H, d-C4alkyl and Ci-C4acyl; or
wherein A is selected from O, S and NRi3a, Ri3a represents H, Ci-C4alkyl or Ci-C4acyl, Ri4 and Ri5 are independently selected from H, Ci-C4alkyl, ORi6, SRi6 and NHRi6, Ri6 represents CrC4alkyl or Ci-C4acyl, Xb represents a bond or CH2, when Xb is bond then Ri7 represents H and when Xb is CH2 then R17 represents H, F, Cl, OH, SH, Ci-C4 alkyl, ORi63, SRi6a or NHRi63 and Ri6a represents Ci-C4alkyl or Ci-C4 acyl; or
wherein A represents O, S, or NRi3b, Ri3b represents H, Ci-C4alkyl, or Ci-C4acyl, Ri4a represents Ci-C4alkyl, ORi6, SRi6 or NHRi6, Ri6 represents Ci-C4alkyl or Ci-C4acyl, Ri8 represents H, F, Cl, OH, SH, Ci-C4 alkyl, ORi6a, SRi6a or NHRi63, Ri6a represents Ci-C4 alkyl or Ci-C4acyl, Xc and Xd independently represent bond or CH2 provided that Xc and Xd do not both represent CH2 and when Xc is bond then Ri7a represents H and when Xc is CH2 then Ri7a represents H, F, Cl, OH, SH, Ci-C4alkyl, ORi6b, SRi6b or NHRi6b, Ri6b represents CrC4 alkyl or
CrC4 acyl and when Xd is bond Ri9 represents H and wherein when Xd represents CH2 then Rig represents H, F, Cl, OH, SH, d-C4 alkyl, C1Ri6C, SRi6c or NHRi6c, and Ri6c represents d- C4alkyl or Ci-C4acyl; or
wherein Ri9b and R2o independently represent H, F, Cl, OH, SH, d-C4 alkyl, ORi6d, SRi6d or NHRi6d, and Ri6d represents Ci-C4alkyl or d-C4 acyl; or
wherein Ri9c represents H, F, Cl, OH, SH, CrC4alkyl, ORi6e, SR16e or NHRi6e, and Ri6e represents d-C4 alkyl or d-C4 acyl; or
wherein R19d and R2Oa independently represent H, F, Cl, OH, SH, d-C4 alkyl, ORi6f,
SRi6f or NHR16f and Ri6f represents d-C4 alkyl or d-C4 acyl ; or
wherein R18a, Ri9e and R2Ob independently represent H, F, Cl, OH, SH, d-C4 alkyl, ORi6g, SRi6g or NHR16g and Ri6g represents CrC4 alkyl or d-C4 acyl; or
wherein R2i, R22 and R23 independently represent H, F, Cl, OR26, SR26, CrC4 alkyl or CN, R26 represents H, d-C4 alkyl or d-C4 acyl, R24 and R25 independently represent H, F, Cl, OH, SH or CrC4 alkyl, provided that at least one of R2i, R22 and R23 is not H or d-C4 alkyl; or
wherein R21a, R22a and R23a are independently selected from H, F, Cl, OR26, SR26, C1- C4 alkyl and CN, R26a represents H, d-C4 alkyl or d-C4 acyl, R24a and R25a independently represent H, F, Cl, OH, SH or d-C4 alkyl and provided that at least one of R2ia, R22a and R23a does not represent H or d-C4 alkyl.
Starter units are suitably provided as the free carboxylic acid, but derivatives that may
be employed include salts and esters.
The aforementioned starter unit substances are either known or may be prepared by a skilled person using conventional methods.
Standard methods known to those of skill in the art may be used to culture the host or recombinant strain in order to produce compounds of formula (I). Such methods include, without limitation, those described in the examples below; additional methods may also be found in Reynolds and Demain, 1997 and references therein.
Compounds of the invention may be isolated using standard methods known to those of skill in the art, including, without limitation, those described in the methods of the examples below. Alternatives to these methods which may also be considered by a person of skill in the art include those as described in Natural Products Isolation (Cannell et al., 1998).
Compounds of formula (I) are useful as pharmaceuticals for example, but without limitation, having potential utility as immunosuppressants, antifungal agents, anticancer agents, neuroregenerative agents, or agents for the treatment of psoriasis, rheumatoid arthritis, fibrosis and other hyperproliferative diseases. In a further aspect, the invention provides for the use of a compound of formula (I) as disclosed herein, in the preparation of a medicament for the prophylaxis and/or treatment of organ rejection after transplantation, autoimmune diseases, inflammatory disorders, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders. In a further aspect, the invention provides for a method of treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders comprising administering a compound of formula (I) to a subject in need thereof. Additionally, the compounds of formula (I) disclosed herein may be used in the preparation of a medicament for the prevention of organ allograft rejection. In a preferred embodiment the compounds of formula (I) are used in the preparation of a medicament for the treatment of autoimmune diseases or inflammatory disorders.
One skilled in the art would be able by routine experimentation to determine the ability of these compounds to inhibit fungal growth (e.g. Baker, H., et al., 1978; NCCLS Reference method for broth dilution antifungal susceptibility testing for yeasts: Approved standard M27-A, 17(9). 1997). Additionally, one skilled in the art would be able by routine experimentation to determine the ability of these compounds to inhibit tumour cell growth, (see Dudkin, L., et al., 2001 ; Yu et al. 2001 ). In a further aspect the compounds of this invention are useful for inducing immunosuppression and therefore relate to methods of therapeutically or prophylactically inducing a suppression of a human's or an animal's immune system for the treatment or prevention of rejection of transplanted organs or tissue, the treatment of autoimmune, inflammatory, proliferative and hyperproliferative diseases (examples include but
are not inclusively limited to autoimmune diseases, diabetes type I, acute or chronic rejection of an organ or tissue transplant, asthma, tumours or hyperprolific disorders, psoriasis, eczema, rheumatoid arthritis, fibrosis, allergies and food related allergies). Such assays are well known to those of skill in the art, for example but without limitation: Immunosuppressant activity - Warner, LM.,et al., 1992, Kahan et al. (1991 ) & Kahan & Camardo, 2001 ); Allografts - Fishbein, T.M., et al., 2002, Kirchner et al. 2000; Autoimmune / Inflammatory / Asthma - Carlson, R.P. et al., 1993, Powell, N. et al., 2001 ; Diabetes I - Rabinovitch, A. et al., 2002; Psoriasis - Reitamo, S. et al., 2001 ; Rheumatoid arthritis - Foey, A., et al., 2002; Fibrosis - Zhu, J. et al., 1999, Jain, S., ef a/., 2001 , Gregory et al. 1993 The ability of the compounds of this invention to induce immunosuppression may be demonstrated in standard tests used for this purpose. In a further aspect the compounds of this invention are useful in relation to antifibrotic, neuroregenerative and anti-angiogenic mechanisms, one skilled in the art would be able by routine experimentation to determine the ability of these compounds to prevent angiogenesis (e.g. Guba, M.,et al., 2002, ). One of skill in the art would be able by routine experimentation to determine the utility of these compounds in stents (e.g. Morice, M. C, et al., 2002). Additionally, one of skill in the art would be able by routine experimentation to determine the neuroregenerative ability of these compounds (e.g. Myckatyn, T.M., et al., 2002, Steiner ef al. 1997)
The compounds of formula (I) are also, or in particular, expected to be useful as a therapeutic or prophylactic agents for one or more of the following conditions: rejection reactions after transplantation of organs or tissues (for example heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, intervertebral disc, trachea, myoblast and cartilage); graft-versus-host reactions following bone marrow transplantation; autoimmune diseases (for example rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes); infections caused by pathogenic microorganisms, in particular fungal infections; inflammatory or hyperproliferative skin diseases or cutaneous manifestations of immunologically-mediated diseases (for example psoriasis, atopic dermatitis, contact dermatitis, eczematoid dermatitis, pyoderma gangrenosum, seborrhoeic dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, rosacea, urticaria, angioedema, vasculitides, erythema, dermal eosinophilia, lupus erythematosus, acne, Netherton syndrome, and alopecia areata); autoimmune or allergic diseases of the eye (for example keratoconjunctivitis, vernal conjunctivitis, allergic conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical keratitis, corneal epithelial dystrophy, keratoleukoma, ocular pemphigus, Mooren's ulcer, scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, keratoconjunctivitis sicca (dry eye), phlyctenule, iridocyclitis, sarcoidosis affecting the eye, endocrine ophthalmopathy); reversible obstructive airway diseases or asthma, in particular
chronic or inveterate asthma (for example late asthma, airway hyperresponsiveness, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, and dust asthma), mucosal or vascular inflammations (for example gastric ulcers, ischaemic or thrombotic vascular injury, ischaemic bowel diseases, enteritis, necrotizing enterocolitis, intestinal damage associated with thermal burns, leukotriene B4-mediated diseases); intestinal inflammations or allergies (for example coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis); food-related allergic diseases with symptomatic manifestation remote from the gastrointestinal tract (for example migraine, rhinitis and eczema); renal diseases (for example interstitial nephritis, Goodpasture's syndrome, haemolytic uraemic syndrome, nephrotic syndrome (for example glomerulonephritis) and diabetic nephropathy); nervous system diseases (for example multiple myositis, Guillain-Barre syndrome, Meniere's disease, multiple neuritis, solitary neuritis, cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and radiculopathy); ischaemic diseases (for example head injury, brain haemorrhage, cerebral thrombosis, cerebral embolism, cardiac arrest, stroke, transient ischemic attack, hypertensive encephalopathy, cerebral infarction); endocrine diseases (for example hyperthyroidism and Basedow's disease); haematic diseases (for example pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anaemia, agranulocytosis, pernicious anaemia, megaloblastic anaemia, and anerythroplasia); bone diseases (for example osteoporosis); respiratory diseases (for example sarcoidosis affecting the respiratory tract, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and idiopathic interstitial pneumonia); skin diseases (for example dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photosensitivity, and cutaneous T-cell lymphoma); circulatory diseases (for example arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa, and myocardosis); collagen diseases (for example scleroderma, Wegener's granulomatosis, and Sjogren's syndrome); adiposis; eosinophilic fasciitis; periodontal diseases (for example damage to gingiva, periodontium, alveolar bone or substantia ossea dentis); male pattern alopecia, alopecia senile; muscular dystrophy; pyoderma and Sezary syndrome; chromosome abnormality-associated diseases (for example Down's syndrome); Addison's disease; Human Immunodeficiency Virus (HIV) infection or AIDS; hypertrophic cicatrix and keloid due to trauma, burn, or surgery.
The compounds of formula (I) may also be useful as a therapeutic or prophylactic agents for Bronchiolitis Obliterans Syndrome (BOS).
The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including topically (for example by inhalation, vaginally, intranasally, or by eye or ear drop), enterally (for example orally or rectally) or parenterally (for example by intravenous, intracavernosal, subcutaneous, intramuscular, intracardiac or
intraperitoneal injection) or via a medical device (for example via a stent). The treatment may consist of a single dose or a plurality of doses over a period of time.
Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more physiologically acceptable diluents or carriers. The diluents or carrier(s) must be "physiologically acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. In some cases, the diluent or carrier will be water or saline which will be sterile and pyrogen free.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformLy and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
Aerosol formulations suitable for administering via inhalation can also be made using methods known in the art. Examples of this include administration of the compounds of the invention by inhalation in the form of a powder (e.g. micronized) or in the form of atomized solutions or suspensions. The aerosol formulation may be placed in a suitable pressurized propellant, and may be used with additional equipment such as nebulizer or inhaler.
For applications to external tissues, for example the mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active agent may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
The compounds of the invention may also be administered using medical devices known in the art. For example, in one embodiment, a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. 5,399,163; U.S. 5,383,851 ; U.S. 5,312,335; U.S. 5,064,413; U.S.
4,941 ,880; U.S. 4,790,824; or U.S. 4,596,556. Examples of well-known implants and modules useful in the present invention include : US 4,487,603, which discloses an implantable micro- infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi- chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.
The compounds can be administered as the sole active agent, or in combination with other pharmaceutical agents, such as other agents that stimulate or inhibit cell proliferation of immune responses. These agents include e.g. cyclosporine, rapamycin, FK506, leflunomide, butenamides, corticosteroids, Doxorubicin, and the like. In such combinations, each active ingredient can be administered either in accordance with its usual dosage range, or at a lower dose level.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents. Pharmaceutical compositions of the invention may optionally contain further active ingredients.
Further aspects of the invention include:
-An engineered strain based on a FK506 or FK520 producing strain in which fkbO or a homologue thereof and optionally further precursor supply genes and/or post-PKS genes have been deleted or inactivated;
-An engineered strain based on a FK506 or FK520 producing strain in which the loading module of the PKS is replaced with an avermectin loading module and optionally the KS1 module of the PKS is replaced with an avermectin KS1 module;
-Such engineered strains wherein the strain is a FK506 or FK520 producing strain which is Streptomyces hygroscopicus subsp hygroscopicus or Streptomyces tsukubaensis;
-Use of such an engineered strain in the preparation of an FK506 or FK520 analogue or a pharmaceutically acceptable salt thereof;
-A process for preparation of a polyketide (i.e. a FK506 or FK520 analogue) which comprises:
(a) feeding a non-natural starter unit to said engineered strain; (b) culturing said strain; and
(c) optionally isolating the polyketide.
Brief description of the Figures
Figure 1 Diagram describing the methodology used for generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
Figure 2 Diagram describing the methodology used for generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
Figure 3 Diagram describing the methodology used for generation of a Streptomyces tsukubaensis strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
Figure 4 Plots of the data generated from analysis of compound levels after dosing
FK506, FK520 or the Pyran FK520 as a single 1 mg/kg Lv. dose (filled
diamonds) or 10mg/kg p.o. dose (open diamonds), with concentration measured in whole blood (mean of triplicates in ng/mL) compared to time after dosing (in hours).
Materials and Methods
Materials
All molecular biology enzymes and reagents were from commercial sources. Cosmid
Supercos-1 was obtained from Stratagene. Vector pKC1139B01was obtained by inserting a linker into pKC1 139 (Bierman et al., 1992). The 674bp BgIW PvuW fragment of pKC1 139 was replaced by the annealing product of oligos B01 and B02 to give the plasmid pKC1 139B01
(5789bp) containing the polylinker below:
Primers 5' gatccactagttcggacgcatatgctggatatccaagtatctagaac BOl (SEQ ID 35)
5' gttctagatacttggatatccagcatatgcgtccgaactagtg B02 (SEQ ID 36) Resulting polylinker
Spel Ndel EcoRV Xbal 5' gatccactagttcggacgcatatgctggatatccaagtatctagaac 3' 3' gtgatcaagcctgcgtatacgacctataggttcatagatcttg 5'
Starter materials
All of the feeds used for starting units were obtained from commercial sources with the exception of tetrahydro-2H-thiopyran-4-carboxylic acid which was synthesised by the method of Strassler et al. 1997
Bacterial strains and growth conditions
Escherichia coli DH 10B (GibcoBRL) was grown in 2xTY medium as described by
Sambrook et al. (1989) and E. coli ET12567(pUZ8002) as described in Paget et al. (1999) in
2xTY medium with kanamycin (25 mg/L) and chloramphenicol (12.5 mg/L). E. coli VCS257 was used for transfection of in vitro packaged cosmids. According to the instructions of
Stratagene's Gigapack® III XL Packaging Extract the strain was kept on LB medium and grown on LB plus 0.2% maltose and 1OmM MgSO4 for transfection. The vector pUC19 was obtained from New England Biolabs. Vector pSET152 is described in Bierman et al., (1992a).
E. coli transformants were selected for with 100 mg/L ampicillin or 50 mg/L apramycin. The avermectin producer Streptomyces avermitilis (DSM41443/) was grown on TSB at 28 ° for genomic DNA isolation.
The FK506 producer Streptomyces tsukubaensis no. 9993 (FERM BP-927) (International Patent Organism Depositary, Tsukuba, Japan) and its derivatives were maintained on medium 1 agar plates or ISP4, ISP3,or ISP2 (see below) at 28 0C.
The FK520 producer, Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822, purchased from DSMZ, Braunschweig, Germany) (known to be equivalent to Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891 ) and its derivatives were maintained on medium 1 agar plates, ISP2, ISP3 or ISP4 (see below) at 28 0C.
Production of FK520 was carried out by fermentation of Streptomyces hygroscopicus subsr. hygroscopicus (DSM 40822), also termed BIOT-4081. A single spore isolate of this strain, terme BIOT-4168, was also used. Streptomyces tsukubaensis no. 9993 (FERM BP-927), also termei
BIOT-31 19 was used for producing FK506. A single spore isolate of this strain, termed BIOT-420f was also used.
Strains were grown on MAM, ISP4, ISP3 or ISP2 agar at 28 0C for 5 - 21 days and used t inoculate seed medium NGY. The inoculated seed medium was incubated with shaking betwee 200 and 300 rpm at 5.0 or 2.5 cm throw at 28 0C for 48 hours. For production of FK520 or FK50ι the fermentation medium PYDG or PYDG+MES buffer (PYDM) were inoculated with 2.5%-10% c the seed culture and incubated with shaking between 200 and 300 rpm with a 5 or 2.5 cm throw ε 28 0C for six days. The culture was then harvested for extraction.
Production of FK520, FK506 or analogues in Shaken Tubes
Spore stocks of BIOT-4081 , BIOT-4168, BIOT-31 19, BIOT-4206 or strains which ar described below were prepared after growth on MAM, ISP4, ISP3 or ISP2, and preserved in 20°/ w/v glycerol in distilled water and stored at -80 0C. Spore stocks were recovered onto plates c MAM, ISP4, ISP3 or ISP2 and incubated for 5-21 days at 28°C. Vegetative cultures (seed culture were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP, plate and inoculating into 7 ml. medium NGY in 50 ml. Polypropylene Centrifuge tubes (cat nc 227261 , purchased from Greiner Bio-One Ltd, Stonehouse, Gloucestershire, UK) with foam plugs or in Erlenmeyer flasks as described below. The culture tubes were incubated at 28°C, 300 rpm (2. cm throw) for 48 h. From the seed culture 0.5 ml. was transferred into 7 ml. production mediur PYDG or PYDG+MES in 50-mL Centrifuge tubes with foam plugs. Cultivation was carried out for days at 28°C and 300 rpm (2.5 cm throw). If necessary a selected starter unit was fed to thi production medium 24 hours post inoculation. Enough feed was dissolved in 0.05 ml. to 0.1 m methanol and added to the culture to give final concentration of 2.12 mM of the feed compound.
Production of FK520, FK506 or analogues in Shaken Flasks
Spore stocks of BIOT-4081 , BIOT-4168, BIOT-31 19, BIOT-4206 or strains which ar described below were prepared after growth on MAM, ISP4, ISP3 or ISP2 agar medium, an preserved in 20% w/v glycerol in distilled water and stored at -80 0C. Spore stocks were recovere onto plates of MAM, ISP4, ISP3 or ISP2 medium and incubated for 5-21 days at 28°C. Vegetativ cultures (seed culture) were prepared by removing 4-10 agar plugs (6 mm in diameter) from thn MAM, ISP4, ISP3 or ISP2 plate and inoculating into 50-250 ml. medium NGY in 250 ml. or 2000 m Erlenmeyer flasks with foam plugs. The seed flasks were incubated at 28°C, 200-250 rpm (5 or 2. cm throw) for 48 h. From the seed culture 2-10% v/v was transferred into 50 or 250 ml. productio medium PYDG (or PYDG + MES) in 250 ml. or 2000 ml. Erlenmeyer flasks with foam plugs Cultivation was carried out for 6 days at 28°C and 200-250 rpm (5 or 2.5 cm throw). If necessary selected starter unit was fed to the production medium 24 hours post inoculation. Enough feed wa dissolved in 0.375 ml. to 1 ml. methanol and added to the culture to give final concentration of 2.1 mM of the feed compound.
Production of FK520, FK506 or analogues in stirred bioreactors
Spore stocks of BIOT-4081 , BIOT-4168, BIOT-31 19, BIOT-4206 or strains which ar described below were prepared after growth on MAM, ISP4, ISP3 or ISP2 agar medium, an preserved in 20% w/v glycerol in distilled water and stored at -80 0C. Spore stocks were recovere onto plates of MAM, ISP4, ISP3 or ISP2 medium and incubated for 5-21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing 5-10 agar plugs (6 mm i diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 200-350 ml. medium NGY i 2 litre Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 28°C, 250 rpr (2.5 cm throw). The entire seed culture in one flask was transferred into 5 litres of medium PYD( containing 0.01-0.05% antifoam SAG 471 , in 7 L Applikon Fermentor. The fermentation mediur was pre-adjusted at pH 6.0-7.0 poststerilization. The fermentation was carried out for 6 days at 2 0C, with starting agitation set at 300-450 rpm, aeration rate at 0.5-0.8 V/V/M and dissolved oxyge (DO) level controlled with the agitation cascade at 20 - 40% air saturation. If required the pH ma be maintained using acid or base addition on demand. For production of analogues of FK520 c FK506, the selected feed (starting unit for biosynthesis of target compound) was fed to thi production medium 12 - 24 hours post inoculation. Enough feed was dissolved in 3 ml. to 5 m methanol and added to the culture to give final concentration of 2 mM of the feed compound. Th amount of methanol does not exceed 1 % of the total volume. Fermentation was continued fc further five days post-feeding
Media Recipes
Water used for preparing media was prepared using Millipore Elix Analytical Grade Wate Purification System
Medium 1: Modified A-medium (MAM) component Source g/L
Wheat starch Sigma 10 g
Corn steep powder Sigma 2.5 g
Yeast extract Difco 3 g
Calcium carbonate Sigma 3 g
Iron sulphate Sigma 0.3 g
BACTO agar Difco 2O g
No pH adjustment is made. Sterilise by autoclaving 1210C, 15 minutes.
Medium 9: R6 (Kieseret al., 2000) component Source
Sucrose Fisher 20O g
Dextrin Avedex 1 O g
Casamino acids Difco i g
MgSO4.7H2O Sigma 0.05 g
ISP Trace Element See below 1 ml_ Solution
K2SO4 Sigma 0.1 g
Water to 70O mL
Add 20 g BACTO agar and a stirrer bar to each 1 L Duran and autoclave at 121 ° for 15 minutes.
After autoclaving add component Source
(previously sterilised individually by autoclaving 1210C, 15 minutes)
0.65 M L-glutamic Sigma 10O mL acid, mono sodium (10.99 g)
salt
0.48 M CaCI2.2H2O Sigma 10O mL (7.06 g)
0.1 M MOPS pH 7.2 Fisher 10O mL (2.09 g)
ISP3 component Source g/L
Oatmeal Tesco 20g
BACTO agar Difco 18g
ISP Trace Element See 1 mL Solution below
Oatmeal is cooked/steamed in the water for 20 mins, strained through a muslin and more water added to replace lost volume. ISP Trace Elements Solution is added and pH adjusted to 7.2 with NaOH. Agar is added before autoclaving at 1210C, 15 minutes.
ISP Trace Elements Solution component Source g
FeSO4.7H2O Sigma 0.1 g
MnCI2.4H2O Sigma 0.1 g
ZnSO4.7H2O Sigma 0.1 g
Distilled water 100 mL
Store in the dark at 40C
2xTY component Source g/L
Tryptone Difco 16g
Yeast extract Difco 10g
NaCI Sigma 5g
BACTO agar Difco 15g
Sterilise by autoclaving 1210C, 15 minutes.
LB
Component Source per L
Tryptone Difco 1 O g
Yeast extract Difco 5 g
NaCI Sigma 1 O g
BACTO agar Difco 15 g
Sterilised by autoclaving 121 0C, 15 min.
TSB
Component Source per L
Bacto Tryptic Soy BD 3O g Broth
Sterilised by autoclaving 121 0C, 15 min.
NGY component Source g/L
Difco Nutrient Broth Difco 8g
Glucose Sigma 10g
Yeast Extract Difco 5g
The medium is adjusted to pH 7.0, with NaOH and then sterilised by autoclaving 1210C, 15 minutes.
PYDG component Source g/L
Peptone from Milk Sigma 15g Solids
Yeast Extract Difco 1.5g
Dextrin Avedex 45g
Glucose Sigma 5g
The medium is adjusted to pH 7.0 with NaOH, and then sterilised by autoclaving 1210C, 15 minutes.
When MES is added to PYDG (PYDG + MES) it is added 21.2g/L prior to pH adjustment
ISP4 component Source g/L
Soluble Starch BDH 10 g
K2HPO4 Sigma 1.O g
MgSO4.7H2O Sigma 1.O g
NaCI RDH 1.O g
(NhU)2SO4 RDH 2.O g
CaCO3 Caltec 2.O g
BACTO agar Difco 2Og
ISP Trace Elements See 1 ml. Solution above
A paste is made using a little cold water and the starch. This is brought up to a volume of 500 ml_. All other ingredients are then added, and the pH of the media is adjusted to pH 7.0-7.4. Sterilise by autoclaving 1210C, 15 minutes.
ISP2 component Source g/L
Yeast extract Difco 4 g
Malt extract Difco 10 g
Glucose Sigma 4 g
Bacto agar Difco 2O g
Sterilise by autoclaving 1210C, 15 minutes.
DNA manipulation and sequencing DNA manipulations, PCR and electroporation procedures were carried out as described in Sambrook et al. (1989).. DNA sequencing was performed as described previously (Gaisser et al., 2000). Genome sequencing was carried out using 454 technology (Margulies et al., 2005) at Cogenics and the University of Cambridge.
Genomic DNA preparation
Strains were grown in shake flasks containing 25 ml. TSB or ISP2 medium at 250 - 300 rpm and 28 0C and harvested after 2 to 3 days. Cell pellets were washed with 10.3% sucrose and frozen at -20 0C until used. The following method was most successful for genomic DNA isolation from S. hygroscopicus, S. tsukubaensis and S. avermitilis. A pellet originating from 12.5 ml. of culture was resuspended in 1 ml. STE buffer (100 mM NaCI, 10 mM Tris HCI pH8, 1 mM EDTA). 20 ml. STE buffer supplemented with 2 mg/mL lysozyme were added and the resuspension incubated for 30 min at 37 0C. 20 DL of RNaseA (10 mg/mL) were added and the mixture incubated for another 30 min at 37 0C. 4.8 mL EDTA (0.1 M final
concentration) were added to stop the reaction. 1.4 ml. 20% SDS were added. After careful mixing the lysate was incubated on ice for 5 min, then extracted with one volume of phenol/chloroform/isoamylalcohol (25:24:1 ) and centrifuged at 2300 g and 4 0C for at least 15 min up to 1 h. Extractions were repeated until no more protein was visible at the interface, followed by a final chloroform/isoamylalcohol (49: 1 ) extraction. The upper phase was precipitated with 1/10 vol. 5 M NaCI and 1 vol. cold isopropanol. After a few min, the DNA was spooled out with a glass rod and washed in ice cold 70% EtOH. After brief drying, the recovered DNA was dissolved in 0.5 - 1 ml. TE 10:1. The proteinase K method (Kieser et al., 2000) was also applied successfully to recover genomic DNA from S. tsukubaensis.
Cosmid library preparation for S. tsukubaensis
A cosmid library of genomic DNA of S. tsukubaensis, was constructed. High molecular weight DNA from several genomic DNA preps was partially digested with BfuC\, an isoschizomer of Sau3A, to a mean size of 30 - 60 kb, ligated to Supercos-1 , packaged into A phage using Gigapack® I I I XL Packaging Extract (Stratagene) and transfected into Escherichia coli VCS257. The titre was 6.7 x 105 cfu / μg vector. DNA of 10 cosmids was isolated and digested with EcoRI to check the insert size which was 40 kb on average. 2000 clones were grown in 96-well microtitre plates (150 μl_ LB AmplOO Kan50 per well) at 37 0C and frozen at -80 0C after mixing wells with 50μL LB/glycerol 1 :1.
FkbO probe preparation
A DIG labeled fkbO probe was used to detect cosmids containing this region of the FK506 biosynthetic cluster. The probe was prepared by PCR using DIG labeled dNTP mix (Roche). It comprises 410 bp of 3'-terminal fkbO sequence. Sequence information for primer design had been obtained by 454 sequencing of BIOT-31 19 - see Fig. 1 and SEQ ID 1. Primer sequences were:
UES2for (Seq ID 2) 5'-CACTCCTTCGATCTCCACGAGCAGGTCGCCACGGGC-S' and UES2rev (Seq ID 3) 5'-ACCCTGCCGTCCTCACGGCACACCACTACCCCACGG-S'. Annealing temperatures between 66 and 71 0C and extension for 20 sec at 68 0C proved to be successful.
Colony hybridization
Thawed microplate-cultures were stamped onto positively charged filter membranes
(Roche) which had been placed on LB AmplOO Kan50 plates. After overnight growth at 37 0C membranes were taken off. Cells were lysed and cell debris removed according to the DIG
Application Manual for Filter Hybridization (Roche). DNA was crosslinked by exposing
membranes to UV (302nm) for 5 min. Membranes were kept between two sheets of filter paper soaked with 2xSSC at 4 0C or used immediately for hybridization. Hybridization was carried out using standard hybridization buffer and DIG labeled fkbO probe (see above) at a hybridization temperature of 68 0C. Stringent washes were performed at 68 0C. The nonradioactive DIG Nucleic Acid Detection kit from Roche was used to identify 5 positive clones on 4 library plates. The procedure followed the instructions of the DIG Application Manual for Filter Hybridization (Roche).
Conjugation of Streptomyces hygroscopicus subsp. hygroscopicus Escherichia coli ET12567, harbouring the plasmid pUZ8002 (Macneil et al., 1992, Paget et al., 1999) was transformed with pAESABI by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) by spore conjugation (Kieser et al. 2000).
Fresh spores were harvested in water from plates of Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) and were heat-shocked at 500C for 10 minutes. These were then mixed with the E. coli donor strain, which had been washed twice with 2xTY, in a ratio of 1 :3 streptomycete to E. coli, and plated on R6 medium, incubating at 28°C. After -20 hr, the plates were overlaid with water containing 2 mg of apramycin sulphate and 1 mg of nalidixic acid and incubation continued at 28°C.
After 3-5 days, apramycin resistant colonies were seen, indicating integration of the pAESABI plasmid into the genome.
Conjugation of Streptomyces tsukubaensis
Escherichia coli ET12567, harbouring the plasmid pUZ8002 (Macneil et al., 1992, Paget et al., 1999) was transformed with pAES10 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces tsukubaensis no. 9993 (FERM BP-927) by spore conjugation (Kieser et a 1.2000).
Fresh spores were harvested in water from plates of Streptomyces tsukubaensis no. 9993 (FERM BP-927) and were heat-shocked at 500C for 10 minutes. These were then mixed with the E. coli donor strain, which had been washed twice with 2xTY, in a ratio of 3:1 Streptomycete to £. coli, and the mixture shaken at 37°C, 200 rpm, 1 " throw for 3 hr. The conjugation mixture was then plated on R6 medium, incubating at 37°C. After -20 hr, the plates were overlaid with water containing 2 mg of apramycin sulphate and 1 mg of nalidixic acid and incubation continued at 37°C.
After 4-6 days, apramycin resistant colonies were seen, indicating integration of the pAESI O plasmid into the genome.
Culture broth sample extraction and analysis
Culture broth (0.9 ml.) is added to an Eppendorf tube (2.0 ml.) and ethyl acetate (0.9 mL) added. The broth is mixed with the solvent for 30 minutes on a shaking platform (vibrax) at 400 rpm. The phases are then separated by centrifugation (10 minutes, 13,200 rpm). An aliquot of the organic layer (0.1 mL) is then transferred to either a clean glass LC-vial or a vial containing 0.005 mg of pimecrolimus (as an internal standard for quantification). The solvent is removed in vacuo and then residue re-dissolved in methanol (1 mL) by gentle agitation on a shaking platform (5 minutes).
Analysis by LCMS The HPLC system comprised an Agilent HP1100 equipped with a Hyperclone ODS2,
C18, 3 micron 4.6 x 150 mm column (Phenomonex). Injection volume 10 μL, oven 500C, A: 0.1 % formic acid, B: 0.1% formic acid in MeCN. 1 mL/min; 0-1 min 65% B; 6.5 min 100% B; 10 min 100% B; 10.05 min 65% B, 12 min 65% B. The HPLC system described above was coupled to a Bruker Daltonics Esquire3000 electrospray mass spectrometer. Positive-negative switching was used over a scan range of 500 to 1000 Dalton.
Alternatively, LC samples that have been spiked with 0.005 mg/mL pimecrolimus were analysed on the same instrument and with the same chromatographic conditions. However the MS was conducted in multiple reaction monitoring mode (MRM mode) in order to quantify the amount of FK analog in the sample. Details of the quantification are:
negative scan mode, m/z = 450-850
MRM setup: transitions [Da] fragmentation amplitude [V] pimecrolimus (IS): 808.4 → 548.3 1.15
FK520 790.5 → 548.3 1.15
FK506 802.4 → 560.3 1.15
all parent ions are isolated with a width of 3 amu.
All FK520 and FK506 analogues can be quantified in this manner, with the parent ion isolated as [M-H]" and the transition to 548.2 (for Fk520 analogues) or 560.2 (for FK506 analogues) used.
The amount of analyte present is then calculated by dividing the integral for the analyte transition (as detailed above) with that for the internal standard, pimecrolmius. This ratio is then compared with a standard calibration curve for FK520 or FK506 up to 100 ng on column with 50 ng on column pimecrolimus.
NMR spectra (1H, 13C, DQF-COSY, TOCSY, HMQC, HMBC, NOESY) of purified material were recorded on a Bruker Advance DRX500 spectrometer which operated at 500 MHz (for proton derived spectra, pro rata for other nuclei) at 27 0C,. Chemical shifts are described in parts per million (ppm) and are referenced to solvent signal e.g. CHCI3 at δπ 7.26 (1H) and CHCI3 at δc 77.0 (13C). J values are given in Hertz (Hz).
Assessment of effect on NFA T gene expression
To assess potency of the compounds on inhibition of the IL2 pathway, the Invitrogen GENEblazer NFAT gene reporter assay was used (Hanson, 2006; Qureshi, 2007) .
The general process of carrying out the assay was as follows: NFAT-bla Jurkat cells were thawed and resuspended in Assay Media (OPTI-MEM, 0.5% dialyzed FBS, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 100 U/mL/100 μg/mL Pen/Strep) to a concentration of 781 ,250 cells/mL. 4 μL of a 10X serial dilution of the test compounds were added to appropriate wells of a TC-Treated assay plate. 32 μL of cell suspension was added to the wells and pre- incubated at 37°C/5% CO2 in a humidified incubator with test compounds for 30 minutes. Anti CD4:CD8 activator at the pre-determined EC80 concentration was added to wells containing the test compounds. The plate was incubated for 5 hours at 37°C/5% CO2 in a humidified incubator. 8 μL of 1 μM Substrate Loading Solution was added to each well and the plate was incubated for 2 hours at room temperature. The plate was read on a fluorescence plate reader.
Assessment of water solubility Water solubility may be tested as follows: A 10 mM stock solution of the sanglifehrin analogue is prepared in 100% DMSO at room temperature. Triplicate 0.01 mL aliquots are made up to 0.5 mL with either 0.1 M PBS, pH 7.3 solution or 100% DMSO in amber vials. The resulting 0.2 mM solutions are shaken, at room temperature on an IKA® vibrax VXR shaker for 6 h, followed by transfer of the resulting solutions or suspensions into 2 mL Eppendorf tubes and centrifugation for 30 min at 13200 rpm. Aliquots of the supernatant fluid are then analysed by the LCMS method as described above.
Assessment of cell permeability
Cell permeability was tested using the BIOCOAT® HTS Caco-2 Assay System developed by BD Biosciences as follows: The test compound was dissolved in DMSO and then diluted further in buffer to produce a final 1 μM dosing concentration. The fluorescence marker lucifer yellow was also included to monitor membrane integrity. Test compound was then applied to the apical surface of Caco-2 cell monolayers and compound permeation into the basolateral compartment is measured. This is performed in the reverse direction (basolateral to apical) to investigate active transport. LC-MS/MS was used to quantify levels of both the test and standard control compounds (such as Propanolol and Acebutolol).
In vivo assessment of pharmacokinetics
In vivo assays may also be used to measure the bioavailability of a compound. Generally, a compound is administered to a test animal (e.g. mouse) both intravenously (i.v.) and orally {p.o.) and blood samples are taken at regular intervals to examine how the plasma (or whole blood) concentration of the drug varies over time. The time course of plasma (or whole blood) concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models.
For FK506 analogues, whole blood was analysed. Compounds were formulated in 5% ethanol / 5% cremophor EL / 90% saline for both p.o. and i.v. administration. Mice were dosed with either 1 mg/kg i.v. or 10mg/kg p.o. of the compound of the invention or the parent compound. Triplicate blood samples were taken at 0.25, 0.5, 2, 8, and 24 hours and the concentration of the compound of the invention or parent compound in the sample was determined via LCMS. The time-course of plasma concentrations was plotted and used to derive area under the whole blood concentration-time curve (AUC - which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation). These values were used to generate the oral bioavailability (F%).
Example 1 - Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift.
1.1 Cloning of DNA homologous to the upstream flanking region of fkbO disruption. Oligos SG165 (SEQ ID NO: 1 ) and SG166 (SEQ ID NO: 2) were used to amplify a 1727 bp region of DNA, aesFK520A (SEQ ID NO: 10) from Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et al., 2000) as the template and hot start KOD DNA polymerase. A 5' extension was designed in each oligo to include the restriction sites present in the genomic
sequence to aid cloning of the amplified fragment (Figure 1 ). This 1727 bp fragment was cloned into pUC19 that had been linearised with Sma\, resulting in plasmid pAESA5 and the insert verified by sequencing.
SG 165 CCAGATCTCGTCGGGCACCTTGAAGTAGGCGAGCCG
BgIW (SEQ ID NO: 1 )
SG 166 CCCTCGAGGTCCGGTGATCCGGTCTTCTCGAAGC Xho\
(SEQ ID NO: 2)
1.2 Cloning of DNA homologous to the downstream flanking region of fkbO disruption. Oligos SG167 (SEQ ID NO: 3) and SG168 (SEQ ID NO: 4) were used to amplify a 2034 bp region of DNA, termed aesFK520B (SEQ ID NO: 1 1 ) from Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et al., 2000) as the template and hot start KOD DNA polymerase. A 5' extension was designed in oligo SG167 to introduce the restriction site to aid cloning of the amplified fragment (Figure 1 ) and SG168 included the restriction site present in the genomic sequence. This 2034 bp fragment was cloned into pUC19 that had been linearised with Sma\, resulting in plasmid pAESBI and the insert verified by sequencing.
SG 167 CCCTCGAGCGCACAGCGCCCTGTCGAGTCCGGCATG
Xho\ (SEQ ID NO: 3)
SG 168 CCGAATTCGAGGTCGAAGCGGGTGAACCAGCGTC
EcoRI
(SEQ ID NO: 4)
The -1.7 kb Bgl\\/Xho\ fragment from pAESA5 and -2.0 kb Xho\/EcoR\ fragment from pAESBI were cloned into the -5.9 kb Sg/ll/EcoRI fragment of pKC1 139 (Bierman et al., 1992) to make pAESABI . pAESABI therefore contained the upstream and downstream regions such that the double crossover event would result in the desired small deletion, introducing a frameshift in the fkbO gene.
1.3 Transformation of Streptomyces hygroscopicus subsp. hygroscopicus
Escherichia coli ET12567, harbouring the plasmid pUZ8002, was transformed with pAESABI by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) by spore conjugation (as described in General Methods). Exconjugants were plated on R6 agar and incubated at 28°C. pAESABI is able to self-replicate in Streptomyces hygroscopicus subsp.
hygroscopicus (DSM 40822) at 28°C. Transformants were subcultured onto MAM plates with apramycin (0.050 mg/mL) at 28°C to ensure the pAESABI plasmid with resistance marker was present. Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured again on to MAM plates with apramycin at 37°C to induce the plasmid to integrate, as the plasmid cannot self-replicate at 37°C. The transformants were then subcultured for four subsequent rounds at 37°C on MAM plates with no antibiotic. The transformants from the fourth subculture on antibiotic free plates were plated for spore harvest on ISP3 medium at 28°C. Serial dilutions were made from the filtered collected spores and were plated on MAM plates to achieve single colonies.
1.4 Screening for secondary crosses
Single colonies were patched in duplicate onto MAM supplemented with 0.050 mg/mL apramycin and MAM containing no antibiotics, and grown at 28 C for 3-4 days. Patches that grew on the no antibiotic plate but did not grow on the apramycin plate were screened to test if the desired double recombination effect had occurred. A 6 mm circular plug from each patch that had lost the marker was used to inoculate individual 50 ml. falcon tubes containing 7 ml_ FK seed medium (See Media Recipes) without antibiotics and grown for 2 days at 28°C, 300 rpm with a 1 inch throw. These were then used to inoculate (0.5 ml. into 7 ml. - 7% inoculum) FK production medium (See Media Recipes) in a 50 ml. falcon tube at 28°C, 300 rpm with a 1 inch throw. After 24 hours, each falcon tube was fed with 0.050 ml. 0.32 M 4-trans- hydroxycyclohexane carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation was continued as before. The cultures were sampled after 6 days growth and analysed by LC-MS, using the methods described above.
14 out of 79 apramycin sensitive strains tested had undergone the desired recombination event to give the disruption of the fkbO gene. These strains produced a metabolite that was 30 amu less than FK520 itself. This compound was shown to be 31-desmethoxyFK520 following isolation and characterisation of that compound. One strain was ultimately selected, designated "AB5-10". This strain was later renamed "BIOT-4131 ".
Example 2 - Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift. 2.1 Cloning of DNA homologous to the upstream flanking region of fkbO disruption.
Oligos SG165 (SEQ ID NO: 1 ) and SG166 (SEQ ID NO: 2) were used to amplify a 1727 bp region of DNA, termed aesFK520A (SEQ ID NO: 10) from Streptomyces hygroscopicus
subsp. hygroscopicus (DSM 40822) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et a/.2000) as the template and hot start KOD DNA polymerase. A 5' extension was designed in each oligo to include the restriction sites present in the genomic sequence to aid cloning of the amplified fragment (Figure 2). This 1727 bp fragment was cloned into pUC19 that had been linearised with Sma\, resulting in plasmid pAESA5 and the insert verified by sequencing.
SG 165 CCAGATCTCGTCGGGCACCTTGAAGTAGGCGAGCCG
BgIW (SEQ ID NO: 1 )
SG 166 CCCTCGAGGTCCGGTGATCCGGTCTTCTCGAAGC
Xho\
(SEQ ID NO: 2)
2.2 Cloning of DNA homologous to the downstream flanking region of fkbO disruption. Oligos SG169 (SEQ ID NO: 5) and SG168 (SEQ ID NO: 6) were used to amplify a 1460 bp region of DNA, termed aesFK520C (SEQ ID NO: 12) from Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et al., 2000) as the template and hot start KOD DNA polymerase. A 5' extension was designed in oligo SG169 to introduce the restriction site to aid cloning of the amplified fragment (Figure 2) and SG168 included the restriction site present in the genomic sequence. This 1460 bp fragment was cloned into pUC19 that had been linearised with Sma\, resulting in plasmid pAESCδ and the insert verified by sequencing.
SG169 CCCTCGAGAACATGGCCCGGGTCATCGGCGCGG
Xho\ (SEQ ID NO: 5) SG 168 CCGAATTCGAGGTCGAAGCGGGTGAACCAGCGTC
EcoRI (SEQ ID NO: 6)
The -1.7 kb Bgl\\IXho\ fragment from pAESA5 and -1.5 kb Xho\/EcoR\ fragment from pAESBI were cloned into the -5.9 kb Bgl\\/EcoR\ fragment of pKC1 139 (Bierman et al., 1992) to make pAESAC4. pAESAC4 therefore contained the upstream and downstream regions such that the double crossover event would result in the desired large deletion of the fkbO gene.
2.3 Transformation of Streptomyces hygroscopicus subsp. hygroscopicus Escherichia coli ET12567, harbouring the plasmid pUZ8002, was transformed with pAESAC4 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) by spore
conjugation (as described in General Methods). Exconjugants were plated on R6 agar and incubated at 28°C. pAESAC4 is able to self-replicate in Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) at 28°C. Transformants were subcultured onto MAM plates with apramycin (0.050 mg/mL) at 28°C to ensure the pAESAC4 plasmid with resistance marker was present. Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured on to MAM plates with apramycin at 37°C to induce the plasmid to integrate, as the plasmid cannot self-replicate at 37°C. The transformants were then subcultured for four subsequent rounds at 37°C on MAM plates with no antibiotic. The transformants from the fourth subculture on antibiotic free plates were plated for spore harvest on ISP3 medium at 28°C. Serial dilutions were made from the filtered collected spores and were plated on MAM plates to achieve single colonies.
2.4 Screening for secondary crosses
Single colonies were patched in duplicate onto MAM supplemented with 0.050 mg/mL apramycin and MAM containing no antibiotics, and grown at 28 °C for 3-4 days. Patches that grew on the no antibiotic plate but did not grow on the apramycin plate were screened to test if the desired double recombination effect had occurred. A 6 mm circular plug from each patch that had lost the marker was used to inoculate individual 50 ml. falcon tubes containing 7 ml_ FK seed medium (See Media Recipes) without antibiotics and grown for 2 days at 28°C, 300 rpm with a 1 inch throw. These were then used to inoculate (0.5 ml. into 7 ml. - 7% inoculum) FK production medium (See Media Recipes) in a 50 ml. falcon tube at 28°C, 300 rpm with a 1 inch throw. After 24 hours, each falcon tube was fed with 0.050 ml. 0.32 M 4-frans-hydroxy cyclohexane carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation was continued as before. The cultures were sampled after 6 days growth and analysed by LC-MS using the methods described above.
15 out of 59 apramycin sensitive strains tested had undergone the desired recombination event to give the disruption of the fkbO gene. When fed as described above these strains produced a metabolite that was 30 amu less than FK520 itself. This compound was shown to be 31-desmethoxyFK520 by comparison with isolated and charaterised material. One of these strains was designated BIOT-4131.
Example 3 - Generation of a Streptomyces tsukubaensis strain in which the fkbO gene has been inactivated by introducing a small deletion inducing a frameshift. 3.1 Cloning of DNA homologous to the upstream flanking region of fkbO disruption.
Oligos AES43 (SEQ ID NO: 6) and AES40 (SEQ ID NO: 7) were used to amplify a 2057 bp region of DNA, termed aesFK506D1 1 (SEQ ID NO: 13) from Streptomyces tsukubaensis no.
9993 (FERM BP-927) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et al., 2000) as the template and hot start KOD DNA polymerase. A 5' extension was designed in each oligo to introduce the restriction sites to aid cloning of the amplified fragment (Figure 3). This 2057 bp fragment was cloned into pUC19 that had been linearised with Sma\, resulting in plasmid pAESδ and the insert verified by sequencing.
AES43 CCAAGCTTGAGCGCCTCGTCCCAGAGCGCGGCCTGGTC
H/ndlll
(SEQ ID NO: 6)
AES40 CCATGCATCGGGACACCGTCCGTGAGCGACACCTCGGCATGACC
(SEQ ID NO: 7)
3.2 Cloning of DNA homologous to the downstream flanking region of fkbO disruption.
Oligos AES41 (SEQ ID NO: 8) and AES42 (SEQ ID NO: 9) were used to amplify a 1985 bp region of DNA, termed aesFK506E3 (SEQ ID NO: 14) from Streptomyces tsukubaensis no. 9993 (FERM BP-927) in a standard PCR reaction (Sambrook and Russell, 2001 ) using genomic DNA (Kieser et al., 2000) as the template and hot start KOD DNA polymerase. A 5' extension was designed in each oligo to introduce the restriction sites to aid cloning of the amplified fragment (Figure 3). This 1985 bp fragment was cloned into pUC19 that had been linearised with Sma\, resulting in plasmid pAES9 and the insert verified by sequencing.
AES41 CCATGCATCCGATGCCGTCGCGGCGCTCTACACGCGGG
(SEQ ID NO: 8)
AES42 CCAGATCTGAAGGGCTCGGCGGTCACACCGGGCAGCGC
BgIW (SEQ ID NO: 9)
The -2.0 kb Hind\\\/Nsi\ fragment from pAESδ and -2.0 kb NsiVBglW I fragment from pAES9 were cloned into the -6.0 kb Hinά\\\/Bgl\\ fragment of pKC1 139 (Bierman et al., 1992) to make pAES10. pAES10 therefore contained the upstream and downstream regions such that the double crossover event would result in the desired small deletion, introducing a frameshift in the fkbO gene.
3.3 Transformation of Streptomyces tsukubaensis
Escherichia coli ET12567, harbouring the plasmid pUZ8002, was transformed with pAES10 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces tsukubaensis no. 9993 (FERM BP-927) by spore conjugation (as described in General Methods). Exconjugants were plated on R6 agar and incubated at 37°C.
pAESIO is not able to self-replicate in Streptomyces tsukubaensis no. 9993 (FERM BP-927) at 37°C and must integrate into the genome. Transformants were subcultured onto MAM plates with apramycin (0.050 mg/mL) at 37°C two to three times, to ensure that the pAESI O plasmid with resistance marker had integrated. Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured for two subsequent rounds at 37°C on MAM plates with no antibiotic and then a final time at 28°C. The transformants from the fourth subculture on antibiotic free plates were plated for spore harvest on ISP4 medium at 28°C. Serial dilutions were made from the filtered collected spores and plated on ISP4 plates at 28°C to achieve single colonies.
3.4 Screening for secondary crosses
Single colonies were patched in duplicate onto ISP4 supplemented with 0.050 mg/mL apramycin and ISP4 containing no antibiotics, and grown at 28 C for 3-4 days. Patches that grew on the no antibiotic plate but did not grow on the apramycin plate were screened to test if the desired double recombination effect had occurred. A 6 mm circular plug from each patch that had lost the marker was used to inoculate individual 50 ml. falcon tubes containing 7 mL NGY (See Media Recipes) without antibiotics and grown for 2 days at 28°C, 300 rpm with a 1 inch throw. These were then used to inoculate (0.5 ml. into 7 ml. - 7% inoculum) PYDG+MES (See Media Recipes) in a 50 ml. falcon tube at 28°C, 300 rpm with a 1 inch throw. After 24 hours, each falcon tube was fed with 0.050 ml. 0.32 M 4-frans-hydroxy cyclohexane carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation will be continued as before. The cultures were sampled after 6 days growth and analysed by LC-MS by the methods described above. Of the 93 patches that were screened, 49 had undergone the desired double recombination event. One of these strains was designated BIOT 4254.
Example 4 - Array feeding to BIOT-4131 with various starter units
Spore stocks of BIOT-4131 were prepared after growth on MAM, ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 mL medium NGY in 50 mL Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h. From the seed culture 0.5 mL was transferred into 7 mL production medium PYDG + MES in 50 mL centrifuge tubes with foam plugs. Cultivation was carried out at 28°C and 300 rpm (2.5 cm throw). For production of FK520 analogues, 0.05 mL of a 0.32 M methanolic solution of the feed compound was added to each tube at 24 hours post inoculation
to give a final concentration of 2.12 mM. Cultivation was continued for additional five days post feeding at 28°C and 300 rpm (2.5 cm throw).
Example 5 - Isolation of 28-des(3-c/s-methoxy-4-frans-hydroxycyclohexyl)-28-(4- tetrahydro-2H-pyran)FK520 (Pyran-FK520)
Spore stocks of BIOT-4131 were recovered onto plates of MAM medium and incubated for 10 days at 28°C. Vegetative cultures (seed culture) were prepared by removing 4-5 agar plugs (6 mm in diameter) from the MAM plate and inoculating into 50 ml. medium NGY in 250 ml. Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 28°C and 300 rpm (2.5 cm throw). From the seed culture 2.5 ml. was transferred into 50 ml. production medium PYDG in 250 ml. Erlenmeyer flasks with foam plug. After 24 hours cultivation at 28°C and 200 rpm (2.5 cm throw) (also at 250 rpm with similar results), 0.33 ml. from a 0.32 M
methanolic solution of tetrahydropyran-4-carboxylic acid was added to each production flask to give a final 2.12 mM concentration of the feed. Cultivation was continued for further five days at 28°C and 200 rpm (2.5 cm throw). The culture broths were pooled (to 3.75 L) and extracted with ethyl acetate three times. The combined organics were taken to dryness under reduced pressure to yield a brown oil (8 g). The oil was dissolved in methanol and silica (10 g) added. The solvent was removed under reduced pressure to impregnate the silica with the crude extract. The impregnated silica was dry packed above silica (1 10 g) in a column with a 5 cm diameter. The column was conditioned with and eluted with ethyl acetate / hexane (1 :1 , 1 litre), ethyl acetate (1 litre). After a 250 ml. pre-fraction, 50 ml. fractions were collected and the target compound identified in fractions 2 - 10. The fractions were combined and taken to dryness (500 mg). This material was impregnated onto silica (4 g) and this material was dry- packed onto silica (20 cm x 2.5 cm diameter) and conditioned with ethyl acetate / hexane (1 :2, 300 ml.) and eluted with ethyl actate / hexane (1 :1 , 300 ml.) , ethyl acetate / hexane (2:1 , 300 ml.) and finally ethyl actate. After a prefraction (150 ml.) 15 ml. fractions were collected and the target compound was identified in fractions 13 to 20. These were combined and taken to dryness under reduced pressure (133 mg). This material was dissolved in 0.9 ml. methanol and purified by preparative HPLC in 2 injections. A phenomenex C18 Luna column (5 micron, 25 cm x 22.5 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 60 % B for 5 minutes following the injection followed by a gradient to 100 % B at 25 minutes. The compound started to elute at 21 minutes. Pure fractions were identified by HPLC-MS and combined. These fractions were taken to dryness under reduced pressure to yield 28-des(3-c/s-methoxy-4-frans- hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520 (also described as Pyran-FK520) as a white solid (56 mg).
Example 6 - Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the loading module of the FK520 PKS (comprising DHCHCA CoA ligase-like domain, enoyl CoA reductase and ACP) is replaced by the loading module of the avermectin PKS (comprising AT and ACP) from Streptomyces avermitilis.
6.1 Cloning of the left flanking sequence located upstream of the FK520 loading module. Oligos PCR1 1 F and PCR1 1 R were used to amplify a 2.19 kb region of DNA from Streptomyces hygroscopicus subsp. hygroscopicus (DSM40822) using genomic DNA template (Kieser et al., 2000) and the KOD Polymerase kit from Novagen. PCR samples were
supplemented with 10% DMSO. Annealing temperatures between 65 and 70 0C and extension for 1 min at 70 0C were used. A 5' extension was designed for each oligo to introduce restriction sites (Spel, Λ/αtel) to aid cloning of the amplified fragment. The Nde\ site of PCR1 1 R comprises the loading module start codon (ATG), and the 3 non-coding bases upstream are mutated to CAT (replacing TCC). The 2.19 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19, resulting in plasmid pUC19 PCR1 1. The insert sequence was verified by sequencing. PCR11 F (Seq ID 18) CGACTAGTGCAGCGCGAGCGTGTTGACGAACATGCCGATCAGG
Spel PCR11 R (Seq I D 19) GGCATATGAACACCTTTCTCTCGACCAACCGCACAACAGCACG
Λfctel
6.2 Cloning of the right flanking sequence located downstream of the FK520 loading module and covering the region containing FK520 KS1 and part of AT1. Oligos PCR1 3F2 and PCR13R were used to amplify a 2.20 kb region of DNA from Streptomyces hygroscopicus subsp. hygroscopicus (DSM40822) using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 65 and 70 0C and extension for 1 min at 70 0C were used. A 5' extension was designed for each oligo to introduce restriction sites {Nhe\, Xba\) to aid cloning of the amplified fragment. The 2.20 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19. A construct was selected with the insert orientated such that the Nhe\ site of the PCR fragment would be close to the Nde\ site of pUC19. This was relevant for the subsequent cloning procedure and the resulting plasmid was named pUC19 PCR13F. The insert sequence was verified by sequencing. PCR13F2 comprises the 5' end of KS1 and the choice of Nhe\ introduces a mutation resulting in the KS1 N-terminal sequence of 'DPLA' instead of the original FK520 KS1 sequence 'DPVA'. PCR13F2 (Seq ID 20)
CTCGGCTCCGGCGACCCGCTAGCGATCGTCGGCATGGCGT
Nhe\ PCR13R (Seq lD 21 )
GCTCTAGACACCGGCTCGGTCACCCAGGCGCTGTCCAC Xba\
6.3 Cloning of a DNA fragment coding for the loading module of avermectin PKS (comprising AT and ACP)
Oligos PCR12F and PCR12R were used to amplify a 1.46 kb region of DNA from Streptomyces avermitilis (DSM41443/) using genomic DNA template (Kieser et al., 2000) and
KOD Polymerase. PCR samples were supplemented with 1 0% DMSO . An nealing temperatures between 65 and 70 0C and extension for 1 min at 70 0C were used. A 5' extension was designed for each oligo to introduce restriction sites (Λ/c/el, Apa\) to aid cloning of the amplified fragment. The 1 .46 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19, resulting in plasmid pUC19 PCR22 and the insert verified by sequencing. PCR12F comprises the 5' end of ave loading module and the choice of Λ/c/el introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS. PCR12R comprises the 5' end of KS1 and as mentioned for PCR13F2 the choice of Nhe\ introduces a mutation resulting in the KS1 N-terminal sequence of 'DPLA' instead of 'DPVA' in the FK520 cluster and 'DPIA' in the avermectin cluster. PCR12F (Seq ID 22)
TTCATATGCAGAGGATGGACGGCGGGGAAGAACCCCGCCCTGCGG
Λ/c/el
PCR12R (Seq ID 23) TTGCTAGCGGGTCGTCCGCCGCTGCCGTGCCTCCGTGGCCGCT
Nhe\
6.4 Assembly of pUS4 which carries the replacement loading module and the two flanking fragments The Spel Λ/c/el fragment of pUC19PCR1 1 was ligated with Spel Λ/c/el cut pKC1 139B01 resulting in pUS1. The Λ/c/el Nhe\ fragment of pUC19PCR12 was ligated with Λ/c/el Nhe\ cut pUC19PCR13F resulting in pUS2. The Λ/c/el Xba\ fragment of pUS2 was then cloned into pUS1 digested with Λ/c/el Xba\, the final plasmid being pUS4.
6.5 Transformation of Streptomyces hygroscopicus subsp. hygroscopicus
Escherichia coli ET12567 (pUZ8002) was transformed with pUS4. This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar, incubated at 37 0C and overlaid with nalidixic acid (25 mg/L) and apramycin (50 mg/L) the next day. pUS4 is not able to self-replicate in Streptomyces hygroscopicus subsp. hygroscopicus at 37 0C and is forced to integrate into the genome by recombination. Six days later, transformants were subcultured on MAM plates containing apramycin (50 mg/L) at 37 0C to ensure the pUS4 plasmid with resistance marker was present. Subculturing for two subsequent rounds at 37 0C on MAM plates without apramycin was carried out to allow secondary recombination. This event would either cause the loss of the plasmid via the second region of homology, not the one by which it had originally integrated resulting in the desired gene replacement; or the loss of the plasmid via the same region of homology as the original integration resulting in a
wildtype revertant. Unexpected recombination events could occur as well. This required single spore isolation of subcultured patches. The transformants were subcultured on MAM plates without apramycin at 28°C and subsequently incubated at 28 0C on ISP3 plates for spore harvest. Serial dilutions were made from the collected spores and plated on MAM plates to achieve single colonies.
6.6 Screening for secondary crosses
Single colonies were patched in duplicate onto MAM supplemented with 50 mg/L apramycin and MAM containing no antibiotics, and grown at 28 0C for 3-4 days. Patches that grew on the no antibiotic plate but did not grow on the apramycin plate were screened for production to test if the desired double recombination event had occurred. A 6 mm circular plug from each patch that had lost the marker was used to inoculate individual 50 ml. falcon tubes containing 7 ml_ FK seed medium (see Media Recipes) without antibiotics and grown for 2 days at 28 0C, 300 rpm, 2.5 cm throw. These were then used to inoculate (0.5 ml. into 7 ml. - 7% inoculum) FK production medium (see Media Recipes) in a 50 ml. falcon tube and incubated at 28 0C, 300 rpm, 2.5 cm throw. The cultures were harvested after 6 days growth and analysed by LC-MS, using the methods described above. 61 out of 93 apramycin sensitive strains tested had undergone the desired recombination event. These strains produced FK520 derivates arising from the use of isobutyrate and 2-methylbutyrate starter units, most of them producing FK520 as well.
Six of these strains were selected and feeding experiments were undertaken. After inoculation of FK production medium (see above), tubes were incubated at 28 0C, 300 rpm, 2.5 cm throw. After 24 h, each falcon tube was fed with 50 μl_ 0.32 M cyclobutyl carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation was continued for five days. Culture extracts were analysed by LC-MS, using the methods described above. All six isolates used cyclobutyl carboxylic acid as starter. One strain was ultimately selected, designated "#1 -9". This strain was later assigned as "BIOT-4225".
Example 7 - Generation of a Streptomyces hygroscopicus subsp. hygroscopicus strain in which the loading module and KS1 of the FK520 PKS are replaced by the loading module and KS1 of the avermectin PKS from Streptomyces avermitilis.
7.1 Cloning of the left flanking sequence located upstream of the FK520 loading module, (see 1.1 ) Plasmid pUC19PCR11 from section 1.1 was used to provide the left flanking region.
7.2 Cloning of the right flanking sequence located downstream of the FK520 loading module + KS1
Oligos PCR23F and PCR23R were used to amplify a 2.15 kb region of DNA from Streptomyces hygroscopicus subsp. hygroscopicus using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 65 and 70 0C and extension for 1 min at 70 0C proved to be most successful. A 5' extension was designed for each oligo to introduce restriction sites (Apa\, Xba\) to aid cloning of the amplified fragment. The 2.15 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19. A construct was selected with the insert orientated such that the Apa\ site of the PCR fragment would be close to the Nde\ site of pUC19. This was relevant for the subsequent cloning procedure and the resulting plasmid was named pUC19 PCR23F. The insert sequence was verified by sequencing. Choosing Apa\ will introduce a silent mutation with the translated code still being 'GP'. PCR23F (Seq ID 24) TTGGGCCCTCGCGTGTGGAGTCGGGTGGTGATGGGTTG
Apa\ PCR23R (Seq ID 25)
TGTCTAGACGCCTCGGCGACCGCGAGCCACTCCAACCG
Xba\
7.3 Cloning of a DNA fragment coding for S. avermitilis loading module and KS1
Oligos PCR12F and PCR22R were used to amplify a 2.75 kb region of DNA from Streptomyces avermitilis using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 65 and 70 0C and extension for 1.5 min at 70 0C proved to be most successful. A 5' extension was designed for each oligo to introduce restriction sites (Λ/αtel, Apa\) to aid cloning of the amplified fragment. The 2.75 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19, resulting in plasmid pUC19 PCR22 and the insert verified by sequencing. PCR12F comprises the 5' end of ave loading module and the choice of Nde\ introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS. PCR22R was designed such that the sequence of the amplified fragment ends slightly downstream of the KS1 gene where Apa\ links to the FK520 sequence. PCR12F (Seq ID 22)
TTCATATGCAGAGGATGGACGGCGGGGAAGAACCCCGCCCTGCGG Λfctel
PCR22R (Seq ID 26)
TTGGGCCCCGGCGCCTCCTCCAAAATCACATGCGCATTCGTGC
Apa\
7.4 Assembly of the two flanking fragments and the replacement module.
The Spe\ Nde\ fragment of pUC19PCR1 1 was ligated with Spe\ Nde\ cut pKC1139B01 resulting in pUS1. Since the pUC19PCR22 insert contains 2 internal Apa\ sites, a third single- cutting restriction enzyme (C/al) was chosen to aid cloning. C/al is affected by dam methylation. The dam" dcm" strain E. coli JM1 10 was transformed with pUC19PCR22 and the isolated plasmid digested with Nde\ C/al and C/al Apa\. A 3-part ligation was performed with the 1.76 kb Nde\ C/al fragment, the 0.98 kb C/al Apa\ fragment and pUC19PCR23F cut with Nde\ Apa\ resulting in plasmid pUS3. The Nde\ Xba\ fragment of pUS3 was then cloned into pUS1 digested with Nde\ Xba\, the final plasmid being pUS5.
7.5 Transformation of Streptomyces hygroscopicus subsp. hygroscopicus
Escherichia coli ET12567 (pUZ8002) was transformed with pUS5 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces hygroscopicus subsp. hygroscopicus by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar, incubated at 37°C and overlaid with nalidixic acid (25 mg/L) and apramycin (50 mg/L) the next day. pUS5 is not able to self- replicate in Streptomyces hygroscopicus subsp. hygroscopicus at 37 0C and is forced to integrate into the genome by recombination. Six days later, transformants were subcultured on MAM plates with apramycin (50 mg/L) at 37 0C to ensure the pUS5 plasmid with resistance marker was present. Subculturing for two subsequent rounds at 37 0C on MAM plates without apramycin was carried out to allow secondary recombination. This event would either cause the loss of the plasmid via the second region of homology, not the one by which it had originally integrated resulting in the desired gene replacement; or the loss of the plasmid via the same region of homology as the original integration resulting in a wildtype revertant. Unexpected recombination events could occur as well. This required single spore isolation of subcultured patches. The transformants were subcultured on MAM plates without apramycin at 28 0C and subsequently incubated at 28 0C on ISP3 plates for spore harvest. Serial dilutions were made from the collected spores and plated on MAM plates to achieve single colonies.
7.6 Screening for secondary crosses
Single colonies were patched in duplicate onto MAM supplemented with 50 mg/L apramycin and MAM containing no antibiotics, and grown at 28 0C for 3 - 4 days. Patches that grew on the no antibiotic plate but did not grow on the apramycin plate were screened to test if the desired double recombination effect had occurred. A 6 mm circular plug from each patch that
had lost the marker was used to inoculate individual 50 ml. falcon tubes containing 7 ml. FK seed medium (see Media Recipes) without antibiotics and grown for 2 days at 28 0C, 300 rpm, 2.5 cm throw. These were then used to inoculate (0.5 ml. into 7 ml. - 7% inoculum) FK production medium (see Media Recipes) in a 50 ml. falcon tube and incubated at 28 0C, 300 rpm, 2.5 cm throw. The cultures were harvested after 6 days growth and analysed by LC-MS, using the methods described above. 2 out of 93 apramycin sensitive strains tested had undergone the desired recombination event. These strains produced FK520 derivates arising from the use of isobutyrate and 2-methylbutyrate starter units (FK520 background). Feeding experiments were undertaken with the two strains to further confirm the integration of the ave loading module sequence. After inoculation of FK production medium (see above), tubes were incubated at 28 0C, 300 rpm, 2.5 cm throw. After 24 h, each falcon tube was fed with 50 μl_ 0.32 M cyclobutyl carboxylic acid to give a final concentration of 2.12 mM acid and shaking incubation was continued for five days. Culture extracts were analysed by LC-MS, using the methods described above. Both isolates designated "#1-7" and "#1-8" incorporated cyclobutyl carboxylic acid as a starter unit. Isolate #1-7 was designated as "BIOT-4230".
Example 8 - Generation of a Streptomyces tsukubaensis strain in which the loading module of the FK506 PKS (comprising DHCHCA CoA ligase-like domain, enoyl CoA reductase and ACP) is replaced by the loading module of the avermectin PKS (comprising AT and ACP) from Streptomyces avermitilis.
8.1 Sequence information for fkbO, P, B of the FK506 biosynthetic cluster of Streptomyces tsukubaensis.
The sequence information of FK506 fkbO,P,B is needed for the construction of ave load variants of BIOT-3119. 454 Sequencing of BIOT-31 19 gave two contigs covering part of the
FK506 cluster including fkbO and fkbP, but neither of them covering fkbB sufficiently. The 5 positive cosmids obtained via hybridization of the BIOT-31 19 cosmid library with an FK506 fkbO probe (see Materials & Methods) were end-sequenced. The alignment of end sequences with the FK520 cluster sequence (AF235504) showed that two of the cosmids contained fkbO, P, B completely. One of them, 3G9, was sequenced (Cambridge University DNA Sequencing
Facility). The sequence relevant for the cloning procedure described in Example 3 and 4 is shown in Fig. 1.
8 .2 C l o n i n g of t h e l eft f l anking sequence located upstream of the FK506 loading module.
Oligos UES4_For and UES4_Rev were used to amplify a 2.27 kb region of DNA from Streptomyces tsukubaensis no. 9993 using cosmid 3G9 template and KOD Polymerase. PCR
samples were supplemented with 10% DMSO. Annealing temperatures of 66 and 71 0C and extension for 1 .5 min at 68 0C were used. A 5' extension was designed for each oligo to introduce restriction sites (Spel, Λ/αtel) to aid cloning of the amplified fragment. The Nde\ site of UES4_For comprises the loading module start codon (ATG), and the 3 non-coding bases upstream are mutated to CAT (replacing TCC). The 2.27 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19, resulting in plasmid 'pUC19 fkbP'O 506 left'. The insert sequence was verified by sequencing.
UES4_For (Seq lD 27) CGACTAGTGCAGCGCGAGCGTGTTGACGAACATGCCGATGAGG
Spel
UES4_Rev (Seq ID 28) GGCATATG
AACGCCTTTCTCTCGGCTGACCGTACGGCAGCACG Λfctel
8.3 C l on i n g of th e ri g h t fl a n king sequence located downstream of the FK506 loading module and covering the region containing FK506 KS1 and part of AT1.
Oligos UES7_For and UES7_Rev were used to amplify a 2.29 kb region of DNA from Streptomyces tsukubaensis no. 9993 using 3G9 cosmid and KOD Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 66 and 71 0C and extension for 1 .5min at 68 0C proved to be successful. A 5' extension was designed for UES7_Rev to introduce an Xba\ site. UES7_For comprises the internal Pvu\ site at the 5' end of ave KS1 followed by the internal Pvu\ site of FK506 KS1 which was mutated to 'CCATCG'. The 2.29 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19 resulting in plasmid 'pUC19 fkbB' 506 right'. The insert sequence was verified by sequencing. UES7_For (Seq ID 29)
TTCGATCGCCATCGTCGGCATGGCCTGCCGACTGCCGGGCGGCGT
Pvu\ UES7_Rev (Seq ID 30)
GCTCTAGACGGATGGGCGCTGCACTCGACGAAGAGGGAGCCGT Xba\
8.4 Cloning of a DNA fragment coding for the loading module of avermectin PKS (comprising AT and ACP)
Oligos PCR12F and UES5_Rev were used to amplify a 1.46 kb region of DNA from Streptomyces avermitilis using genomic DNA template (Kieser et al., 2000) and KOD
Polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 66 and 71 0C and extension for 1 .5 min at 68 0C were used. A 5' extension was designed for PCR12F to introduce an Nde\ site, whereas UES5_Rev comprises the internal Pvu\ site at the 5'-end of ave KS1. The 1.46 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19, resulting in plasmid 'pUC19 ave load middle' and the insert verified by sequencing. PCR12F comprises the 5' end of ave loading module and the choice of Nde\ introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS. UES5_Rev comprises the 5' end of ave KS1 and introduces a mutation resulting in the KS1 N-terminal sequence of 'EPIA' instead of 'DPIA' in the avermectin cluster and as 'EPIA in the FK506 cluster (see 3.5).
PCR12F (Seq ID 22)
TTCATATGCAGAGGATGGACGGCGGGGAAGAACCCCGCCCTGCGG
Λfctel UES5_Rev (Seq lD 31 )
TTCGATCGGTTCGTCCGCCGCTGCCGTGCCTCCGTGGCCGCTGGGTG Pvu\
8.5 Assembly of the two flanking fragments and the replacement module. The Spe\ Nde\ fragment of 'pUC19 fkbP'O 506 left' was ligated with Spe\ Nde\ cut pKC1139B01 resulting in pUS1 1. A 3-fragment ligation was performed with the 4.72 kb Nde\ Xba\ fragment of pUS1 1 , the 1.46 kb Nde\ Pvu\ fragment of 'pUC19 ave load middle' and the 2.29 kb Pvu\ Xba\ fragment of 'pUC19 fkbB' 506 right'. The resulting plasmid was named pUS14.
8.6 Transformation of Streptomyces tsukubaensis
Escherichia coli ET12567 (pUZ8002) was transformed with pUS14 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces tsukubaensis by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar and incubated at 37°C. pUS14 is not able to self- replicate in Streptomyces tsukubaensis at 37 0C and must integrate into the genome. Transformants were subcultured onto MAM plates with apramycin (100 mg/L) and nalidixic acid (50 mg/L) at 37 0C and subcultured again onto MAM plates with apramycin (50 mg/L) and nalidixic acid (25 mg/L) at 37 0C, and once more onto MAM plates containing apramycin (50 mg/L), to ensure that the pUS14 plasmid with resistance marker had integrated. Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured for three subsequent rounds at 37 0C on MAM plates with no antibiotic and a final
time at 28 0C. The transformants from the last subculture on antibiotic free plates were plated for spore harvest on ISP4 medium at 28 0C. Serial dilutions were made from the filtered collected spores and plated on MAM plates to achieve single colonies.
8.7 Screening for secondary crosses
Single colonies were patched in duplicate onto MAM supplemented with 50 mg/L apramycin and MAM containing no antibiotics, and grown at 28 0C for 7 - 8 days. A selection of patches that grew on the no antibiotic plate but did not grow on the apramycin plate were screened to test if the desired double recombination event had occurred. A 6 mm circular plug from each patch that had lost the marker was used to inoculate individual 50 ml. falcon tubes containing 7 ml. NGY (See Media Recipes) without antibiotics and grown for 2 days at 28 0C, 300 rpm, 2.5 cm throw. These were then used to inoculate (0.5 ml. into 7 ml. - 7% inoculum) PYDG+MES medium (See Media Recipes) in a 50 ml. falcon tube at 28 0C, 300 rpm, 2.5 cm throw. After 24 h, each falcon tube was fed with 50 μl_ 0.32 M cyclobutyl carboxylic acid, to give a final concentration of 2 mM acid, and shaking incubation was continued as before. The cultures were sampled after 6 days total growth and analysed by LC-MS. A total of 26 out of 51 isolates had undergone the desired double recombination event.
A selection of isolates were re-tested in triplicate and ultimately a strain was selected as the FK506 ave-load strain. This strain was designated as "BIOT-4276".
Example 9 - Generation of a Streptomyces tsukubaensis strain in which the loading module and KS1of the FK506 PKS are replaced by the loading module and KS1 of the avermectin PKS from Streptomyces avermitilis.
9.1 Cloning of the left flanking sequence located upstream of the FK506 loading module, (see 3.1 ) Plasmid 'pUC19 fkbP'O 506 left' from section 3.2 was used to provide the left flanking region.
9.2 Cloning of the right flanking sequence located downstream of the FK506 loading module + KS1 (within FK506 fkbB)
Oligos UES10_For and UES10_Rev were used to amplify a 2.23 kb region of DNA from Streptomyces tsukubaensis using cosmid 3G9 template and KOD polymerase. PCR samples were supplemented with 10% DMSO. Annealing temperatures between 66 and 71 0C and extension for 1 .5 min at 68 0C were used. A 5' extension was designed for each oligo to introduce restriction sites (Sfo\, Xba\) to aid cloning of the amplified fragment. The 2.23 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19 resulting in plasmid 'pUC19 fkbB" 506 right'. The insert sequence was verified by sequencing. The choice of Sfo\
as restriction site immediately downstream of fkbB KS1 introduces a mutation (GTG to GCG) with the translated code changing from VPEVS' to 'APEVS' (see 4.4).
UES10_For (Seq lD 32) TTGGCGCCGGAGGTTTCGGTCTCTCCCACAGAGTCGTCCGGTG
Sfol UES10_Rev (Seq lD 33)
GCTCTAGAGGGCGGGCATATCGGTGAGGTCGGTGAGGTCGTCC
Xba\
9.3 Cloning of a DNA fragment coding for S. avermitilis loading module and KS1
Oligos PCR12F and UES9_Rev were used to amplify a 2.75 kb region of DNA from Streptomyces avermitilis (DSM41443J using genomic DNA template (Kieser et al., 2000) and KOD Polymerase. PCR samples were su pplemented with 1 0% DMSO. Anneal ing temperatures between 66 and 71 0C and extension for 1 .5 min at 68 0C proved to be most successful. A 5' extension was designed for each oligo to introduce restriction sites (Λ/c/el, Afe\) to aid cloning of the amplified fragment. The 2.75 kb PCR fragment was ligated with Sma\ digested and SAP-treated pUC19, resulting in plasmid 'pUC19 ave load/KS1 middle' and the insert verified by sequencing. PCR12F comprises the 5' end of ave loading module and the choice of Λ/c/el introduces a mutation resulting in the N-terminal sequence of the loading module being 'MQR' instead of 'VQR' in the avermectin PKS. Through the introduction of an Afe\ site downstream of KS1 and the subsequent blunt end ligation (Afe\/Sfo\) with the right hand Fk506 fragment, the translated code of the sequence following KS1 would be 'EAP' (as with avermectin PKS) rather than 'GVP' (as with FK506 PKS). PCR12F (Seq ID 22)
TTCATATGCAGAGGATGGACGGCGGGGAAGAACCCCGCCCTGCGG
Λ/c/el UES9_Rev (Seq ID 34)
GGAGCGCTTCCTCCAAAATCACATGCGCATTCGTGCCGCCGATCC Afe\
9.4 Assembly of pUS16 which carries the replacement module and the two flanking fragments The Spel Λ/c/el fragment of 'pUC 19 fkbP'O 506 left' was ligated with Spel Λ/c/el cut pKC1139B01 resulting in pUS1 1. A 3-fragment ligation was performed with the 4.72 kb Λ/c/el Xba\ fragment of pUS1 1 , the 2.75 kb Λ/c/el Afe\ fragment of 'pUC19 ave load/KS1 middle' and the 2.23 kb Pvu\ Xba\ fragment of 'pUC19 fkbB" 506 right'. The resulting plasmid was named pUS16.
9.5 Transformation of Streptomyces tsukubaensis
Escherichia coli ET12567 (pUZ8002) was transformed with pUS16 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Streptomyces tsukubaensis by spore conjugation (as described in Materials & Methods). Exconjugants were plated on R6 agar and incubated at 37°C. pUS16 is not able to self- replicate in Streptomyces tsukubaensis at 37 0C and must integrate into the genome. Transformants were subcultured onto MAM plates with apramycin (100 mg/L) and nalidixic acid (50 mg/L) at 37 0C and subcultured again onto MAM plates with apramycin (50 mg/L) and nalidixic acid (25 mg/L) at 37 0C, and once more onto MAM plates containing apramycin (50 mg/L), to ensure that the pUS16 plasmid with resistance marker had integrated. Subculturing to allow secondary recombination was carried out as follows: the transformants were subcultured for three subsequent rounds at 37 0C on MAM plates with no antibiotic and a final time at 28 0C. The transformants from the last subculture on antibiotic free plates were plated for spore harvest on ISP4 medium at 28 0C. Serial dilutions were made from the filtered collected spores and plated on MAM plates to achieve single colonies.
9.6 Screening for secondary crosses
Single colonies were patched in duplicate onto MAM supplemented with 50 mg/L apramycin and MAM containing no antibiotics, and grown at 28 0C for 7 - 8 days. A selection of patches that grew on the no antibiotic plate but did not grow on the apramycin plate were screened to test if the desired double recombination event had occurred. A 6 mm circular plug from each patch that had lost the marker was used to inoculate individual 50 mL falcon tubes containing 7 mL NGY (See Media Recipes) without antibiotics and grown for 2 days at 28 0C, 300 rpm with a 2.5 cm throw. These were then used to inoculate (0.5 mL into 7 mL - 7% inoculum)
PYDG+MES medium (See Media Recipes) in a 50 mL falcon tube at 28 0C, 300 rpm with a 2.5 cm throw. After 24 h, each falcon tube was fed with 50 mL 0.32 M cyclobutyl carboxylic acid, to give a final concentration of 2 mM acid, and shaking incubation was continued as before.
The cultures were sampled after 6 days total growth and analysed by LC-MS. A total of 18 out of 42 isolates had undergone the desired double recombination event. One strain was designated "BIOT-4278".
Example 10 - Array feeding to BIOT-4254 with various starter units
Spore stocks of BIOT-4254 were prepared after growth on MAM , ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C.
Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 ml. medium NGY in 50 ml. Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h. From the seed culture 0.5 ml. was transferred into 7 ml. production medium PYDG + MES in 50 ml. centrifuge tubes with foam plugs. Cultivation was carried out at 28°C and 300 rpm (2.5 cm throw). For production of FK506 analogues, 0.05 ml. of a 0.32 M methanolic solution of the feed compound was added to each tube at 24 hours post inoculation to give a final concentration of 2.12 mM. Cultivation was continued for additional five days post feeding at 28°C and 300 rpm (2.5 cm throw).
Example 11 - Biological data - In vitro evaluation of potency with a NFAT gene reporter.
Compounds were analysed in the NFAT reporter assay (see general methods). IC50 values were additionally generated for FK506 and FK520 as controls.
Example 12 - Biological data - In vitro evaluation of cell permeability and efflux.
A caco-2 cell permeability assay was used, as described in the general methods
From the above data, it can be seen that both FK506 and FK520 show high levels of efflux, possibly due to the action of a transporter, such as Pgp. Meanwhile, 28-des(3-c/s-methoxy-4- frans-hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520 showed much higher apical to basolateral transport and lower basolateral to apical transport, suggesting that it is more
permeable and less of a substrate for efflux, predicting improved and more consistent oral bioavailability.
Example 13 - Biological data - In vivo evaluation of PK.
To evaluate the pharmacokinetics of FK506, FK520 and 28-des(3-c/'s-methoxv-4-frans- hydroxycyclohexyl)-28-(4-tetrahydro-2H-pyran)FK520 in vivo, the mouse PK methodology was used as described in the general methods.
Dose levels of 1 mg/kg PO and 10mg/kg iv were used, the data is plotted in figure 4.
PK parameters were calculated as follows:
As can be seen, the oral bioavailability (F%) of the Pyran FK520 is higher than that for both FK506 and FK520, whilst the half life (T1/2) is shorter.
Example 14 - Array feeding to BIOT-4225 with various starter units
Spore stocks of BIOT-4225 were prepared after growth on MAM, ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 ml. medium NGY in 50 ml. Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h. From the seed culture 0.5 ml. was transferred into 7 ml. production medium PYDG + MES in 50 ml. centrifuge tubes with foam plugs. Cultivation was carried out at 28°C and 300 rpm (2.5 cm throw). For production of FK520 analogues, 0.05 ml. of a 0.32 M methanolic solution of the feed compound was added to each tube at 24 hours post inoculation to give a final concentration of 2.12 mM. Cultivation was continued for additional five days post feeding at 28°C and 300 rpm (2.5 cm throw).
Example 15 - Array feeding to BIOT-4276 with various starter units
Spore stocks of BIOT-4276 were prepared after growth on MAM , ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at -80 0C. Spore stocks were recovered onto plates of MAM medium and incubated for 5 - 21 days at 28°C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 7 ml. medium NGY in 50 ml. Centrifuge tubes with foam plugs. The culture tubes were incubated at 28°C, 300 rpm (2.5 cm throw) for 48 h. From the seed culture 0.5 ml. was transferred into 7 ml. production medium PYDG + MES in 50 ml. centrifuge tubes with foam plugs. Cultivation was carried out at 28°C and 300 rpm (2.5 cm throw). For production of FK506 analogues, 0.05 ml. of a 0.32 M methanolic solution of the feed compound was added to each tube at 24 hours post inoculation to give a final concentration of 2.12 mM. Cultivation was continued for additional five days post feeding at 28°C and 300 rpm (2.5 cm throw).
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Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.
All documents referred to herein, including patents and patent applications, are incorporated by reference in their entirety.
Claims
1 , A compound of formula (I)
wherein
Xa represents bond or CH2;
R2 represents -CH3, -CH2CH3, Or -CH2CH=CH2;
R3 represents H2 or =0;
Ri is selected from the group consisting of
wherein R10, Rn and R12 are independently selected from H, F, Cl, OR13, SR13 and NHR13 and R13 is selected from H, CrC4alkyl and Ci-C4acyl; and
wherein A is selected from O, S and NR13a, R13a represents H, CrC4alkyl or Ci-C4acyl, R14 and R15 are independently selected from H, CrC4alkyl, OR16, SR16 and NHR16, R16 represents Ci-C4alkyl or CrC4acyl, Xb represents a bond or CH2, when Xb is bond then R17 represents H and when Xb is CH2 then R17 represents H, F, Cl, OH, SH, C1-C4 alkyl, OR163, SR163 or NHR163 and R163 represents CrC4alkyl or C1-C4 acyl; and
wherein A represents O, S, or NR13b, R1Sb represents H, Ci-C4alkyl, or Ci-C4acyl, R143 represents Ci-C4alkyl, OR16, SR16 or NHR16, R16 represents CrC4alkyl or Ci-C4acyl, R1S represents H, F, Cl, OH, SH, C1-C4 alkyl, OR163, SR163 or NHR163, R163 represents C1-C4 alkyl or Ci-C4acyl, Xc and Xd independently represent bond or CH2 provided that Xc and Xd do not both represent CH2 and when Xc is bond then R173 represents H and when Xc is CH2 then R173 represents H, F, Cl, OH, SH, CrC4alkyl, OR16b, SR16b or NHR16b, R16b represents C1-C4 alkyl or C1-C4 acyl and wherein when Xd is bond R-ig represents H and when Xd represents CH2 then R19 represents H, F, Cl, OH, SH, C1-C4 alkyl, OR16c, SR16c or NHR16c, and R16c represents C1- C4alkyl or Ci-C4acyl; and
wherein R19b and R20 independently represent H, F, Cl, OH, SH, C1-C4 alkyl, OR16d, SR16d or NHR16d, and R16d represents CrC4alkyl or C1-C4 acyl;. and
(E) wherein R19c represents H, F, Cl, OH, SH, d-C4alkyl, ORi6e, SRi6e or NHR16e, and Ri6e represents CrC4 alkyl or d-C4 acyl; and
wherein R19d and R2Oa independently represent H, F, Cl, OH, SH, d-C4 alkyl, ORi6f, SRi6f or NHR16f and Ri6f represents d-C4 alkyl or d-C4 acyl ; and
wherein R18a, Ri9e and R2Ob independently represent H, F, Cl, OH, SH, d-C4 alkyl, ORi6g, SRi6g or NHR16g and Ri6g represents d-C4 alkyl or d-C4 acyl; and
wherein R2i, R22 and R23 independently represent H, F, Cl, OR26, SR26, CrC4 alkyl or CN, R26 represents H, d-C4 alkyl or d-C4 acyl, R24 and R25 independently represent H, F, Cl, OH, SH or CrC4 alkyl, provided that at least one of R2i, R22 and R23 is not H or d-C4 alkyl; and wherein R2ia, R22a and R23a are independently selected from H, F, Cl, OR263, SR263, C1- C4 alkyl and CN, R26a represents H, d-C4 alkyl or d-C4 acyl, R24a and R25a independently represent H, F, Cl, OH, SH or d-C4 alkyl and provided that at least one of R2ia, R22a and R23a does not represent H or d-C4 alkyl; and physiologically functional derivatives thereof.
2. A compound according to claim 1 wherein R2 represents -CH2CH=CH2.
3. A compound according to claim 1 wherein R2 represents -CH2CH3.
4. A compound according to any one of claims 1 to 3 wherein R3 represents =0.
5. A compound according to any one of claims 1 to 4 wherein Xa represents a bond.
6. A compound according to any one of claims 1 to 4 wherein Xa represents CH2.
7. A compound according to any one of claims 1 to 6 wherein R1 represents [G].
8. A compound according to any one of claims 1 to 6 wherein R1 represents [J].
9. A compound according to any one of claims 1 to 6 wherein R1 represents [B].
10. A compound according to any one of claims 1 to 6 wherein R1 represents [E].
1 1. A compound according to any one of claims 1 to 6 wherein R1 represents [D].
12. A compound according to any one of claims 1 to 6 wherein R1 represents [A].
13. A compound according to claim 1 selected from:
and physiologically functional derivatives thereof.
14. A compound according to claim 1 selected from:
and physiologically functional derivatives thereof.
15. A compound according to any one of claims 1 to 14 for use as a pharmaceutical.
16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14 together with one or more physiologically acceptable diluents or carriers.
17. A compound according to any one of claims 1 to 14 for use in the treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders.
18. Use of a compound according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders.
19. A method of treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders which comprises administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 14.
20. A method of producing a compound according to any one of claioms 1 to 14 which comprises feeding an appropriate non-natural starter acid to an FK506 or FK520 producing strain, culturing the strain and optionally isolating the compounds thereafter.
21. A method of producing a compound of formula (I) according to any one of claims 1 to 14 comprising:
(a) generating a recombinant strain of a FK506 or FK520 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene has been deleted or inactivated, or in which the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; and
(b) feeding a non-natural starter unit to said recombinant strain; (c) culturing said strain; and
(d) optionally isolating compounds of formula (I).
22. A method of producing a compound of formula (I) according to any one of claims 1 to 14 comprising: (a) feeding a non-natural starter unit to a recombinant strain of a FK506 or FK520 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene has been deleted or inactivated, or in which the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; (b) culturing said strain; and
(c) optionally isolating compounds of formula (I).
23. A method according to claim 21 or 22 wherein the precursor supply gene that has been deleted or inactivated is fkbO.
24. A method of producing a compound of formula (I) according to any one of claims 1 to 14 comprising:
(a) generating a recombinant strain of a non-FK506 or FK520 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene is absent or nonfunctional or the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS; and
(b) feeding a non-natural starter unit to said recombinant strain;
(c) culturing said strain; and (d) optionally isolating compounds of formula (I).
25. A method of producing a compound of formula (I) according to any one of claims 1 to 14 comprising:
(a) feeding a non-natural starter unit to a recombinant strain of a non-FK506 or FK520 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which at least one precursor supply gene is absent or non-functional or the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS;
(b) culturing said strain; and
(c) optionally isolating compounds of formula (I).
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US11066416B2 (en) | 2016-02-04 | 2021-07-20 | The Johns Hopkins University | Rapafucin derivative compounds and methods of use thereof |
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