WO2011021036A1 - Analogues de polycétides et leurs procédés de production - Google Patents

Analogues de polycétides et leurs procédés de production Download PDF

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WO2011021036A1
WO2011021036A1 PCT/GB2010/051364 GB2010051364W WO2011021036A1 WO 2011021036 A1 WO2011021036 A1 WO 2011021036A1 GB 2010051364 W GB2010051364 W GB 2010051364W WO 2011021036 A1 WO2011021036 A1 WO 2011021036A1
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alkyl
crc
compound according
acyl
strain
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PCT/GB2010/051364
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Barrie Wilkinson
Christine Martin
Steven Moss
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Biotica Technology Limited
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • the present invention relates to the use of the genes for biosynthesis of the allyl extender unit of FK506, the generation of novel FK506 and FK520 analogues and strains generated by manipulating one or more of these extender unit genes and optionally feeding non-natural extender units, or crotonic acid precursors thereof, to these strains and to the 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.
  • FK525 Hatanaka H, et al., 1989
  • FK523 Hatanaka, H., et al., 1988
  • 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
  • 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
  • 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 ef a/., 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.
  • FK506 analogues altered in the side-chain at C21 may demonstrate modulated calcineurin binding.
  • FK506 and FK520 analogues with modulated calcineurin binding may be useful for reduction of toxicity and may demonstrate improved activities in diseases dependent upon immunophillin ligands, including, but not limited to, FKBP12, FKBP51 , FKBP52 and FKBP13, this may include increased neuroprotection, neuroregeneration and reduced neurotoxicity.
  • 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 altered
  • 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
  • the complete biosynthetic cluster of FK506 is described. This includes the genes dedicated to provision of the allyl extender unit at C21 of FK506 which comprises some or all of ORF1 to ORF9 in Table 2. Analysis of gene function indicates a pathway in which a 3-carbon unit derived from propionyl-CoA is condensed with a 2-cabron unit derived from malonyl-CoA to give a 5-carbon chain. Selection and covalent attachment of at least one of these units is performed by ORF9, a dual acyltransferase-acylcarrier protein. The enzyme responsible for condensation is the ketosynthase ORF8.
  • the 3-carbon unit is most likely modified to an acrylic acid derivative prior to condensation by the action of an acyl-CoA dehydrogenase, most probably ORF6 but possibly ORF2.
  • an oxygenase such as ORF3.
  • the resulting acylcarrier protein- or coenzyme-A linked 5-carbon acid is modified to a crotonic acid derivative by the action of 'house-keeping' enzymes from the fatty acid biosynthetic pathway, specifically a beta- ketoacylreductase and a dehydratase.
  • Other possible enzymes involved in this chemistry include the short chain dehydrogenase/reductase ORF2.
  • the final step of biosynthesis is a reductive carboxylation catalysed by the ccr homologue ORF7 to a give a 3-carbon (allyl) substituted malonic acid thioester derivative.
  • Provision of the allyl extender unit may be engineered out of the cell by, for example, disruption of, or deletion of, one or more of the genes responsible for the provision of the allyl extender unit. Exogenous compounds which are accepted into this extender position may then be fed.
  • the FK506 cluster ccr gene (ORF7) may be retained in order to carboxylate the fed acid or ester appropriately for incorporation into the PKS.
  • a heterologous ccr gene may be expressed in the engineered organism in order to allow incorporation of a broader set of extender units.
  • FK506 analogues at C21 may be combined with previous strategies for the production of FK506 and FK520 analogues, of engineering a strain that cannot provide the natural starter unit and feeding exogenous acids or esters and generating engineered strains where 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.
  • strains optionally having been mutated by classical methods or targeted inactivation or deletion of one or more genes responsible for post-PKS modification, and/or mutated by classical methods or targeted inactivation or deletion of one or more precursor supply genes, including bkd genes (Ward et al., 1999) and homologues thereof.
  • X a represents CH 2 , S, O, fused cyclopropyl unit and X b represents a bond;
  • Z represents keto or CH 2 ;
  • Ri represents a moiety selected from:
  • V W X Y R 2 represents H, alkyl, halo, hydroxyl or thiol
  • R 3 represents H, Ci -4 alkyl, halo, hydroxyl or thiol
  • R 4 represents H, Ci -4 alkyl, halo, hydroxyl or thiol
  • R 5 represents OMe, Me or H
  • R 6 represents OMe, Me or H
  • R 8 represents OH
  • R 9 represents H, OH, halo, thiol, CrC 4 alkyl
  • Rio, Rn and Ri 2 independently represent F, Cl, CrC 4 alkyl, ORi 3 , SRi 3 or NHRi 3 and Ri 3 represents H, CrC 4 alkyl or CrC 4 acyl, wherein two or three of Rio-Ri 2 are CrC 4 alkyl;
  • Ri 3 and Ri 4 independently represent H, F, Cl, CrC 4 alkyl, ORi 5 , SRi 5 or NHRi 5 and Ri 5 represents H, CrC 4 alkyl or CrC 4 acyl;
  • Ri6 Ri 7 and Ri 8 independently represent H, F, Cl, CrC 4 alkyl, ORi 9 , SRi 9 or NHRi 9 and Ri 9 represents H, CrC 4 alkyl or CrC 4 acyl,
  • R 20 and R 2 i independently represent H, F, Cl, CrC 4 alkyl, OR 22 , SR 22 or NHR 22 and R 22 represents H, CrC 4 alkyl or CrC 4 acyl,
  • R 23 and R 24 independently represent H, F, Cl, CrC 4 alkyl, OR 25 , SR 25 or NHR 25 and R 25 represents H, CrC 4 alkyl or CrC 4 acyl;
  • R 26 represents H, F, Cl, CrC 4 alkyl, OR 27 , SR 27 or NHR 27 , and R 27 represents H, CrC 4 alkyl or
  • R 28 and R 29 independently represent H, F, Cl, CrC 4 alkyl, OR 30 , SR 30 or NHR 30 and R 30 represents H, CrC 4 alkyl or CrC 4 acyl ;
  • R 3 i R 32 and R 33 independently represent H, F, Cl, CrC 4 alkyl, OR 34 , SR 34 or NHR 34 and R 34 represents H, CrC 4 alkyl or CrC 4 acyl;
  • R35 R36 and R 37 independently represent H, F, Cl, CrC 4 alkyl, OR 38 , SR 38 or NHR 38 and R 38 represents H, CrC 4 alkyl or CrC 4 acyl, save that OR 41 shall not represent OH;
  • R 39 R 40, R 4 i and R 42 independently represent H, F, Cl, CrC 4 alkyl, OR 43 , SR 43 or NHR 43 and R 43 represents H, CrC 4 alkyl or CrC 4 acyl,
  • R 44 and R 45 independently represent H, F, Cl, CrC 4 alkyl, OR 46 , SR 46 or NHR 46 and R 46 represents H, CrC 4 alkyl or CrC 4 acyl;
  • R 47 , R 48 and R 49 are independently selected from H, F, Cl, OR 50 , SR 50 and NHR 50 and R 50 is selected from H, Ci-C 4 alkyl and Ci-C 4 acyl;
  • A is selected from O, S and NR 5 i
  • R 5 i represents H, Ci-C 4 alkyl or Ci-C 4 acyl
  • R 52 and R 53 are independently selected from H, Ci-C 4 alkyl
  • R 54 represents Ci-C 4 alkyl or CrC 4 acyl
  • X c represents a bond or CH 2
  • R 54 represents H and when X c is
  • R 54 represents H, F, Cl, OH, SH, Ci-C 4 alkyl, OR 55 , SR 55 or NHR 55 and R 55 represents Ci-C 4 alkyl or Ci-C 4 acyl;
  • B represents O, S, or NR 6 O
  • R 6 o represents H, Ci-C 4 alkyl, or Ci-C 4 acyl
  • R 56 represents d-
  • R 62 represents Ci-C 4 alkyl or C r C 4 acyl
  • X d and X e independently represent bond or CH 2 provided that X d and X e do not both represent CH 2 and when X d is bond then R 57 represents H and when X e is CH 2 then R 57 represents H, F, Cl, OH,
  • R 63 represents CrC 4 alkyl or CrC 4 acyl and when X e is bond R 59 represents H and wherein when X e represents CH 2 then R 59 represents H, F, Cl, OH,
  • R 65 and R 66 independently represent H, F, Cl, OH, SH, C r C 4 alkyl, OR 67 , SR 67 or NHR 67 , and
  • R 67 represents Ci-C 4 alkyl or CrC 4 acyl
  • R 68 represents H, F, Cl, OH, SH, C r C 4 alkyl, OR 69 , SR 69 or NHR 69 , and R 69 represents C r C 4 alkyl or CrC 4 acyl;
  • R 70 and R 7i independently represent H, F, Cl, OH, SH, C r C 4 alkyl, OR 72 , SR 72 or NHR 72 and
  • R 72 represents CrC 4 alkyl or CrC 4 acyl ;
  • R 73 , R 74 and R 75 independently represent H, F, Cl, OH, SH, C r C 4 alkyl, OR 76 , SR 76 or NHR 76 and R 76 represents CrC 4 alkyl or CrC 4 acyl;
  • R 77 , R 78 and R 79 independently represent H, F, Cl, OR 82 , SR 82 , CrC 4 alkyl or CN, R 82 represents H, CrC 4 alkyl or CrC 4 acyl, R 80 and R 8 i independently represent H, F, Cl, OH, SH or CrC 4 alkyl, provided that at least one of R 77 , R 78 and R 79 is not H or CrC 4 alkyl;
  • R 83 , R 84 and R 85 are independently selected from H, F, Cl, OR 88 , SR 88 , C1-C4 alkyl and CN, R 88 represents H, CrC 4 alkyl or CrC 4 acyl, R 86 and R 87 independently represent H, F, Cl, OH, SH or CrC 4 alkyl and provided that at least one of R 83 , R 84 and R 85 does not represent H or CrC 4 alkyl;
  • 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.
  • the present invention provides strains containing hybrid PKS, with a loading module conferring avermectin-like chain initiation, and the rest of the PKS conferring FK506/FK520-like chain processing and termination.
  • the present invention provides processes for production of FK506 and FK520 analogues defined by structure (I) above.
  • 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.
  • extender unit(s) includes references to carboxylic acids or esters fed to cells that are then further processed by the addition of CoA or an acylcarrier protein, and/or also by reductive carboxylation, halogenation or any other cellular process that converts the fed compound into a functional extender unit for the PKS.
  • auxiliary gene(s) includes references to modifying genes, precursor supply genes or both modifying genes and precursor supply genes.
  • 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.
  • 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
  • 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 incorporated by that PKS.
  • avermectin -I ike 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.
  • acyl means an alkyl group in which the first carbon atom is a carbonyl moiety.
  • C1-4 acyl groups examples include C2-4 acyl groups such as -COMe and -COEt, especially COMe.
  • C2-4 acyl groups such as -COMe and -COEt, especially COMe.
  • CHO i.e. C1 acyl 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 ethanedioic
  • 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 a bond
  • Z 0.
  • R 5 represents OMe
  • R 6 represents OMe
  • R 2 represents H. In one embodiment R 3 represents H.
  • R 4 represents H.
  • the alkyl or alkenyl group of R 7 may optionally be substituted by one to three e.g. one or two or three halogen atoms. In one embodiment the alkyl or alkenyl group of R 7 is not substituted by halogen atoms. In one embodiment the alkyl or alkenyl group of R 7 is substituted by one halogen atom. In one embodiment the alkyl or alkenyl group of R 7 is substituted by two halogen atoms. In one embodiment the alkyl or alkenyl group of R 7 is substituted by three halogen atoms.
  • R 7 is a linear group.
  • R 7 is a branched group.
  • R 7 may for example be selected from CH 3 , CH 2 CH 2 CI, CH 2 CH 2 F,
  • 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]. In another embodiment R 1 represents [L]. In another embodiment R 1 represents [M]. In another embodiment R 1 represents [N]. In another embodiment R 1 represents [O]. In another embodiment R 1 represents [P]. In another embodiment R 1 represents [Q]. In another embodiment R 1 represents [R]. In another embodiment R 1 represents [S]. In another embodiment R 1 represents [T]. In another embodiment R 1 represents [U]. In another embodiment R 1 represents [V]. In another embodiment R 1 represents [W]. In another embodiment R 1 represents [X]. In another embodiment R 1 represents [Y].
  • Compounds of formula (I) may be produced by preventing the in vivo production of the natural extender unit for example but not restricted to interrupting or deleting one or more essential genes such as ORF8 (the KS) or ORF 6 (the AT/ACP), and feeding exogenous carboxylic acids (or derivatives thereof) which can be incorporated; or preventing the in vivo production of the natural extender unit as above while retaining the function of the ccr gene either in its natural position or by providing it in trans for example at an attachment site, and feeding exogenous carboxylic acids (or derivatives thereof) which can be incorporated; or preventing the in vivo production of the natural extender unit as above while retaining the function of a ccr for example a heterologous ccr which may have a different substrate tolerance for example but not limited to the nove chloro-ccr gene of salinosporamide, salG (Liu et al 2009) and feeding exogenous acids which can be incorporated.
  • ORF8 the
  • fkbO is required for the provision of the natural starter unit, 4,5-di- hydroxycyclohex-1-ene carboxylic acid.
  • Disruption, deletion or otherwise removing the function of FkbO (for example but not limited to a frameshift deletion or the use of UV mutagenesis followed by screening for a non-producer of FK506 that is restored to production by feeding exogenous 4,5-di-hydroxycyclohex-1-ene carboxylic acid to the culture medium) leads to a strain that can be used to make FK506 or FK520 analogues by feeding exogenous analogues of the natural starter unit to the production medium.
  • the loading modules of avermectin and avermectin-like PKSs consist of an AT and an ACP domain.
  • the strain is mutated to inactivate or delete 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 branched chain keto acid, such as bkd (Ward et al., 1999).
  • the strain may be mutated to inactivate or delete of one or more genes responsible for biosynthesis of pipecolic acid, such as fkbL (Wu et al., 2000).
  • Pipecolic acid 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).
  • a deletion may be the removal of a small part of a gene or most of a gene or all of a gene and it may be done in- frame, or it may be a frameshift. This equally applies to engineering methods to remove starter unit supply and to the use of alternative junctions to join the avermectin loading module to the rapamycin PKS.
  • an avermectin-like loading module may be transferred to the FK506 or FK520 cluster by taking just the Acyltransferase (AT) and Acyl Carrier Protein (ACP) domains from the avermectin PKS and joining to the FK506/FK520 PKS before the first ketosynthase (KS) domain.
  • the junction may be made between the KS from the avermectin PKS and the first extender AT from the FK506/FK520 PKS. Analogous possibilities are discussed in WO 98/01546.
  • the splice junction in the hybrid PKS between the avermectin load and the FK506/FK520 PKS may be before, within or after the KS of the first extension module.
  • part or all of the KS of the first extension domain of the recombinant strain is from the avermectin PKS.
  • the loading module (optionally together with part or all of the KS of the first extension domain) of an avermectin-like PKS may be used in place of the avermectin loading module, such as those loading modules found in the
  • a precursor supply gene or genes such as 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 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.
  • 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).
  • 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 extender units into FK506 and FK520 analogues said method comprising removing the ability of the producing strain to provide the extender unit and feeding alternative compounds which may be incorporated, possibly following modification in vivo to become a suitable substrate for the PKS.
  • the present invention provides a method for additionally incorporating non-natural starter acids into FK506 and FK520 analogues, said method comprising either deleting or inactivating the ability of the cell to produce the natural starter unit, for example by targeted deletion or inactivation of fkbO, or by replacing the natural
  • FK506/FK520 loading module with the loading module of avermectin or an avermectin-like PKS and feeding starter units to this strain which optionally contains chromosomal DNA in which the precursor supply gene has been deleted or inactivated.
  • Suitable gene systems which express FK506/FK520 homologues 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;
  • the precursor supply gene which is deleted or inactivated is preferably a gene whose product is involved in branched chain keto acid formation, such as a bkd gene or homologue.
  • the gene system is preferably the FK506 or FK520 cluster.
  • the precursor supply gene deleted or inactivated is more preferably one or more of the bkd genes.
  • fkbM and/or fkbl are deleted or inactivated in addition to one or more of the bkd genes.
  • 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)
  • the present invention provides a method of producing compounds of formula (I) comprising:
  • strains may be further engineered so as not to produce the natural starter unit.
  • strains may be further engineered so that the gene fkbO is deleted or inactivated.
  • strains may be further engineered so that the natural loading module is replaced by the loading module from the avermectin or an avermectin like PKS.
  • one or more starter genes have been deleted or inactivated which produce 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.
  • 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. ascomyceticus ATCC 55276, Streptomyces hygroscopicus subsp.
  • 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).
  • 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).
  • 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 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.
  • 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.
  • C21 extender units and starter units are suitably provided as the free carboxylic acid, but derivatives that may be employed include salts and esters.
  • C21 extender and 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 arthritis, 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.
  • 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).
  • assays are well known to those of skill in the art, for example but without limitation: Immunosuppressant activity - Warner, IM., et al., 1992, Kahan et al.
  • 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, ).
  • 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).
  • 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 a/. 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
  • 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
  • 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 thrombo
  • 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.
  • HIV Human Immunodeficiency Virus
  • 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
  • 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.
  • lactose lactose
  • starch a cellulose
  • milk sugar or high molecular weight polyethylene glycols.
  • 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
  • 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
  • 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. 4,941 ,880; U.S. 4,790,824; or U.S. 4,596,556.
  • 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.
  • 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.
  • 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.
  • compositions of the invention may optionally contain further active ingredients.
  • Such an engineered strain which strain is further engineered so that the natural loading module is replaced by the loading module from the avermectin or an avermectin like PKS.
  • extender unit genes have been deleted or inactivated and optionally one or more starter unit
  • -A process for producing a polyketide which comprises culturing such an engineered strain in the presence of a non-natural extender unit (and if appropriate a non-natural starter unit) and optionally isolating said polyketide.
  • Escherichia coli DH10B GibcoBRL
  • E. coli JM1 10 New England Biolabs
  • 2xTY medium as described by Sambrook et al. (2001 ).
  • E. coli ET12567(pUZ8002) was grown as described by 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 MgSO 4 for transfection. E. coli transformants were selected for with ampicillin (100 mg/L), kanamycin (50 mg/L), apramycin (50 mg/L).
  • the avermectin producer Streptomyces avermitilis (DSM41443J was grown on TSB at 28 0 C 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.
  • BIOT-4168 Single spore isolates of both strains, termed BIOT-4168 (containing the genes for FK520 biosynthesis) and BIOT-4206 (containing the genes for FK506 biosynthesis), were used for strain construction.
  • BIOT-4081 , BIOT-4168, BIOT-3119, BIOT-4206 or strains which are described below were prepared after growth on MAM, ISP4, ISP3 or ISP2 agar medium, and preserved in 20% (w/v) glycerol and stored at -80 0 C. Spores were recovered on plates of MAM, ISP4, ISP3 or ISP2 medium and incubated for 5-21 days at 28 0 C.
  • Vegetative cultures were prepared by removing 5-10 agar plugs (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculation of 200 - 350 ml. medium NGY in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 h at 28 0 C, 250 rpm (2.5 cm throw). The entire seed culture in one flask was transferred into 5 L PYDG containing 0.01-0.05% antifoam SAG 471 , in 7 L Applikon Fermentor. The fermentation medium was pre-adjusted at pH 6.0-7.0 post-sterilization.
  • the fermentation was carried out for 6 days at 28 0 C, with starting agitation set at 300-450 rpm, aeration rate at 0.5-0.8 v/v/m and dissolved oxygen (DO) level controlled with the agitation cascade at 20 - 40% air saturation. If required the pH may be maintained using acid or base addition on demand.
  • the selected feed (providing the starter unit for biosynthesis of target compound) was fed to the production medium 12 - 24 h post inoculation.
  • the feed compound was dissolved in 3 - 5 ml. methanol and added to the culture to give final concentration of 2 mM of the feed compound, the amount of methanol not exceeding 1 % of the total volume. Fermentation was continued for further five days post-feeding.
  • Water used for preparing media was prepared using Millipore ENx Analytical Grade Water Purification System.
  • Oatmeal is cooked/steamed in the water for 20 min, 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 min.
  • the medium is adjusted to pH 7.0, with NaOH and then sterilised by autoclaving 121 0 C, 15 min.
  • the medium is adjusted to pH 7.0 with NaOH, and then sterilised by autoclaving 121 0 C, 15 min.
  • MES MES
  • PYDG + MES PYDG + MES
  • 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 min.
  • Genomic DNA preparation was carried out as described in Sambrook et al. (2001 ). PCR was performed according to the instructions of the KOD Polymerase kit (Novagen). 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
  • 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 Karl 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® III 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 Ampl OO Kan50 per well) at 37 0 C and frozen at -80 0 C after mixing wells with 50 ⁇ L LB/glycerol 1 :1.
  • Escherichia coli ET12567 (pUZ8002) (Macneil et al., 1992, Paget et al., 1999) was transformed with pKC1 139B01 -derived plasmids by electroporation to generate the E. coli donor strains for spore conjugation (Kieser et al., 2000). Fresh spores were harvested in water from plates of Streptomyces tsukubaensis (BIOT-4206 or Biot-31 19). Spore suspensions were heat-shocked at 50 0 C for 10 min. They were then mixed with the E. coli donor strain, which had been washed twice with 2xTY, in a ratio of 3:1 Streptomycete to E.
  • Culture broth (0.9 ml.) were extracted with ethyl acetate (0.9 ml.) in a 2 ml. Eppendorf tube.
  • the broth was mixed with the solvent for 15 min on a shaking platform (vibrax) at 400 rpm.
  • the phases are then separated by centrifugation (2 min, 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 5 ⁇ g of pimecrolimus (as an internal standard for quantification).
  • the solvent is removed in vacuo (3 min) and then re-dissolved in methanol (1 ml.) by gentle agitation on a shaking platform (5 min).
  • 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 ⁇ l_, oven 50 0 C, 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.
  • LC samples that have been spiked with 0.005 mg/mL pimecrolimus were analysed on the same instrument and with the same chromatographic conditions.
  • 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 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. CHCI 3 at ⁇ H 7.26 ( 1 H) and CHCI 3 at ⁇ c 77.0 ( 13 C). J values are given in Hertz (Hz).
  • Genomic DNA was isolated from S. hygroscopicus subsp. hygroscopicus (DSM 40822 assigned BIOT-4081 ) and Streptomyces tsukubaensis (FermBP927 assigned BIOT-3119) using standard protocols described in Kieser et al., (2000).
  • DNA sequencing of cosmids was carried out by the sequencing facility of the Biochemistry Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QW using standard procedures.
  • a draft genome sequence was obtained using 454 technology by the sequencing facility of the Biochemistry Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QW.
  • the initial draft genome was generated using a whole 454 chip and this data was subsequently improved by a further Vi chip and assembly of contigs carried out using PHredPHrap.
  • a cosmid library was prepared using isolated genomic DNA of the FK506-producing strain Streptomyces tsukubaensis (BIOT-31 19) using standard methods.
  • the genomic DNA was partially digested with BfuC ⁇ , dephosphorylated and ligated to supercos 1 cut with Xba ⁇ , dephosporylated with Shrimp Alkaline Phosphatase (Roche) and subsequently digested with BamYW. This was packaged into ⁇ phage used to transfect E. coli VCS257 and a library of 2000 clones generated.
  • Probes were made as decribed below for screening Streptomyces tsukubaensis (BIOT- 3119) to identify the FK506 cluster. Plasmid pAES2#2 was used as PCR template. It was obtained by cloning a 1.9kb PCR amplified fkbO fkbP fragment from Streptomyces
  • BIOT-3119 tsukubaensis into pUC19. This fragment had been recruited from BIOT-3119 genomic DNA using primers derived from FK520 fkbO and fkbP sequences.
  • cosmid 4D7 was selected for complete sequencing and covers the regions from 63048 to 109611 bp in the full sequence (SEQ ID No: 7) see table 1.
  • Cosmid 4D7 has an insert of ⁇ 46kb and overlaps with the G9 sequence by ca. 2kb.
  • Cosmid 4D7 contains the genes for biosynthesis of the allyl malonate extension unit and following a region of low homology to data there is a siderophore cluster.
  • Table 1 Regions of cluster sequence covered by contigs and cosmids
  • Table 2 Open reading frame predictions and gene assignments for FK506 cluster
  • fkbC and all genes downstream of fkbG were assigned based on glimmer predictions of ORFs with BLASTX / BLASTP predictions of functions.
  • Streptomyces hygroscopicus analysis of the enzymatic domains in the modular polyketide synthase. Gene 169: 9-16.
  • LL-F28249 antibiotic complex a new family of antiparasitic macrocyclic lactones. Isolation, characterization and structures of LL-F28249 alpha, beta, gamma, lambda. J. Antibiot. 41(4): 519-529.
  • Antascomicinc A, B, C, D and E Novel FKBP12 binding compounds from a
  • Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis. Arthritis Res. 4(1):64-70. Epub 2001
  • Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Its effect on cellular, growth factor and cytokine response in injured vessels.
  • 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)
  • Streptomyces hygroscopicus analysis of genes flanking the polyketide synthase.
  • A mycophenolate mofetil, and sirolimus (rapamycin) inhibit allergen-induced proliferation and IL-5 production by PBMCs from atopic asthmatic patients.
  • Rabinovitch A., Suarez-Pinzon, W.L., Shapiro, A.M., Rajotte, R.V., Power, R. (2002).
  • the biosynthetic gene cluster for the polyketide immunosuppressant rapamycin is the biosynthetic gene cluster for the polyketide immunosuppressant rapamycin.
  • rapamycin the discovery of SDZ RAD. Transplantation Proceedings 30: 2192-2194. Sehgal, S. N., Baker, H., and Vezina, C. (1975) Rapamycin (AY-22,989), a new antifungal antibiotic II. Fermentation, isolation and characterization. The Journal of Antibiotics 28:
  • actinorhodin production by rpsL encoding ribosomal protein S12 mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).
  • 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.

Abstract

Cette invention concerne des composés de formule (I) et leur utilisation en thérapie. Dans la formule (I), les variables sont telles que définies dans la description.
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Cited By (4)

* Cited by examiner, † Cited by third party
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WO2011068341A2 (fr) * 2009-12-03 2011-06-09 (주)제노텍 Gène et protéine pour la biosynthèse de composés tricycliques
WO2012089349A3 (fr) * 2010-12-31 2012-12-27 Acies Bio D.O.O. Nouveaux composés polycétides et leurs procédés de fabrication
US20130230559A1 (en) * 2010-08-24 2013-09-05 Ewha University - Industry Collaboration Foundatio Novel tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same
CN114591286A (zh) * 2021-04-12 2022-06-07 南京大学 新型大环内酯化合物acautalides A-C及其制备方法和应用

Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4475196A (en) 1981-03-06 1984-10-02 Zor Clair G Instrument for locating faults in aircraft passenger reading light and attendant call control system
US4486194A (en) 1983-06-08 1984-12-04 James Ferrara Therapeutic device for administering medicaments through the skin
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4596556A (en) 1985-03-25 1986-06-24 Bioject, Inc. Hypodermic injection apparatus
US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
US4941880A (en) 1987-06-19 1990-07-17 Bioject, Inc. Pre-filled ampule and non-invasive hypodermic injection device assembly
EP0427680A1 (fr) * 1989-11-09 1991-05-15 Sandoz Ltd. Composés tricycliques contenant des hétéroatomes
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
WO1993013663A1 (fr) 1992-01-17 1993-07-22 Abbott Laboratories Procede permettant d'effectuer la biosynthese de polycetides specifiques
US5312335A (en) 1989-11-09 1994-05-17 Bioject Inc. Needleless hypodermic injection device
US5383851A (en) 1992-07-24 1995-01-24 Bioject Inc. Needleless hypodermic injection device
WO1997002358A1 (fr) 1995-07-06 1997-01-23 The Leland Stanford Junior University Synthese de polycetides, exempte de cellule
WO1998001546A2 (fr) 1996-07-05 1998-01-15 Biotica Technology Limited Polyketides et leur synthese
WO2000000618A2 (fr) 1998-06-29 2000-01-06 Biotica Technology Limited Polycetides et leur synthese
WO2000001827A2 (fr) 1998-07-06 2000-01-13 Biotica Technology Limited Polycetides, preparation et matieres destinees a etre utilisees dans lesdits polycetides
US6150513A (en) 1998-09-16 2000-11-21 Kosan Biosciences, Inc. Polyketide synthase enzymes and recombinant DNA constructs therefor
WO2002014482A2 (fr) 2000-08-14 2002-02-21 Biotica Technology Limited Polycétides et leur synthèse
WO2004007709A2 (fr) 2002-07-16 2004-01-22 Biotica Technology Limited Production de polyketides et autres produits naturels

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4475196A (en) 1981-03-06 1984-10-02 Zor Clair G Instrument for locating faults in aircraft passenger reading light and attendant call control system
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4486194A (en) 1983-06-08 1984-12-04 James Ferrara Therapeutic device for administering medicaments through the skin
US4596556A (en) 1985-03-25 1986-06-24 Bioject, Inc. Hypodermic injection apparatus
US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
US4941880A (en) 1987-06-19 1990-07-17 Bioject, Inc. Pre-filled ampule and non-invasive hypodermic injection device assembly
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
EP0427680A1 (fr) * 1989-11-09 1991-05-15 Sandoz Ltd. Composés tricycliques contenant des hétéroatomes
US5312335A (en) 1989-11-09 1994-05-17 Bioject Inc. Needleless hypodermic injection device
WO1993013663A1 (fr) 1992-01-17 1993-07-22 Abbott Laboratories Procede permettant d'effectuer la biosynthese de polycetides specifiques
US5383851A (en) 1992-07-24 1995-01-24 Bioject Inc. Needleless hypodermic injection device
US5399163A (en) 1992-07-24 1995-03-21 Bioject Inc. Needleless hypodermic injection methods and device
WO1997002358A1 (fr) 1995-07-06 1997-01-23 The Leland Stanford Junior University Synthese de polycetides, exempte de cellule
WO1998001546A2 (fr) 1996-07-05 1998-01-15 Biotica Technology Limited Polyketides et leur synthese
WO2000000618A2 (fr) 1998-06-29 2000-01-06 Biotica Technology Limited Polycetides et leur synthese
WO2000001827A2 (fr) 1998-07-06 2000-01-13 Biotica Technology Limited Polycetides, preparation et matieres destinees a etre utilisees dans lesdits polycetides
US6150513A (en) 1998-09-16 2000-11-21 Kosan Biosciences, Inc. Polyketide synthase enzymes and recombinant DNA constructs therefor
WO2002014482A2 (fr) 2000-08-14 2002-02-21 Biotica Technology Limited Polycétides et leur synthèse
WO2004007709A2 (fr) 2002-07-16 2004-01-22 Biotica Technology Limited Production de polyketides et autres produits naturels

Non-Patent Citations (116)

* Cited by examiner, † Cited by third party
Title
"Methods in Biotechnology", vol. 4, 1998, HUMANA PRESS, article "Natural Products Isolation"
APARICIO, J.F.; MOLNAR, I.; SCHWECKE, T.; KONIG, A.; HAYDOCK, S.F.; KHAW, L.E.; STAUNTON, J.; LEADLAY, P.F.: "Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase", GENE, vol. 169, 1996, pages 9 - 16
ARMSTRONG, V.W.; OELLERICH, M.: "New developments in the immunosuppressive drug monitoring of cyclosporine, tacrolimus and azathioprine", CLINICAL BIOCHEMISTRY, vol. 34, no. 1, 2001, pages 9 - 16
BAKER, H.; SIDOROWICZ, A.; SEHGAL, S.N.; VÉZINA, C.: "Rapamycin (AY-22,989), a new antifungal antibiotic", IN VITRO AND IN VIVO EVALUATION. JOURNAL OFANTIBIOTICS, vol. 31, 1978, pages 539 - 545
BIERMAN, M.; LOGAN, R.; O'BRIEN, K.; SENO, E.T.; NAGARAJA RAO, R.; SCHONER, B.E.: "Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp", GENE, vol. 116, 1992, pages 43 - 49
BLANC, V.; GIL, P.; BAMAS-JACQUES, N.; LORENZON, S.; ZAGOREC, M.; SCHLEUNIGER, J.; BISCH, D.; BLANCHE, F.; DEBUSSCHE, L.; CROUZET,: "Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylamino-L-phenylalanine precursor of pristinamycin I", MOLECULAR MICROBIOLOGY, vol. 23, 1997, pages 191 - 202
BLANC, V.; LAGNEAUX, D.; DIDIER, P.; GIL, P.; LACROIX, P.; CROUZET, J.: "Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase", JOURNAL OF BACTERIOLOGY, vol. 177, 1995, pages 5206 - 5214
CANNELL ET AL., NATURAL PRODUCTS ISOLATION, 1998
CAO, W.; MOHACSI, P.; SHORTHOUSE, R.; PRATT, R.; MORRIS, R.E.: "Effects of rapamycin on growth factor-stimulated vascular smooth muscle cell DNA synthesis. Inhibition of basic fibroblast growth factor and platelet-derived growth factor action and antagonism of rapamycin by FK506", TRANSPLANTATION, vol. 59, no. 3, 1995, pages 390 - 395
CARLSON, R.P.; HARTMAN, D.A.; TOMCHEK, L.A.; WALTER, T.L.; LUGAY, J.R.; CALHOUN, W.; SEHGAL, S.N.; CHANG, J.Y.: "Rapamycin, a potential disease-modifying antiarthritic drug", J. PHARMACOL. EXP. THER., vol. 266, no. 2, 1993, pages 1125 - 38
CARTER, G.T.; NIETSCHE, J.A.; HERTZ, M.R.; WILLIAMS, D.R.; SIEGEL, M.M.; MORTON, G.O.; JAMES, J.C.; BORDERS, D.B.: "LL-F28249 antibiotic complex: a new family of antiparasitic macrocyclic lactones. Isolation, characterization and structures of LL-F28249 alpha, beta, gamma, lambda", J. ANTIBIOT., vol. 41, no. 4, 1988, pages 519 - 529
CHAMBRAUD, B.; RADANYI, C.; CAMONIS, J.H.; SHAZAND, K.; RAJKOWSKI, K.; BAULIEU, E.E.: "FAP48, a new protein that forms specific complexes both immunophilins FKBP59 and FKBP12. Prevention by the immunosuppressant drugs FK506 and rapamycin", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, 1996, pages 32923 - 32929
CHANG, J.Y.; SEHGAL, S.N.; BANSBACH, C.C.: "FK506 and rapamycin: novel pharmacological probes of the immune response", TRENDS IN PHARMACOLOGICAL SCIENCES, vol. 12, 1991, pages 218 - 223
CHEN, J.; ZHENG, X.F.; BROWN, E.J.; SCHREIBER, S.L.: "Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 92, 1995, pages 4947 - 4951
CHOI, S-S.; HUR, Y-A.; SHERMAN, D.H.; KIM, E-S.: "Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T-cell specific immunomodulator from Streptomyces sp", CK4412 MICROBIOLOGY, vol. 153, 2007, pages 1095 - 1102
CHUNG L ET AL: "DELETION OF RAPQONML FROM THE RAPAMYCIN GENE CLUSTER OF STREPTOMYCES HYGROSCOPICUS GIVES PRODUCTION OF THE 16-O-DESMETHYL-27-DESMETHOXY ANALOG", JOURNAL OF ANTIBIOTICS, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION, TOKYO, JP, vol. 54, no. 3, 1 March 2001 (2001-03-01), pages 250 - 256, XP001194490, ISSN: 0021-8820 *
DILELLA, A.G.; CRAIG, R.J.: "Exon organization of the human FKBP-12 gene: correlation with structural and functional protein domains", BIOCHEMISTRY, vol. 30, 1991, pages 8512 - 8517
DU, L.C.; SÁNCHEZ, C.; CHEN, M.; EDWARDS, D.J.; SHEN, B.: "The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus A TCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase", CHEMISTRY & BIOLOGY, vol. 7, 2000, pages 623 - 642
DUDKIN, L.; DILLING, M.B.; CHESHIRE, P.J.; HARWOOD, F.C.; HOLLINGSHEAD, M.; ARBUCK, S.G.; TRAVIS, R.; SAUSVILLE, E.A.; HOUGHTON, P: "Biochemical correlates of mTOR inhibition by the rapamycin ester CCI-779 and tumor growth inhibition", CLIN. CANCER RES., vol. 7, no. 6, 2001, pages 1758 - 64
DUMONT, F.; STARUCH, M.J.; KOPRAK, S.L.; SIEKIERKA, LIN, C.S.; HARRISON, R.; SEWELL, T.; KINDT, V.M.; BEATTIE, T.R.; WYVRATT, M.;: "The immunosuppressive and toxic effects of FK506 are mechanistically related: Pharmacology of a novel antagonist of FK506 and Rapamycin", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 176, 1992, pages 751 - 760
DUTTON, C. J.; S. P. GIBSON ET AL.: "Novel avermectins produced by mutational biosynthesis", J. ANTIBIOT., vol. 44, no. 3, 1991, pages 357 - 365
FANG, J.; ZHANG, Y.; HUANG, L.; JIA, X.; ZHANG, Q.; ZHANG, X.; TANG, G.; LIU, W.: "Cloning and characterisation of the tetrocarcin A gene cluster from Micromonospora chalcae NRRL 11289 reveals a highly conserved strategy for tetronate biosynthesis in spirotetronate antibiotics", J. BACT., vol. 190, no. 17, 2008, pages 6014 - 6025
FEHR, T.; SANGLIER, J-J.; SCHULER, W.; GSCHWIND, L.; PONELLE, M.; SCHILLING, W.; WIOLAND, C.: "Antascomicinc A, B, C, D and E: Novel FKBP12 binding compounds from a Micromonospora strain", J. ANTIBIOT., vol. 49, no. 3, 1996, pages 230 - 233
FINDLAY J.A; RADICS, L., CANADIAN JOURNAL OF CHEMISTRY, vol. 58, 1980, pages 579
FISHBEIN, T.M.; FLORMAN, S.; GONDOLESI, G.; SCHIANO, T.; LELEIKO, N.; TSCHERNIA, A.; KAUFMAN, S.: "Intestinal transplantation before and after the introduction of sirolimus", TRANSPLANTATION, vol. 73, no. 10, 2002, pages 1538 - 42
FOEY, A.; GREEN, P.; FOXWELL, B.; FELDMANN, M.; BRENNAN, F.: "Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis", ARTHRITIS RES., vol. 4, no. 1, 2002, pages 64 - 70
GAISSER, S.; REATHER, J.; WIRTZ, G.; KELLENBERGER, L.; STAUNTON, J.; LEADLAY, P.F.: "A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea", MOLECULAR MICROBIOLOGY, vol. 36, 2000, pages 391 - 401
GALAT, A.: "Sequence diversification of the FK506-binding proteins in several different genomes", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 267, 2000, pages 4945 - 4959
GARRITY, G.M.; HEIMBUCH, B.K.; MOTAMEDI, H.; SHAFIEE, A.: "Genetic relationships among Actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin", JOURNAL OF INDUSTRIAL MICROBIOLOGY, vol. 1 2, no. 1, 1993, pages 42 - 47
GREGORY, C.R.; HUIE, P.; BILLINGHAM, M.E.; MORRIS, R.E.: "Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Its effect on cellular, growth factor and cytokine response in injured vessels", TRANSPLANTATION, vol. 55, no. 6, 1993, pages 1409 - 1418
GUBA, M.; VON BREITENBUCH, P.; STEINBAUER, M.; KOEHL, G.; FLEGEL, S.; HORNUNG, M.; BRUNS, C.J.; ZUELKE, C.; FARKAS, S.; ANTHUBER,: "Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor", NATURE MEDICINE, vol. 8, 2002, pages 128 - 135
HAMILTON, G.S.; STEINER, J.P.: "Immunophilins: Beyond immunosuppression", JOURNAL OF MEDICINAL CHEMISTRY, vol. 41, 1998, pages 5119 - 5143
HATANAKA H; KINO T; ASANO M; GOTO T; TANAKA H; OKUHARA M: "FK-506 related compounds produced by Streptomyces tsukubaensis No. 9993", J. ANTIBIOT. (TOKYO), vol. 42, no. 4, 1989, pages 620 - 2
HATANAKA, H.; KINO, T.; MIYATA, S.; INAMURA, N.; KURODA, A.; GOTO, T.; TANAKA, H.; OKUHARA, M.: "FR-900520 and FR-900523, novel immunosuppressants isolated from a Streptomyces. II. Fermentation, isolation and physico-chemical and biological characteristics", J. ANTIBIOT. (TOKYO), vol. 41, no. 11, 1988, pages 1592 - 601
HENDRICKSON, B.A.; ZHANG, W.; CRAIG, R.J.; JIN, Y.J.; BIERER, B.E.; BURAKOFF, S.; DILELLA, A.G.: "Structural organization of the genes encoding human and murine FK506-binding protein (FKBP)13 and comparison to FKBP1", GENE, vol. 134, 1993, pages 271 - 275
HOPWOOD, D.A.: "Genetic contributions to understanding polyketide synthases", CHEMICAL REVIEWS, vol. 97, 1997, pages 2465 - 2497
HOSTED, T.J.; BALTZ, R.H.: "Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus", JOURNAL OF BACTERIOLOGY, vol. 179, 1997, pages 180 - 186
HUNG, D.T.; JAMISON, T.F.; SCHREIBER, S.L.: "Understanding and controlling the cell cycle with natural products", CHEMISTRY & BIOLOGY, vol. 3, 1996, pages 623 - 639
HUNG, D.T.; SCHREIBER, S.L.: "cDNA cloning of a human 25 kDa FK506 and rapamycin binding protein", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 184, 1992, pages 733 - 738
JAIN, S.; BICKNELL, G.R.; WHITING, P.H.; NICHOLSON, M.L.: "Rapamycin reduces expression of fibrosis-associated genes in an experimental model of renal ischaemia reperfusion injury", TRANSPLANT PROC., vol. 33, no. 1-2, 2001, pages 556 - 8
JIN, Y.J.; BURAKOFF, S.J.; BIERER, B.E.: "Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, 1992, pages 10942 - 10945
KAHAN, B.D.; CAMARDO, J.S.: "Rapamycin: Clinical results and future opportunities", TRANSPLANTATION, vol. 72, 2001, pages 1181 - 1193
KAHAN, B.D.; CHANG, J.Y.; SEHGAL, S.N.: "Preclinical evaluation of a new potent immunosuppressive agent, rapamycin", TRANSPLANTATION, vol. 52, 1991, pages 185 - 191
KAPTURCZAK, H.U.; MEIER-KRIESCHE; KAPLAN, B.: "Pharmacology of Calcineurin Antagonists", TRANSPLANTATION PROCEEDINGS, vol. 36, no. 2S, 2004, pages 25S - 32S
KHAW, L.E.; BOHM, G.A.; METCALFE, S.; STAUNTON, J.; LEADLAY, P.F.: "Mutational biosynthesis of novel rapamycins by a strain of Streptomyces hygroscopicus NRRL 5491 disrupted in rapL, encoding a putative lysine cyclodeaminase", JOURNAL OF BACTERIOLOGY, vol. 180, 1998, pages 809 - 814
KIESER, T.; BIBB, M.J.; BUTTNER, M.J.; CHATER, K.F.; HOPWOOD, D.A.: "Practical Streptomyces Genetics", 2000, JOHN INNES FOUNDATION
KIRBY, B.; GRIFFITHS,C.E.M.: "Psoriasis: the future", BRITISH JOURNAL OF DERMATOLOGY, vol. 144, 2001, pages 37 - 43
KIRCHNER, G.I.; WINKLER, M.; MUELLER L.; VIDAL, C.; JACOBSEN, W.; FRANZKE, A.; WAGNER, S.; BLICK, S.; MANNS M.P.; SEWING K.-F.: "Pharmacokinetics of SDZ RAD and cyclosporin including their metabolites in seven kidney graft patients after the first dose of SDZ RAD", BRITISH JOURNAL OF CLINICAL PHARMACOLOGY, vol. 50, 2000, pages 449 - 454
KONIG, A.; SCHWECKE, T.; MOTNAR, I.; BOHM, G.; LOWDEN, P.A.S.; STAUNTON, J.; LEADLAY, P.F.: "The pipecolate-incorporating enzyme for the biosynthesis of the immunosuppressant rapamycin. Nucleotide sequence analysis, disruption and heterologus expression of rapP from Streptomyces hygroscopicus", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 247, 1997, pages 526 - 534
KONZ, D.; M. A. MARAHIEL: "How do peptide synthetases generate structural diversity?", CHEMISTRY & BIOLOGY, vol. 6, no. 2, 1999, pages R39 - R48
LIU, Y.; HAZZARD, C.; EUSTAQUIO, A.S.; REYNOLDS, K.; MOORE B.S.: "Biosynthesis of Salinosporamide from a,D-Unsaturated Fatty Acids: Implications for Extending Polyketide Synthase Diversity", J. AM. CHEM. SOC., 2009
LOWDEN, P. A. S.: "Ph.D. Dissertation", 1997, UNIVERSITY OF CAMBRIDGE, article "Studies on the biosynthesis of rapamycin"
LOWDEN, P.A.S.; BOHM, G.; STAUNTON, J.; LEADLAY, P.F.: "The nature of the starter unit for the rapamycin polyketide sythase", ANGEWANDTE CHEMIE, vol. 35, 1996, pages 2249 - 2251
LOWDEN, P.A.S.; WILKINSON, B.; BOHM, G.A.; HANDA, S.; FLOSS, H.G.; LEADLAY, P.F.; STAUNTON, J.: "Origin and true nature of the starter unit for the rapamycin polyketide synthase", ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, vol. 40, 2001, pages 777 - 779
LYONS, W.E.; GEORGE, E.B.; DAWSON, T.M.; STEINER, J.P.; SNYDER, S.H.: "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, vol. 91, 1994, pages 3191 - 3195
MACNEIL, D.J.; GEWAIN, K.M.; RUBY, C.L.; DEZENY, G.; GIBBONS, P.H.; MACNEIL, T.: "Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector", GENE, vol. 111, 1992, pages 61 - 68
MAES, B.D.; VANRENTERGHEM, Y.F.CH.: "Cyclosporine: Advantages Versus Disadvantages Vis-a-vis Tacrolimus", TRANSPLANTATION PROCEEDINGS, vol. 36, no. 2S, 2004, pages 40S - 49S
MARAHIEL, M.A.; STACHELHAUS, T.; MOOTZ, H.D.: "Modular peptide synthetases involved in nonribosomal peptide synthesis", CHEMICAL REVIEWS, vol. 97, 1997, pages 2651 - 2673
MARCH, J: "Advanced Organic Chemistry - Reactions, Mechanisms and Structure", WILEY INTERSCIENCE
MARGULIES ET AL.: "Genome sequencing in microfabricated high-density picolitre reactors", NATURE, vol. 437, 2005, pages 376 - 380
MEINGASSNER, J.G.; GRASSBERGER, M.; FAHRNGRUBER, H.; MOORE, H.D.; SCHUURMAN, H.; STUTZ., A.: "A novel anti-inflammatory drug, SDZ ASM 981, for the topical and oral treatment of skin diseases: in vivo pharmacology", BRITISH JOURNAL OF DERMATOLOGY, vol. 137, 1997, pages 568 - 576
MOLNÁR, I.; APARICIO, J.F.; HAYDOCK, S.F.; KHAW, L.E.; SCHWECKE, T.; K6NIG, A.; STAUNTON, J.; LEADLAY, P.F.: "Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase", GENE, vol. 169, 1996, pages 1 - 7
MORICE, M.C.; SERRUYS, P.W.; SOUSA, J.E.; FAJADET, J.; BAN HAYASHI, E.; PERIN, M.; COLOMBO, A.; SCHULER, G.; BARRAGAN, P.; GUAGLIU: "RAVEL Study Group. Randomized Study with the Sirolimus-Coated Bx Velocity Balloon-Expandable Stent in the Treatment of Patients with de Novo Native Coronary Artery Lesions. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization", N. ENG.1 J. MED., vol. 346, no. 23, 2002, pages 1773 - 80
MOTAMEDI H ET AL: "Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosupressants FK506 and FK520", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC; US, vol. 178, no. 17, 1 July 1996 (1996-07-01), pages 5243 - 5248, XP002137077, ISSN: 0021-9193 *
MOTAMEDI H ET AL: "Integrative vectors for heterologous gene expression in Streptomyces spp", GENE, ELSEVIER, AMSTERDAM, NL, vol. 160, no. 1, 4 July 1995 (1995-07-04), pages 25 - 31, XP004042173, ISSN: 0378-1119, DOI: DOI:10.1016/0378-1119(95)00191-8 *
MOTAMEDI, H.; CAI, S.J.; SHAFIEE, A.; ELLISTON, K.O.: "Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 244, 1997, pages 74 - 80
MOTAMEDI, H.; SHAFIEE, A.: "The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 256, 1998, pages 528 - 534
MOTAMEDI, H.; SHAFIEE, A.; CAI, S.J.; STREICHER, S.L.; ARISON, B.H.; MILLER, R.R.: "Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520", JOURNAL OF BACTERIOLOGY, vol. 178, 1996, pages 5243 - 5248
MURAMATSU, H.; MOKHTAR, S.I.; KATSUOKA, M.; EZAKI, M.: "Phylogenetic Analysis of Immunosuppressant FK506-Producing Streptomycete Strains", ACTINOMYCETOLOGICA, vol. 19, 2005, pages 33 - 39
MYCKATYN, T.M.; ELLIS, R.A.; GRAND, A.G.; SEN, S.K.; LOWE, J.B. 3RD; HUNTER, D.A.; MACKINNON, S.E.: "The effects of rapamycin in murine peripheral nerve isografts and allografts", PLAST. RECONSTR. SURG., vol. 109, no. 7, 2002, pages 2405 - 17
NAVIA, M.A.: "Protein-drug complexes important for immunoregulation and organ transplantation", CURRENT OPINION IN STRUCTURAL BIOLOGY, vol. 6, 1996, pages 838 - 847
NCCLS REFERENCE METHOD FOR BROTH DILUTION ANTIFUNGAL SUSCEPTIBILITY TESTING FOR YEASTS: APPROVED STANDARD M27-A, vol. 17, no. 9, 1997
NGHIEM, P.; PEARSON, G.; LANGLEY, R.G.: "Tacrolimus and pimecrolimus: From clever prokaryotes to inhibiting calcineurin and treating atopic dermatitis", JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY, vol. 46, 2002, pages 228 - 241
OKAZAKI, T.; ONO, M.; AOKI, A.; FUKUDA, R.: "Milbemycins, a new family of macrolide antibiotics: producing organism and its mutants", J. ANTIBIOT. (1983), vol. 36, no. 4, 1983, pages 438 - 441
OLIYNYK, M.; STARK, C.B.W.; BHATT, A.; JONES, M.A.; HUGHES-THOMAS, Z.A.; WILKINSON, C.; OLIYNYK, Z.; DEMYDCHUK, Y.; STAUNTON, J.;: "Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization", MOL. MICRO., vol. 49, no. 5, 2003, pages 1179 - 1190
PAGET, M.S.B.; CHAMBERLIN, L.; ATRIH, A.; FOSTER, S.J.; BUTTNER, M.J.: "Evidence that the extracytoplasmic function sigma factor CyE is required for normal cell wall structure in Streptomyces coelicolor A3(2)", JOURNAL OF BACTERIOLOGY, vol. 181, 1999, pages 204 - 211
PAIVA, N.L.; DEMAIN, A.L.; ROBERTS, M.F.: "Incorporation of acetate, propionate, and methionine into rapamycin By Streptomyces hygroscopicus", JOURNAL OF NATURAL PRODUCTS, vol. 54, 1991, pages 167 - 177
PAIVA, N.L.; DEMAIN, A.L.; ROBERTS, M.F.: "The immediate precursor of the nitrogen- containing ring of rapamycin is free pipecolic acid", ENZYME AND MICROBIAL TECHNOLOGY, vol. 15, 1993, pages 581 - 585
PATTERSON, C.E.; SCHAUB, T.; COLEMAN, E.J.; DAVIES E.C.: "Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein", MOLECULAR BIOLOGY OF THE CELL, vol. 11, 2000, pages 3925 - 3935
PFEIFER, B.A.; ADMIRAAL, S.J.; GRAMAJO, H.; CANE, D.E.; KHOSLA, C.: "Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli", SCIENCE, vol. 291, 2001, pages 1790 - 1792
POWELL, N.; TILL, S.; BUNGRE, J.; CORRIGAN, C.: "The immunomodulatory drugs cyclosporin A, mycophenolate mofetil, and sirolimus (rapamycin) inhibit allergen-induced proliferation and IL-5 production by PBMCs from atopic asthmatic patients", J. ALLERGY CLIN. IMMUNOL., vol. 108, no. 6, 2001, pages 915 - 7
RABINOVITCH, A.; SUAREZ-PINZON, W.L.; SHAPIRO, A.M.; RAJOTTE, R.V.; POWER, R.: "Combination therapy with sirolimus and interleukin-2 prevents spontaneous and recurrent autoimmune diabetes in NOD mice", DIABETES, vol. 51, no. 3, 2002, pages 638 - 45
RAWLINGS, B.J.: "Type I polyketide biosynthesis in bacteria (Part A)", NATURAL PRODUCT REPORTS, vol. 18, 2001, pages 190 - 227
REITAMO, S.; SPULS, P.; SASSOLAS, B.; LAHFA, M.; CLAUDY, A.; GRIFFITHS, C.E.: "Br. J. Dermatol.", vol. 145, 2001, SIROLIMUS EUROPEAN PSORIASIS STUDY GROUP, article "Efficacy of sirolimus (rapamycin) administered concomitantly with a subtherapeutic dose of cyclosporin in the treatment of severe psoriasis: a randomized controlled trial", pages: 438 - 45
REYNOLDS, K.A.; DEMAIN, A.L.: "Biotechnology of Antibiotics", 1997, article "Rapamycin, FK506 and Ascomycin-related Compounds", pages: 497 - 520
ROSEN, M.K.; SCHREIBER, S.L.: "Natural products as probes of cellular function: studies of immunophilins", ANGEWANDTE CHEMIE-INTERNATIONAL EDITION IN ENGLISH, vol. 31, 1992, pages 384 - 400
ROY, J.N.; BARAMA, A.; POIRIER, C.; VINET, B.; ROGER, M.: "Cyp3A4, Cyp3A5, and MDR-1 genetic influences on tacrolimus pharmacokinetics in renal transplant recipients", PHARMACOGENETICS, vol. 16, no. 9, 2006, pages 659 - 665
ROYMANS, D.; SLEGERS, H.: "Phosphaditidylinositol 3-kinases in tumor progression", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 268, 2001, pages 487 - 498
SAMBROOK, J.; FRITSCH, E.F.; MANIATIS, T.: "Molecular cloning: a laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK, J.; RUSSELL, D.W.: "Molecular cloning: a laboratory manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SCHREIBER, S.L.; CRABTREE, G.R.: "The mechanism of action of cyclosporine A and FK506", IMMUNOLOGY TODAY, vol. 13, 1992, pages 136 - 142
SCHWARZER, D.; MARAHIEL, M.A.: "Multimodular biocatalysts for natural product assembly", NATURWISSENSCHAFTEN, vol. 88, 2001, pages 93 - 101
SCHWECKE, T.; APARICIO, J.F.; MOLNÁR, I.; K6NIG, A.; KHAW, L.E.; HAYDOCK, S.F.; OLIYNYK, M.; CAFFREY, P.; CORTÉS, J.; LESTER, J.: "The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OFAMERICA, vol. 92, 1995, pages 7839 - 7843
SEDRANI, R.; COTTENS, S.; KALLEN, J.; SCHULER, W.: "Chemical modifications of rapamycin: the discovery of SDZ RAD", TRANSPLANTATION PROCEEDINGS, vol. 30, 1998, pages 2192 - 2194
SEHGAL, S.N.; BAKER, H.; VÉZINA, C.: "Rapamycin (AY-22,989), a new antifungal antibiotic . Fermentation, isolation and characterization", THE JOURNAL OF ANTIBIOTICS, vol. 28, 1975, pages 727 - 733
SHAFIEE A ET AL: "CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF TWO FK506 ANALOGS PRODUCED BY TARGETED GENE DISRUPTION IN STREPTOMYCES SP. MA6548", JOURNAL OF ANTIBIOTICS, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION, TOKYO, JP, vol. 50, no. 5, 1 January 1997 (1997-01-01), pages 418 - 423, XP000906741, ISSN: 0021-8820 *
SHEPHERD, P.R; WITHERS, D.J.; SIDDLE K.: "Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling", BIOCHEMICAL JOURNAL, vol. 333, 1998, pages 471 - 490
SHIMA, J.; HESKETH, A.; OKAMOTO, S.; KAWAMOTO, S.; OCHI, K.: "Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2)", JOURNAL OF BACTERIOLOGY, vol. 178, 1996, pages 7276 - 7284
SIGAL, N.H.; DUMONT, F.; DURETTE, P.; SIEKIERKA, J.J.; PETERSON, L.; RICH, D.; DUNLAP, B.E.; STARUCH, M.J.; MELINO, M.R.; KOPRAK,: "Is Cyclophilin Involved in the Immunosuppressive and Nephrotoxic Mechanism of Action of Cyclosporin A?", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 173, 1991, pages 619 - 628
SIGAL, N.H.; DUMONT, F.J.: "Cyclosporine A, FK-506, and rapamycin: pharmacological probes of lymphocyte signal transduction", ANNUAL REVIEW OF IMMUNOLOGY, vol. 10, 1992, pages 519 - 560
STAUNTON, J.; WEISSMAN, K.J.: "Polyketide biosynthesis: a millennium review", NATURAL PRODUCT REPORTS, vol. 18, 2001, pages 380 - 416
STEINER, J.P.; HAMILTON, G.S.; ROSS, D. T.; VALENTINE, H.L.; GUO, H.; CONNOLLY, M.A.; LIANG, S.; RAMSEY, C.; LI, J.-H.J.; HUANG, W: "Neutrophic immunophilin ligands stimulate structural and functional recovery in neurodegenerative animal models", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 94, 1997, pages 2019 - 2024
STRÄSSLER, C.; LINDEN, A.; HEIMGARTNER, H.: "Novel heterospirocyclic 3-amino-2H-azirines as synthons for heterocyclic a-amino acids", HELV. CHIM. ACTA., vol. 80, 1997, pages 1528 - 1554
TAKIGUCHI, Y.; MISHIMA, H.; OKUDA, M.; TERAO, M.; AOKI, A.; FUKUDA, R.: "Milbemycins, a new family of macrolide antibiotics: fermentation, isolation and physico-chemical properties", J. ANTIBIOT., vol. 33, no. 10, 1980, pages 1120 - 7
TAMURA, S.; TOKUNAGA, Y.; IBUKI, R.; AMIDON, G.L.; SEZAKI, H.; YAMASHITA, S.: "The site specific transport and metabolism of Tacrolimus in the rat small intestine", THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 306, 2003, pages 310 - 316
VAN DUYNE, G.D.; STANDAERT, R.F.; KARPLUS, P.A.; SCHREIBER, S.L.; CLARDY, J.: "Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin", JOURNAL OF MOLECULAR BIOLOGY, vol. 229, 1993, pages 105 - 124
VILELLA-BACH, M.; NUZZI, P.; FANG, Y.M.; CHEN, J.: "The FKBP12-rapamycin-binding domain is required for FKBP12-rapamycin-associated protein kinase activity and G1 progression", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, 1999, pages 4266 - 4272
WALLER, J.R.; NICHOLSON, M.L.: "Molecular mechanisms of renal allograft fibrosis", BRITISH JOURNAL OF SURGERY, vol. 88, 2001, pages 1429 - 1441
WARD, D.E.; ROSS, RP.; VAN DER WEIJDEN, C.C.; SNOEP, J.L.; CLAIBORNE, A.L.: "Catabolism of branched-chain .alpha.-keto acids in Enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route", J. BACT., vol. 181, no. 17, 1999, pages 5433 - 5442
WARNER, L.M.; ADAMS, L.M.; CHANG, J.Y.; SEHGAL, S.N.: "A modification of the in vivo mixed lymphocyte reaction and rapamycin's effect in this modeI.Cin", IMMUNOL. IMMUNOPATHOL., vol. 64, no. 3, 1992, pages 242 - 7
WEI, X.; LIANG, Y.; ZHENG, Y.: "Enhancement and Selective Production of Oligomycin through Inactivation of Avermectin's Starter Unit in Streptomyces avermitilis", BIOTECHNOLOGY LETTERS, vol. 28, no. 12, 2006, pages 911 - 916
WILKINSON, B.; FOSTER, G.; RUDD, B.A.M.; TAYLOR, N.L.; BLACKABY, A.P.; SIDEBOTTOM, P.J.; COOPER, D.J.; DAWSON, M.J.; BUSS, A.D.; G: "Novel octaketide macrolides related to 6- deoxoerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase", CHEMISTRY & BIOLOGY, vol. 7, 2000, pages 111 - 117
WU, K.; CHUNG, L.; REVILL, W.P.; KATZ, L.; REEVES, C.D.: "The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units", GENE, vol. 251, 2000, pages 81 - 90
YEM, A.W.; TOMASSELLI, A.G.; HEINRIKSON, R.L.; ZURCHER-NEELY, H.; RUFF, V.A.; JOHNSON, R.A.; DEIBEL, M.R.: "The Hsp56 component of steroid receptor complexes binds to immobilized FK506 and shows homology to FKBP-12 and FKBP-13", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, 1992, pages 2868 - 2871
YU, K.; TORAL-BARZA, L.; DISCAFANI, C.; ZHANG, W.G.; SKOTNICKI, J.; FROST, P.; GIBBONS, J.J.: "mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer", ENDOCRINE-RELATED CANCER, vol. 8, 2001, pages 249 - 258
ZHU, J.; WU J.; FRIZELL, E.; LIU, S.L.; BASHEY, R.; RUBIN, R.; NORTON, P.; ZERN, M.A.: "Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis", GASTROENTEROLOGY, vol. 117, no. 5, 1999, pages 1198 - 204

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