HRP990124A2 - Active hedgehog protein conjugate, process for its production and use - Google Patents
Active hedgehog protein conjugate, process for its production and use Download PDFInfo
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- HRP990124A2 HRP990124A2 HR98107911.4A HRP990124A HRP990124A2 HR P990124 A2 HRP990124 A2 HR P990124A2 HR P990124 A HRP990124 A HR P990124A HR P990124 A2 HRP990124 A2 HR P990124A2
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- Croatia
- Prior art keywords
- hedgehog
- acid
- protein
- hydrophobic
- shh
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Description
Izum se odnosi na konjugat hedgehog proteina s povišenom aktivnošću, na postupak za njegovu proizvodnju i na njegovu terapeutsku upotrebu. The invention relates to a hedgehog protein conjugate with increased activity, to a process for its production and to its therapeutic use.
Pod hedgehog (hh) proteinima podrazumijeva se porodicu izlučenih signalnih proteina koji su odgovorni za tvorbu brojnih struktura u embriogenezi (J.C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al. , Nature 83 (1996) 407, M.J. Bitgood et al. , Curr. Bioi. 6 (1996) 296, A. Vortkamp et al. , Science 273 (1996) 613, C.J. Lai et al., Development 121 (1995) 2349). Tijekom biosinteze 20 kD N-terminalna domena i 25 kD C-terminalna domena dobiju se nakon odcjepljenje signalne sekvence i autokatalitičkog cijepanja. U proteinu koji nastaje prirodnim putem N-terminalna domena se modificira s kolesterolom na svom C kraju nakon odcjepljenja C-terminalne domene (J.A. Porter et al., Science 274 (1996) 255-259). U višim oblicima života porodica hh sastavljena je od najmanje tri člana tj. sunčanog/ indijskog i pustinjskog hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Razlike u aktivnosti rekombinantno proizvedenih hedgehog proteina opažene su nakon proizvodnje u prokariotima i eukariotima (M. Hynes et al. , Neuron 15 (1995) 35-44 i T. Nakamura et al. , Biochem. Biophys. Res . Comm. 237 (1997) 465-469. Hedgehog (hh) proteins are a family of secreted signaling proteins that are responsible for the formation of numerous structures in embryogenesis (J.C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M.J. Bitgood et al., Curr. Bioi. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996) 613, C.J. Lai et al., Development 121 ( 1995) 2349). During biosynthesis, the 20 kD N-terminal domain and the 25 kD C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. In the naturally occurring protein, the N-terminal domain is modified with cholesterol at its C terminus after cleavage of the C-terminal domain (J.A. Porter et al., Science 274 (1996) 255-259). In higher life forms, the hh family is composed of at least three members, i.e. sun/Indian and desert hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of recombinantly produced hedgehog proteins have been observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997) ) 465-469.
Hynes et al usporedili su aktivnost hh proteina u supernatantu transformiranih humanih embrionskih bubrežnih 293 stanica (eukariotski hh) s hh proteinom kojeg je proizvela E. coli i našli su četverostruko višu aktivnost hh iz supernatanta bubrežne stanične linije. Kao razlog za tu povišenu aktivnost razmatran je mogući dodatni dopunski faktor koji se umnaža samo u eukariotskim stanicama, post-translacijska modifikacija, različit N-kraj, budući da hh izoliran iz E. coli sadrži 50% hh koji nosi dvije dodatne N-terminalne amino kiseline (Gly-Ser) ili je skraćen za 5 -6 amino kiselina, ili više stanje agregacije (npr. vezanjem na nikal agarozna zrnca). Hynes et al compared hh protein activity in the supernatant of transformed human embryonic kidney 293 cells (eukaryotic hh) with hh protein produced by E. coli and found a fourfold higher activity of hh from the supernatant of a renal cell line. As the reason for this increased activity, a possible additional complement factor that is reproduced only in eukaryotic cells, post-translational modification, different N-terminus, since hh isolated from E. coli contains 50% of hh carrying two additional N-terminal amino acids, was considered. acid (Gly-Ser) or shortened by 5-6 amino acids, or a higher state of aggregation (eg by binding to nickel agarose beads).
Nakamura et al. usporedili su aktivnost shh u supernatantu transfomiranih fibroplasta iz pilećeg embrija s shh fuzijskim proteinom izoliranim iz E. coli koji još uvijek ima N-terminalni polihistidinski dio. Shh u supernatantu fibroplasta ima sedmerostruko višu aktivnost u usporedbi s očišćenim E. coli proteinom s obzirom na stimulaciju alkalne fosfataze (AP) u C3H10T 1/2 stanicama. Pretpostavilo se je da do povišene aktivnosti dolazi zbog sinergizma hh s molekulama kao što su koštani morfogenetički proteini (BMPs) koji su prisutni samo u supernatantu eukariotskih stanica i u kombinaciji s hh uzrokuju jaču indukciju AP-a. Nakamura et al. compared shh activity in the supernatant of transformed chicken embryo fibroplasts with an shh fusion protein isolated from E. coli that still has the N-terminal polyhistidine portion. Shh in fibroplast supernatant has a sevenfold higher activity compared to purified E. coli protein with respect to alkaline phosphatase (AP) stimulation in C3H10T 1/2 cells. It was assumed that the increased activity occurs due to the synergism of hh with molecules such as bone morphogenetic proteins (BMPs) which are present only in the supernatant of eukaryotic cells and in combination with hh cause a stronger induction of AP.
Kinto et al., FEBS Letters, 404 (1997) 319-323 opisali su da fibroplasti koji izlučuju hh induciraju ektopijsku tvorbu kosti kod i.m. implatacije na koleganu. Medutim, takova aktivnost nije poznata za izolirani hh protein. Kinto et al., FEBS Letters, 404 (1997) 319-323 reported that hh-secreting fibroblasts induce ectopic bone formation in i.m. implations on the colleague. However, such activity is not known for the isolated hh protein.
Cilj izuma je osigurati hh proteine (polipeptide) koji u usporedbi s poznatim oblicima imaju značajno poboljšanu aktivnost. The aim of the invention is to provide hh proteins (polypeptides) which, compared to known forms, have significantly improved activity.
Taj cilj postignut je s rekombinantno proizvedenim hedgehog proteinom koji je na umjetni način učinjen lipofilnim. Takova liofilizacija postiže se ponajprije pomoću kemijske modifikacije. Takav hedgehog konjugat sadrži ponajprije dodatni polipeptid koji je kovalentno povezan (prednosno na C kraju i/ili N kraju) i koji je sastavljen od 10 - 30 ponajprije hidrofobnih amino kiselina i/ili onih amino kiselina koje tvore transmembranske helikse. Dodatni polipeptid posebno prednosno sadrži 2-12 lizina i/ili arginina, ali ne sadrži polihistidinski dio koji bi bio prikladan za čišćenje konjugata na stupcu Ni helata. Također je prednosno kovalentno vezati (ponajprije na C kraju i/ili na N kraju) 1-4 alifatska, zasićena ili nezasićena ugljikovodična ostatka s duljinom lanca od 8 -24 C atoma ili steroide s lipofilnim (hidrofobnim) djelovanjem. Prednosno je, nadalje, na hh proteine kovalentno vezati hidrofobne tio spojeve, takove kao što su posebno tiokolesterol i tioalkani, tioalkeni, preko disulfidnog mosta nastalog oksidacijom (posebno na C kraju i/ili N-kraju i u tom slučaju na N-kraju cisteina). This goal was achieved with a recombinantly produced hedgehog protein that was artificially rendered lipophilic. Such lyophilization is primarily achieved by chemical modification. Such a hedgehog conjugate primarily contains an additional polypeptide that is covalently linked (preferably at the C end and/or N end) and that is composed of 10-30 preferably hydrophobic amino acids and/or those amino acids that form transmembrane helices. The additional polypeptide particularly preferably contains 2-12 lysines and/or arginines, but does not contain a polyhistidine moiety that would be suitable for purifying the conjugate on a Ni chelate column. It is also preferable to covalently bind (preferably at the C end and/or at the N end) 1-4 aliphatic, saturated or unsaturated hydrocarbon residues with a chain length of 8-24 C atoms or steroids with lipophilic (hydrophobic) action. Furthermore, it is preferable to covalently attach hydrophobic thio compounds to hh proteins, such as especially thiocholesterol and thioalkanes, thioalkenes, via a disulfide bridge formed by oxidation (especially at the C-end and/or N-end and in this case at the N-end of cysteine). .
Protein je hidrofobiziran takovim lipofiliziranjem ostataka koji poboljšava njegovu interakciju s lipidnim membranama eukariotskih stanica, posebno stanica sisavaca. The protein is hydrophobized by such lipophilization of residues that improves its interaction with the lipid membranes of eukaryotic cells, especially mammalian cells.
Konačno, kao lipofilizirani protein prema izumu podrazumijeva se hidrofobizirani protein koji u usporedbi s nemodificiranim proteinom ima povećanu površinsku hidrofobnost koja povisuje njegov afinitet prema nepolarnim molekulama ili amfifilima. Porast stupnja lipofilnosti proteina može se izmjeriti pomoću stupnja inegracije u lipidni sloj kako su opisali Haque, Z. et al., J. Agric. Food Chem. 30 (1982), 481. Metode za hidrofobno (lipofilizirajuće) modificiranje proteina opisali su, na primjer, Haque, Z. et al., u J. Agric. Food Chem. 31 (1983) 1225-1230; Webb, R.J. et al., Biochemistry 37 (1998) 673-679; Hancock, J.F./ Cell 63 (1990) 133-139; A Practicai guide to membrane protein purification, Izd. G.V. Jagow, Hermann Schagger (1994), (poglavlje 16, str. 535-554). Finally, a lipophilized protein according to the invention means a hydrophobized protein which, compared to an unmodified protein, has an increased surface hydrophobicity that increases its affinity towards non-polar molecules or amphiphiles. The increase in the degree of lipophilicity of the protein can be measured by the degree of integration into the lipid bilayer as described by Haque, Z. et al., J. Agric. Food Chem. 30 (1982), 481. Methods for hydrophobic (lipophilizing) modification of proteins are described, for example, by Haque, Z. et al., in J. Agric. Food Chem. 31 (1983) 1225-1230; Webb, R.J. et al., Biochemistry 37 (1998) 673-679; Hancock, J.F. / Cell 63 (1990) 133-139; A practical guide to membrane protein purification, Ed. G.V. Jagow, Hermann Schagger (1994), (chapter 16, pp. 535-554).
Iznenađujuće se je pokazalo da takovi lipofilizirani proteini (koji se u nastavku također označavaju i kao hedgehog konjugati (hh konjugati)) pokazuju drastično povišenu aktivnost, ponajprije najmanje deseterostruku, posebno prednosno od 103-105-struko u usporedbi s nemodificiranim hedgehog proteinima (npr. nakon citoplazmičke ekspresije u E. coli) posebno u farmaceutskoj formulaciji i in vitro. K tome, posebno je iznenađujuće da se takovi hedgehog konjugati prema izumu mogu upotrijebiti posebno prednosno za lokalnu terapiju ponajprije na kostima, na kralježnici, na nervnim stanicama (kod nervnih lezija ili kod neurodegenerativnih bolesti) ili u mišićnom tkivu. Surprisingly, it has been shown that such lipophilized proteins (which are also referred to below as hedgehog conjugates (hh conjugates)) show a drastically increased activity, preferably at least ten-fold, especially preferably 103-105-fold compared to unmodified hedgehog proteins (e.g. after cytoplasmic expression in E. coli) especially in pharmaceutical formulation and in vitro. In addition, it is particularly surprising that such hedgehog conjugates according to the invention can be used particularly advantageously for local therapy, primarily on bones, on the spine, on nerve cells (in case of nerve lesions or in neurodegenerative diseases) or in muscle tissue.
Od Yang et al., Development 124 (1997) 4393-4404 poznato je da se za farmaceutski učinkovito djelovanje in vivo visoke lokalne koncentracije hedgehog proteina na strani djelovanja u tijelu moraju održavati tijekom perioda od najmanje 16 sati. Sistem nosača za tu svrhu opisali su Yang et al., t.j. kromatografsko sredstvo Affigel CM napunjeno s hedgehog proteinom, Ni-agarozu su opisali Marti et al. u Nature 375 (1995) 322-325 ili Affigel Blue kojeg su upotrijebili Lopez-Martinez et al. u Curr. Biol. 5 (1995) 791-796, ili čestice heparin agaroze, koje su oni upotrijebili, i koje su manje prikladne za farmaceutsku primjenu, jer su one imunogene i mogu uzrokovati upalne reakcije. It is known from Yang et al., Development 124 (1997) 4393-4404 that for a pharmaceutically effective action in vivo, high local concentrations of hedgehog protein on the side of action in the body must be maintained for a period of at least 16 hours. A carrier system for this purpose was described by Yang et al., i.e. Chromatographic agent Affigel CM loaded with hedgehog protein, Ni-agarose was described by Marti et al. in Nature 375 (1995) 322-325 or Affigel Blue used by Lopez-Martinez et al. in Curr. Biol. 5 (1995) 791-796, or heparin agarose particles, which they used, and which are less suitable for pharmaceutical use, because they are immunogenic and can cause inflammatory reactions.
Konjugati prema izumu služe kao nove aktivne tvari za proizvodnju farmaceutskih oblika za davanje. Općenito, vezanje ima za posljedicu poboljšani farmakokinetički profil hedgehog proteina. Hidrofobni ugljikovodični ostatak ima za posljedicu lokalizaciju hedgehog proteina na membrani ciljnih stanica/ dodatno za olakšavanje integracije u unutrašnjost stanice, a iznad svega ima za posljedicu bitno dulju prisutnost na površini stanice što je optimalno za farmakološki učinak. The conjugates according to the invention serve as new active substances for the production of pharmaceutical forms for administration. In general, binding results in an improved pharmacokinetic profile of the hedgehog protein. The hydrophobic hydrocarbon residue results in the localization of the hedgehog protein on the membrane of target cells/in addition to facilitating integration into the interior of the cell, and above all results in significantly longer presence on the cell surface, which is optimal for pharmacological effect.
Konjugati prema izumu ne moraju se nužno dodatno vezati na nosač za polagano oslobađanje. Konjugati prema izumu su također visoko aktivni na strani djelovanja u tijelu bez usporenog oslobađanja tijekom dugog perioda uzrokovanog nosačem (nekoliko dana). Unatoč tome, upotreba farmaceutskog sastava prikladna je za lokalnu primjenu hedgehog konjugata prema izumu koji sadrži konjugat prema izumu zajedno s nosećom matricom. Noseća matrica služi uglavnom za olakšavanje lokalne aplikacije, dajući posebno takav farmaceutski sastav s minimalnom viskoznošću prikladnom za iokalnu aplikaciju. Farmaceutski sastav je prednosno puferiran u pH području između pH 4 i 9 i sadrži jedan ili više neionskih detergenata, kao što su detergenti tipa polisorbata ili polioksietilena (npr. Tween® 20, Tween® 80, Triton® X-100), oktilglukozidni ili ionski detergenti, kao natrijev deoksiholat, natrijev holat, natrijev taurodeoksiholat. Conjugates according to the invention do not necessarily need to be additionally bound to a sustained release carrier. The conjugates according to the invention are also highly active on the side of action in the body without delayed release over a long period caused by the carrier (several days). Nevertheless, the use of a pharmaceutical composition is suitable for local administration of the hedgehog conjugate according to the invention containing the conjugate according to the invention together with a carrier matrix. The carrier matrix serves mainly to facilitate topical application, providing particularly such a pharmaceutical composition with minimal viscosity suitable for topical application. The pharmaceutical composition is preferably buffered in a pH range between pH 4 and 9 and contains one or more nonionic detergents, such as polysorbate or polyoxyethylene type detergents (eg Tween® 20, Tween® 80, Triton® X-100), octylglucoside or ionic detergents, such as sodium deoxycholate, sodium cholate, sodium taurodeoxycholate.
U prednosnoj izvedbi umnaža se hh protein koji dodatno sadrži 10 - 30 uglavnom hidrofobnih amino kiselina na N kraju i/ili na C kraju budući da se one također ugrađuju u membrane stanica [Webb et al., Biochemistry 37 (1998) 673-679, Skolnick et al., Biol. Membranes (1996) 536-554; izd.: Merz & Roux]. Kao hidrofobne amino kiseline u smislu izuma podrazumijevaju se amino kiseline koje imaju negativnu slobodnu energiju u prijelazu iz vodene faze u hidrofobno/organsku fazu. Nadalje N-terminalne i/ili C-terminalne sekvence, za koje se zna da tvore transmembranske helikse, kao npr. M28 peptid ili onaj koji ulazi u interakciju kao heliks s površinom membrana, kao npr. maginin 2 (Skolnick et al., 1966), također su prikladni za povišenje aktivnosti hh proteina. In a preferred embodiment, the hh protein is amplified which additionally contains 10-30 mostly hydrophobic amino acids at the N end and/or at the C end since they are also incorporated into cell membranes [Webb et al., Biochemistry 37 (1998) 673-679, Skolnick et al., Biol. Membranes (1996) 536-554; ed.: Merz & Roux]. Hydrophobic amino acids in the sense of the invention mean amino acids that have a negative free energy in the transition from the aqueous phase to the hydrophobic/organic phase. Furthermore, N-terminal and/or C-terminal sequences, which are known to form transmembrane helices, such as the M28 peptide or one that interacts as a helix with the membrane surface, such as maginin 2 (Skolnick et al., 1966 ), are also suitable for increasing hh protein activity.
U daljnjoj prednosnoj izvedbi N kraj i/ili C kraj hedgehog proteina modificiran je s polipeptidnim ostatkom koji sadrži 2-12 lizina i/ili arginina. U tom slučaju može se izostaviti modifikaciju s ugljikovodičnim ostatkom. In a further preferred embodiment, the N terminus and/or C terminus of the hedgehog protein is modified with a polypeptide residue containing 2-12 lysines and/or arginines. In this case, the modification with the hydrocarbon residue can be omitted.
Alifatski, zasićeni ili nezasićeni ugljikovodični ostatak s hidrofobnim djelovanjem i lancem duljine 8-24, ponajprije 10-24, a najbolje 12-18 C atoma, je prednosno ostatak zasićene ili jednostruko nezasićene do višestruko nezasićene masne kiselina ili ostatak alkilnog alkohola, po potrebi prekinut s atomom kisika ili sumpora ili s karbonilnom skupinom. Posebno prednosne zasićene masne kiseline jesu kaprinska kiselina, laurinska kiselina, miristinska kiselina, palmitinska kiselina, stearinska kiselina, arahidinska kiselina i beheninska kiselina. Prednosne jednostruko nezasićene masne kiseline jesu miristinska kiselina, palmitoleinska kiselina i oleinska kiselina. Posebno prednosne višestruko nezasićene masne kiseline jesu linoleinska kiselina, linolenska kiselina i arahidonska kiselina. Ostaci takovih masnih kiselina vezani su na reaktivne skupine proteina ponajprije preko esterske veze, preko kiselinskog amida, disulfidne ili tioesterske veze. An aliphatic, saturated or unsaturated hydrocarbon residue with a hydrophobic effect and a chain length of 8-24, preferably 10-24, and preferably 12-18 C atoms, is preferably a residue of a saturated or monounsaturated to polyunsaturated fatty acid or an alkyl alcohol residue, interrupted if necessary with an oxygen or sulfur atom or with a carbonyl group. Particularly preferred saturated fatty acids are capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid. Preferred monounsaturated fatty acids are myristic acid, palmitoleic acid and oleic acid. Particularly preferred polyunsaturated fatty acids are linoleic acid, linolenic acid and arachidonic acid. The residues of such fatty acids are attached to the reactive groups of proteins primarily through an ester bond, through an acid amide, disulfide or thioester bond.
Brojni hidrofobni ugljikovodični lanci mogu se prikladno kontrolirati po protinskoj molekuli pomoću uvjeta reakcije (npr. razrjeđivanjem) ili odabirom amino kiseline koju će se modificirati. Na primjer shh sadrži tri cisteina čiji N-terminalni cistein je posebno reaktivan. U tom slučaju reakcija može dovesti do N-terminainog cisteina koji je modificiran s jednim ili s više hidrofobnih ugljikovodičnih ianaca. Takoder se mogu statistički modificirati dva ili gotovo sva tri cisteina. lako je pri modificiranju druge amino kiseline prednosno modificirati definirane amino kiseline, za farmaceutski sastav se također mogu upotrijebiti derivatizirani hedgehog proteini u kojima postoji statistička razdioba modifikacija ugljikovodičnog lanca od pribl. 1 do pribl. 4 lanca po molekuli. lako je prikladan veći broj ugljikovodičnih lanaca po molekuli, time opada topivost farmaceutskog sastava i to može dovesti do smetnji u strukturi aktivnog trodimenzionalnog proteina. Kod povezivanja s alkilnim skupinama dugačkog lanca (C14-C24) prednosno je vezati samo 1-2 ugljikova lanca, a kad se povezuju lanci kratkog ugljikovg lanca, tada je međutim prednosno vezati 2-3 alkilne skupine. The number of hydrophobic hydrocarbon chains can be conveniently controlled per protein molecule by reaction conditions (eg, dilution) or by selecting the amino acid to be modified. For example shh contains three cysteines whose N-terminal cysteine is particularly reactive. In this case, the reaction can lead to N-terminal cysteine modified with one or more hydrophobic hydrocarbon groups. Two or almost all three cysteines can also be statistically modified. it is easy to modify another amino acid, it is preferable to modify defined amino acids, derivatized hedgehog proteins can also be used for the pharmaceutical composition, in which there is a statistical distribution of modifications of the hydrocarbon chain of approx. 1 to approx. 4 chains per molecule. a larger number of hydrocarbon chains per molecule is easily suitable, thereby decreasing the solubility of the pharmaceutical composition and this can lead to disturbances in the structure of the active three-dimensional protein. When connecting with long-chain alkyl groups (C14-C24), it is preferable to connect only 1-2 carbon chains, and when connecting short carbon chain chains, it is preferable to connect 2-3 alkyl groups.
U prednosnoj izvedbi derivatizacija također može obuhvatiti povezivanje dvaju hidrofobnih ugljikovodičnih lanaca na jednu amino kiselinu. To se može postići, na primjer, povezivanjem diglicerida masne kiseline na amino kiselinu. In a preferred embodiment, derivatization can also include linking two hydrophobic hydrocarbon chains to one amino acid. This can be achieved, for example, by linking a diglyceride of a fatty acid to an amino acid.
Kako su hedgehog proteini vrlo nestabilni, u prednosnoj izvedbi za povezivanje se upotrebljava hedgehog protein u kojem je zaštićena SH skupina N-terminalnog cisteina. Proizvod SH povezivanja se zatim dobije redukcijom zaštićenih SH skupina neposredno prije ili tijekom postupka povezivanja. SH skupinu spomenutog cisteina prednosno je zaštititi tvorbom homolognog hedgehog disulfida ili miješanog disulfida (npr. s GSH ili β-merkaptoetanola). As hedgehog proteins are very unstable, in a preferred embodiment hedgehog protein is used for connection, in which the SH group of the N-terminal cysteine is protected. The SH coupling product is then obtained by reduction of the protected SH groups immediately before or during the coupling procedure. It is preferable to protect the SH group of the mentioned cysteine by forming a homologous hedgehog disulfide or a mixed disulfide (eg with GSH or β-mercaptoethanol).
Tiolne zaštitne skupine su poznate u struci, i uključuju ali nisu ograničene samo na trifeniimetil (tritil) i s-t-butil, s-p-nitrobenzil i s-p-metoksi-benzii (vidi npr. Green i Wuts, Protective Groups in Organic Synthesis, drugo izdanje, John Wiley & Sons, New York (1991), i Atherton et al., The Peptides, Gross i Meienhofer, izd. Academic Press, New York (1983), svezak 19, 1-38). Takovi tiolno zaštićeni ili homo-dimerizirani hedgehog proteini su dragocjeni intermedijati za proizvodnju SH-modificiranih hedgehog proteina i predstavljaju glavne predmete predloženog izuma. Thiol protecting groups are known in the art, and include but are not limited to triphenylmethyl (trityl) and s-t-butyl, s-p-nitrobenzyl, and s-p-methoxy-benzyl (see, e.g., Green and Wuts, Protective Groups in Organic Synthesis, Second Edition, John Wiley & Sons, New York (1991), and Atherton et al., The Peptides, Gross and Meienhofer, eds Academic Press, New York (1983), Volume 19, 1-38). Such thiol-protected or homo-dimerized hedgehog proteins are valuable intermediates for the production of SH-modified hedgehog proteins and represent the main objects of the proposed invention.
Izum se nadalje odnosi na upotrebu hedgehog proteina u kojima su SH skupine N-terminalnog cisteina zaštićene ili homo-dimerizirane kao postojanih intermedijata za proizvodnju SH-modificiranih hedgehog proteina. U tom postupku odcjepljuje se zaštitnu skupinu ili se odcjepljuje homologni dimer i reagira s aktiviranim sredstvom za derivatizaciju. The invention further relates to the use of hedgehog proteins in which the SH groups of the N-terminal cysteine are protected or homo-dimerized as stable intermediates for the production of SH-modified hedgehog proteins. In this process, the protective group is cleaved or the homologous dimer is cleaved and reacted with an activated derivatization agent.
Za povezivanje SH-zaštićenih hedgehog proteina prednosne su slijedeće metode: The following methods are preferred for connecting SH-protected hedgehog proteins:
I. Redukcija disulfida ili odcjepljenje zaštitne skupine u mješavini za povezivanje ponajprije kad se povezivanje vrši preko imidazolida ili CoA derivata. I. Reduction of the disulfide or removal of the protective group in the coupling mixture, preferably when the coupling is carried out via an imidazolide or CoA derivative.
II. Redukcija disulfida, izolacija nezaštićenih monomera u kiseloj sredini i izravno povezivanje u neutralnom području upotrebom aktiviranog disulfida (npr. piridil-SS) kao sredstva za povezivanje. II. Reduction of disulfide, isolation of unprotected monomers in acidic medium and direct coupling in neutral region using activated disulfide (eg pyridyl-SS) as coupling agent.
Pokazalo se je da do dvostrukog aciliranja na N-terminalnom cisteinu dolazi uglavnom pri povezivanju preko imidazolida ili CoA derivata (pribl. 60 - 70%) i u tom slučaju povezani hidrofobni spoj prisutan je u jednom slučaju vezan kao kiselins-ki amid, a u drugom slučaju on je povezan kao tioester. Selektivno odcjepljenje hidrofobnog spoja vezanog u obliku tioestera omogućuje sintezu jednostruko hidrofobiziranog hedgehog proteina. Takovo selektivno odcjepljenje provodi se s redukcijskim sredstvom kao što je DTE ili hidroksilamin. It has been shown that double acylation on the N-terminal cysteine occurs mainly when connected via an imidazolide or CoA derivative (approx. 60 - 70%) and in this case the connected hydrophobic compound is present in one case bound as an acid amide, and in the other case it is linked as a thioester. Selective cleavage of the hydrophobic compound bound in the form of a thioester enables the synthesis of a single hydrophobic hedgehog protein. Such selective cleavage is carried out with a reducing agent such as DTE or hydroxylamine.
Povezivanje preko aktiviranog disulfida, kao što su na primjer piridil-SS derivati, dovodi do monoalkilacije. Opisani postupak prema izumu omogućuje brojne hidrofobne tvari kao steroide, ugljikove lance (na primjer masnih kiselina) koji sadrže tiolne skupine koji se povezuju na hedgehog proteine. Coupling via an activated disulfide, such as, for example, pyridyl-SS derivatives, leads to monoalkylation. The described process according to the invention enables numerous hydrophobic substances such as steroids, carbon chains (for example fatty acids) containing thiol groups that connect to hedgehog proteins.
Slijedeći daljnji predmet izuma je postupak za proizvodnju SH-modificiranog hedgehog proteina ili njegovog N-terminalnog fragmenta, ponajprije fragmenta koji uglavnom sadrži N-terminalnu domenu, koji je naznačen time da je tiolna skupina N-terminalnog cisteina u hedgehog proteinu zaštićena, na taj način proizveden protein se izolira i nakon odcjepljenja zaštitne skupine hedgehog protein se derivatizira na N-terminalnom cisteinu, prednosno kovalentnim povezivanjem hidrofobnog spoja, kao što je masna kiselina ili steroid, na SH skupinu. Takovo povezivanje je prednosno reverzibilno i može se preusmjeriti fiziolškim redukcijskim uvjetima koji postoje u stanicama sisavaca, što in vivo ima za posljedicu tvorbu ne-hidrofobiziranog hedgehog proteina. A further subject of the invention is a method for the production of SH-modified hedgehog protein or its N-terminal fragment, preferably a fragment that mainly contains the N-terminal domain, which is characterized by the fact that the thiol group of the N-terminal cysteine in the hedgehog protein is protected, thus the produced protein is isolated and after removal of the protective group the hedgehog protein is derivatized at the N-terminal cysteine, preferably by covalently linking a hydrophobic compound, such as a fatty acid or a steroid, to the SH group. Such binding is preferably reversible and can be redirected by physiological reducing conditions existing in mammalian cells, which in vivo results in the formation of a non-hydrophobic hedgehog protein.
Daljnji glavni predmet izuma je postupak za povezivanje hidrofobnih spojeva na hedgehog proteine ili njegovih fragmenata preko SH skupine N-terminalnog cisteina, koji je naznačen time da je tiolna skupina N-terminalnog cisteina u hedgehog proteinu zaštićena, spomenuta SH skupina se reducira i taj hedgehog protein reagira s aktiviranim derivatom hidrofobnog spoja čime nastaje tiolna veza između hedgehog proteina i hidrofobnog spoja. U prednosnoj izvedbi može se dodatnu molekulu hidrofobnog spoja povezati na hedgeehog protein pomoću amidne veze. Another main subject of the invention is a method for connecting hydrophobic compounds to hedgehog proteins or its fragments via the SH group of the N-terminal cysteine, which is characterized by the fact that the thiol group of the N-terminal cysteine in the hedgehog protein is protected, the mentioned SH group is reduced and that hedgehog protein reacts with the activated derivative of the hydrophobic compound, which creates a thiol bond between the hedgehog protein and the hydrophobic compound. In a preferred embodiment, an additional molecule of the hydrophobic compound can be linked to the hedgehog protein by means of an amide bond.
Imidazolidni i CoA derivati hidrofobnih spojeva prikladni su, na primjer, kao aktivirani hidrofobni spojevi. Takovi aktivirani spojevi upotrebljavaju se ponajprije po metodi I (vidi gore). Imidazolide and CoA derivatives of hydrophobic compounds are suitable, for example, as activated hydrophobic compounds. Such activated compounds are preferably used according to method I (see above).
Piridil disulfidni derivati hidrofobnih spojeva prikladni su također kao aktivirani hidrofobni spojevi. Pyridyl disulphide derivatives of hydrophobic compounds are also suitable as activated hydrophobic compounds.
Takovi aktivirani spojevi upotrebljavaju se ponajprije po metodi II (vidi gore). Such activated compounds are preferably used according to method II (see above).
Prirodne ili neprirodne zasićene i jednostruko nezasićene ili višestruko nezasićene masne kiseline i posebno zasićene masne kiseline s lancem duljine 4-18, ponajprije 8-18 ugljikovih atoma ili steroidi upotrebljavaju se ponajprije za aciliranje hedgehog proteina imidazolidnim postupkom, pri čemu se dobiju konjugati u kojima ugljikovodični ostaci imaju duljinu lanca od 8-24 C atoma. Natural or unnatural saturated and monounsaturated or polyunsaturated fatty acids and especially saturated fatty acids with a chain length of 4-18, preferably 8-18 carbon atoms or steroids are used primarily for the acylation of hedgehog proteins by the imidazolide method, whereby conjugates are obtained in which the hydrocarbon residues have a chain length of 8-24 C atoms.
Za povezivanje hidrofobnih spojeva s hedgehog proteinima postupkom pomoću piridil disulfida, upotrebljavaju se ponajprije prirodni ili ne-prirodni zasićeni i jednostruko nezasićeni ili višestruko nezasićeni merkaptoalkani, a posebno zasićeni ugljikovi lanci koji sadrže tiolnu skupinu duljine 8-24 C atoma, merkapto steroidi, a posebno tiokolesterol. For the connection of hydrophobic compounds with hedgehog proteins using pyridyl disulfide, natural or non-natural saturated and monounsaturated or polyunsaturated mercaptoalkanes are primarily used, and especially saturated carbon chains containing a thiol group with a length of 8-24 C atoms, mercapto steroids, and especially thiocholesterol.
Za postupak prema izumu prednosna je upotreba hedgehog proteina pri koncentraciji od 0,01-10 mg/ml, posebno prednosno 3 mg/ml. Koncentracija soli je ponajprije 0-2 mola/l. Upotrebljava se ponajprije natrijev klorid. Molarni omjer reagenta za vezanje na protein prikladno je od 1:2 do 20:1. Prednosni puferi su Hepes pufer, fosfatni pufer i MES pufer. Reakcije povezivanja provode se ponajprije u pH području između 4,5 i 8,5, ponajprije pri pH 6,5-7,5. Vrijeme reakcije ovisi o primijenjenim uvjetima i reakcija pripravljanja dipalmitoiliranog shh traje prikladno od 5 minuta do 5 dana. Reakcija se provodi ponajprije u temperaturnom području između 0 i 40°C. Pri niskim temperaturama, ponajprije pri temperaturama ispod 10°C, npr. pri 4°C nizak je udio sporednih proizvoda. For the procedure according to the invention, it is preferable to use hedgehog protein at a concentration of 0.01-10 mg/ml, especially preferably 3 mg/ml. The salt concentration is preferably 0-2 mol/l. Sodium chloride is primarily used. The molar ratio of protein binding reagent is suitably from 1:2 to 20:1. Preferred buffers are Hepes buffer, phosphate buffer and MES buffer. The coupling reactions are preferably carried out in the pH range between 4.5 and 8.5, preferably at pH 6.5-7.5. The reaction time depends on the applied conditions and the reaction to prepare dipalmitoylated shh lasts conveniently from 5 minutes to 5 days. The reaction is preferably carried out in the temperature range between 0 and 40°C. At low temperatures, especially at temperatures below 10°C, for example at 4°C, the proportion of secondary products is low.
Za postupak prema izumu posebno je koristan dodatak detergenata. Takovi reagenti mogu biti, na primjer, ionski ili ne-ionski detergenti. Prednosni su amfoterni detergenti. Prednosne su koncentracije od pribl. 0/5-3% (v/v). The addition of detergents is particularly useful for the process according to the invention. Such reagents can be, for example, ionic or non-ionic detergents. Amphoteric detergents are preferred. Concentrations of approx. 0/5-3% (v/v).
Ugljikovodični lanac ili ugljikovodični lanci ili steroidi povezuju se prikladno na reaktivne skupine skupine proteina,- na primjer na slobodne hidroksi, merkapto, karboksi ili amino skupine, preko amidne veze, esterske, disulfidne ili tioesterske veze. Takovi postupci su poznati stručnjaku i opisani su na primjer u Wong, S.S., Chemistry of Protein Conjugation and Cross Linking, CRC Press, Boca Raton, Fl., SAD, 1993. Na primjer, masne kiseline mogu se povezati kao tioesteri s koenzimom A (npr. palmitoil koenzim A) preko sukcinamidnog estera ili N-maleimidnim poveivanjem (npr. N-hidroksi sulcinimidni ester palmitinske kiseline) preko anhidrida masne kiseline, imidazolida masne kiseline ili kiselinskog klorida. The hydrocarbon chain or hydrocarbon chains or steroids are conveniently linked to reactive groups of the protein group, for example to free hydroxy, mercapto, carboxy or amino groups, via an amide bond, an ester, disulphide or thioester bond. Such procedures are known to those skilled in the art and are described, for example, in Wong, S.S., Chemistry of Protein Conjugation and Cross Linking, CRC Press, Boca Raton, Fl., USA, 1993. For example, fatty acids can be linked as thioesters to coenzyme A ( eg palmitoyl coenzyme A) via succinamide ester or by N-maleimide addition (eg N-hydroxy sulcinimide ester of palmitic acid) via fatty acid anhydride, fatty acid imidazolide or acid chloride.
Postupke povezivanja za palmitoil-CoA, stearoil-CoA ili miristoil-CoA opisali su, na primjer, Ross et al., J. Neurosci. Res 21 (1988) 35-44, Bizzozero et al., J. Biol. Chem. 262 (1987) 2138-2145, a za tubulin su opisali Ozols, J. et al., Molec. Biol. of the Cell 8 (1997) 637-645. Derivatizacija s anhidridima masnih kiselina opisana je, na primjer, za ovalbumin (Segawa, A., et a., Int. Archs Allergy appl. Immun. 66 (1981) 189-199) ili za peptide (Yadav, S.P. et al., Biochem. Biophys. Res. Comm. 205 (1994) 1688-1695). Također postoje brojni primjeri za aciliranje sa sukcinimidnim esterima masnih kiselina, npr. za kazein (Haque, Z. et al., J. Agric. Food Chem. 31 (1983) 1225-1230), (Haque, Z. et al., Agric. Biol. Chem. 46 (1982) 579-599). N-terminalno povezivanje na cistein može se također izvršiti preko aldehidne skupine na partneru fuzije (npr. palmitoil-Cys-CHO) (Lui et al., Proc. Natl. Acad. Sci. USA 91 (1994) 6584-6588). N-terminalno povezivanje na serin može se postići pretvorbom u aldehidnu skupinu, reakcijom s hidrazidom (npr. palmitoil-Cys hidrazidom) i stabilizacijom nastalog hidrazona (npr. redukcijom s NaBH3CN) (Gaertner et al., Bioconjugate Chem. 3 (1992) 262-268) . Coupling procedures for palmitoyl-CoA, stearoyl-CoA, or myristoyl-CoA are described, for example, by Ross et al., J. Neurosci. Res 21 (1988) 35-44, Bizzozero et al., J. Biol. Chem. 262 (1987) 2138-2145, and for tubulin described by Ozols, J. et al., Molec. Biol. of the Cell 8 (1997) 637-645. Derivatization with fatty acid anhydrides has been described, for example, for ovalbumin (Segawa, A., et al., Int. Archs Allergy appl. Immun. 66 (1981) 189-199) or for peptides (Yadav, S.P. et al., Biochem. Biophys. Res. Comm. 205 (1994) 1688-1695). There are also numerous examples for acylation with succinimide esters of fatty acids, eg for casein (Haque, Z. et al., J. Agric. Food Chem. 31 (1983) 1225-1230), (Haque, Z. et al., Agric. Biol. Chem. 46 (1982) 579-599). N-terminal linkage to cysteine can also be accomplished via an aldehyde group on the fusion partner (eg, palmitoyl-Cys-CHO) (Lui et al., Proc. Natl. Acad. Sci. USA 91 (1994) 6584-6588). N-terminal coupling to serine can be achieved by conversion to an aldehyde group, reaction with a hydrazide (e.g. palmitoyl-Cys hydrazide) and stabilization of the resulting hydrazone (e.g. reduction with NaBH3CN) (Gaertner et al., Bioconjugate Chem. 3 (1992) 262 -268).
Hidrofobni ugljikovodični lanac povezuje se ovisno o kemiji povezivanja na primjer u obliku etera, tioetera, estera, tioestera, disulfida ili amida na bočne skupine reaktivnih amino kiselina serina, treonina, glutaminske kiseline, aspartinske kiseline, cisteina, arginina ili lizina. Metode za specifično povezivanje na pojedinačne amino kiseline opisao je Wong, S.S., u Chemistry of Protein Conjugation and Cross Linking, CRC Press Inc., Boca Raton, Fl, SAD (1993) i Lundblad u Techniques in Protein Modification (1995). The hydrophobic hydrocarbon chain is connected depending on the chemistry of connection, for example in the form of ether, thioether, ester, thioester, disulfide or amide to the side groups of reactive amino acids serine, threonine, glutamic acid, aspartic acid, cysteine, arginine or lysine. Methods for specific linkage to individual amino acids are described by Wong, S.S., in Chemistry of Protein Conjugation and Cross Linking, CRC Press Inc., Boca Raton, Fl, USA (1993) and Lundblad in Techniques in Protein Modification (1995).
U prednosnoj izvedbi izuma, hidrofobni spojevi se solubiliziraju u organskom otapalu ili u mješavini organskog otapala i vode koja sadrži ponajprije više od 10% (v/v) organskog otapala. Takova organska otapala jesu ponajprije dioksan, tetrahidrofuran ili izopropanol. Za povezivanje hidrofobnog spoja i hedgehog proteina takove otopine hidrofobnih spojeva pomiješaju se s otopinom hedgehog proteina koja sadrži detergent, ponajprije u njegovom zaštićenom obliku, na takav način da mješavina sadrži 10% ili manje organskog otapala. Nađeno je da otopine hedgeog proteina koje sadrže više od 10% organskog otapala dovode do taloženja i/ili denaturiranja hedgehog proteina. In a preferred embodiment of the invention, the hydrophobic compounds are solubilized in an organic solvent or in a mixture of an organic solvent and water which preferably contains more than 10% (v/v) of the organic solvent. Such organic solvents are primarily dioxane, tetrahydrofuran or isopropanol. To bind the hydrophobic compound and the hedgehog protein, such solutions of the hydrophobic compounds are mixed with a detergent-containing solution of the hedgehog protein, preferably in its protected form, in such a way that the mixture contains 10% or less of organic solvent. Hedgehog protein solutions containing more than 10% organic solvent were found to precipitate and/or denature the hedgehog protein.
U daljnjoj prednosnoj izvedbi tiokolesterol se povezuje na tiolnu skupinu u dotičnom N-terminalnom cisteinu pomoću oksidativno nastalog disulfidnog mosta u prisutnosti detergenta za solubilizaciju, kao što je posebno natrijev deoksiholat, natrijev holat, natrijev taurodeoksiholat, oktil glikozid ili Triton® X-100. Nasuprot N-terminalnom hh fragmentu, koji je modificiran prirodnim putem na C kraju s holesterolom, ovim postupkom dobije se hh oblik koji sadrži tiokolesterol na N kraju. Taj oblik ima sličnu povećanu aktivnost prema prirodnom obliku, ali se može proizvesti mnogo jednostavnije i u većim količinama. Zbog citoplazmične nepostojanosti disulfidnog mosta taj oblik hh nema ili ima samo mali imunogenski utjecaj. In a further preferred embodiment, thiocholesterol is linked to the thiol group in the respective N-terminal cysteine by means of an oxidatively formed disulfide bridge in the presence of a solubilizing detergent, such as in particular sodium deoxycholate, sodium cholate, sodium taurodeoxycholate, octyl glycoside or Triton® X-100. In contrast to the N-terminal hh fragment, which is naturally modified at the C end with cholesterol, this procedure results in an hh form containing thiocholesterol at the N end. This form has similar increased activity to the natural form, but can be produced much more simply and in larger quantities. Due to the cytoplasmic instability of the disulfide bridge, this form of hh has no or only a small immunogenic effect.
Da se povisi topivost lipofilno modificiranih hh proteina dodatno se prednosno provodi derivatizacija i/ili naknadno čišćenje ili farmaceutska formulacija u prisutnosti topivih anionskih polisaharida kao što su suramin ili heparin. In order to increase the solubility of lipophilic modified hh proteins, derivatization and/or subsequent purification or pharmaceutical formulation in the presence of soluble anionic polysaccharides such as suramin or heparin is preferably carried out additionally.
Kao aktivnost u smislu izuma podrazumijeva se aktivnost alkalne fosfataze koja može inducirati polipeptid u stanicama sisavca (aktivnost u pokusu s alkalnom fosfatazom). Po toj metodi mišja stanična linija fibroplasta uzgaja se u mediju koji sadrži fetalni goveđi serum. Zatim se doda sterilno filtrirani uzorak, stanice liziraju nakon pribl. 5 dana i u staničnom lizatu utvrdi se alkalnu fosfatazu odcjepljenjem kromogenog supstrata (pNP, p-nitrofenol) (J. Asahina, Exp. Cell. Res. 222 (1996) 38-47 i T. Nakamura (1997)). Activity in the sense of the invention means alkaline phosphatase activity that can induce a polypeptide in mammalian cells (activity in the alkaline phosphatase experiment). According to this method, the mouse fibroblast cell line is grown in a medium containing fetal bovine serum. A sterile filtered sample is then added, the cells are lysed after approx. 5 days and alkaline phosphatase was determined in the cell lysate by splitting off the chromogenic substrate (pNP, p-nitrophenol) (J. Asahina, Exp. Cell. Res. 222 (1996) 38-47 and T. Nakamura (1997)).
Prema izumu kao hedgehog protein podrazumijeva se izlučeni signalni protein (19 kD N-terminalna signalna domena) koji je odgovoran za tvorbu brojnih struktura u embriogenezi. Posebno prednosno upotrebljavaju se sunčani, indijski ili pustinjski hh (Fietz M. et al., Development (Suppl.) (1994) 43-51). Upotrebljava se ponajprije hh protein sa sekvencom kako je opisana u banci podataka EMBL pod brojem L38518. Proteini hedgehog porodice pokazuju izrazitu homologiju njihovih sekvenci amino kiselina, što je razlog da oni ponajprije umnažaju one nukleinske kiseline koje kodiraju za hedgehog proteine koji su 80% ili više homologni s gore spomenutom sekvencom sunčanog hedgehog proteina (shh). Proteinsku homologiju može se odrediti pomoću računalnog programa Gap ili BestFit (Sveučilište u Wisconsinu; Needleman i Wunsch, J. Mol. Biol. 48 (1970) 443-453; Smith i Watermann, Adv. Appl. Math. 2 (1981) 482-489). According to the invention, hedgehog protein is a secreted signal protein (19 kD N-terminal signal domain) which is responsible for the formation of numerous structures in embryogenesis. Sunny, Indian or desert hh are particularly preferably used (Fietz M. et al., Development (Suppl.) (1994) 43-51). The hh protein with the sequence as described in the EMBL database under the number L38518 is primarily used. The proteins of the hedgehog family show a marked homology of their amino acid sequences, which is the reason that they primarily amplify those nucleic acids that code for hedgehog proteins that are 80% or more homologous to the above-mentioned sequence of the sunny hedgehog protein (shh). Protein homology can be determined using the computer programs Gap or BestFit (University of Wisconsin; Needleman and Wunsch, J. Mol. Biol. 48 (1970) 443-453; Smith and Watermann, Adv. Appl. Math. 2 (1981) 482- 489).
Humani sunčani hedgehog prekurzorski protein sastavljen je od sekvence amino kiselina 1 - 462 opisane u banci podataka EMBL pod brojem L38518. Amino kiseline 1 -23 predstavljaju signalni peptid, amino kiseline 24 - 197 predstavljaju zrelu signalnu domenu, amino kiseline 32 -197 predstavljaju signalnu domenu skraćenu za osam amino kiselina i amino kiseline 198 - 462 predstavljaju samo-preradivačku C-terminalnu domenu nakon autoproteolitskog odcjepljenja. Kao prve amino kiseline (N kraj) ili zadnje amino kiseline (C kraj) N-terminalne signalne domene 24-197 podrazumijevaju se prema izumu prema N kraju ili C kraju hh proteina, na kojem se prednosno odvija povezivanje. Prednosno je povezivanje na jednu ili na nekoliko amino kiselina od prvih ili posljednjih 10 amino kiselina. Posebno prednosno je povezivanje na prvu ili drugu amino kiselinu N-kraja N-terminalne domene (AA 24 ili 25) ili na posljednju ili na pretposljednju amino kiselinu C kraja N-terminalne domene (AA 196 ili 197) . U hedgehog konjugatima prema izumu lipofilne skupine ili ugljikovodični lanac (lanci) posebno prednosno su povezani na N-terminalnu domenu hh proteina, a proizvod povezivanja je posebno tioester ili amid N-terminalnog cisteina na položaju 24 s laurinskom, miristinskom, palmitinskom, stearinskom ili oleinskom kiselinom ili steroid ili je to hh protein na koji je tiokolesterol ili merkaptoalkan/-alken povezan preko disulfidnog mosta. Proizvodnja nemodificiranog hh proteina provodi se prednosno rekombinantno primjenom metoda koje su poznate stručnjacima, ponajprije u prokariotskom (npr. E. coli) sistemu ekspresije. Hedgehog protein se proizvodi prednosno rekombinantno kao fuzijski protein u topivom obliku, izolira se iz supernatanta stanične kulture ili se nakon lize stanica domaćina (ponajprije N kraj) fuzijski dio (npr. poliHys, streptavidin, itd.) odcijepi pomoću sekvencno specifične proteaze kao što je enterokinaza, a slobodna tiolna skupina N-terminalnog cisteina se zaštiti reakcijom s tiolnim zaštitnim reagentom ili dimerizacijom hedgehog proteina disulfidnim premoštavanja na spomenutom cisteinu. The human sunny hedgehog precursor protein consists of the sequence of amino acids 1 - 462 described in the EMBL data bank under number L38518. Amino acids 1 - 23 represent the signal peptide, amino acids 24 - 197 represent the mature signal domain, amino acids 32 - 197 represent the signal domain shortened by eight amino acids and amino acids 198 - 462 represent the self-processing C-terminal domain after autoproteolytic cleavage. As the first amino acids (N end) or the last amino acids (C end) of the N-terminal signal domain 24-197 are meant according to the invention to the N end or the C end of the hh protein, where binding preferably takes place. Linkage to one or several amino acids of the first or last 10 amino acids is preferred. Particularly preferred is the connection to the first or second amino acid of the N-end of the N-terminal domain (AA 24 or 25) or to the last or the penultimate amino acid of the C end of the N-terminal domain (AA 196 or 197). In the hedgehog conjugates according to the invention, lipophilic groups or hydrocarbon chain(s) are especially preferably connected to the N-terminal domain of the hh protein, and the connection product is especially a thioester or amide of the N-terminal cysteine at position 24 with lauric, myristic, palmitic, stearic or oleic acid or steroid or it is a hh protein to which thiocholesterol or mercaptoalkane/-alkene is linked via a disulfide bridge. The production of unmodified hh protein is preferably carried out recombinantly using methods known to experts, primarily in a prokaryotic (eg E. coli) expression system. Hedgehog protein is produced preferably recombinantly as a fusion protein in soluble form, isolated from cell culture supernatant, or after lysis of host cells (preferably the N terminus) the fusion part (e.g. polyHys, streptavidin, etc.) is cleaved off with a sequence-specific protease such as enterokinase, and the free thiol group of the N-terminal cysteine is protected by reaction with a thiol protection reagent or dimerization of the hedgehog protein by disulfide bridging on said cysteine.
Farmaceutski sastav prema izumu sadrži farmakološki učinkovitu dozu hh konjugata i prednosno se može dati lokalno. Prednosna upotreba konjugata prema izumu je u kombinaciji s drugim hedgehog protinima iz porodice hedgehog ili faktora rasta kostiju, kao što su koštani morfogenetički proteini (BMPs) (Wozney et a., Cell. Mol. Biol. of Bone, Bone Morphogenetic Proteins and their Gene Expression (1993) Academic Press Inc., 131-167) ili paratiroidni hormoni (Karablis et al., Genes and Development 8 (1994) 277-289) ili inzulinu slični faktori rasta (IGF-I ili II) ili tranformirajući faktori rasta (TGF-β). The pharmaceutical composition according to the invention contains a pharmacologically effective dose of the hh conjugate and can preferably be administered locally. A preferred use of the conjugate according to the invention is in combination with other hedgehog proteins from the hedgehog family or bone growth factors, such as bone morphogenetic proteins (BMPs) (Wozney et al., Cell. Mol. Biol. of Bone, Bone Morphogenetic Proteins and their Gene Expression (1993) Academic Press Inc., 131-167) or parathyroid hormones (Karablis et al., Genes and Development 8 (1994) 277-289) or insulin-like growth factors (IGF-I or II) or transforming growth factors ( TGF-β).
U daljnjoj prednosnoj izvedbi, prednost se daje farmaceutskom sastavu hedgehog konjugata prema izumu koji sadrži suramin i on se može korisno upotrijebiti. In a further preferred embodiment, a pharmaceutical composition of the hedgehog conjugate according to the invention containing suramin is preferred and can be advantageously used.
U prednosnoj izvedbi farmaceutski sastav hedgehog konjugat koncentracijom od 0,01-10 mg/ml, ponajprije 0/01 do 1 mg/ml. In a preferred embodiment, the hedgehog conjugate pharmaceutical composition has a concentration of 0.01-10 mg/ml, preferably 0/01 to 1 mg/ml.
U prednosnoj izvedbi farmaceutski sastav dodatno sadrži farmaceutski prihvatljiv pufer koje je biološki kompatibilan prednosno u području između pH 4 i pH 10, posebno prednosno u području između pH 6 i 9, a naročito pri pH vrijednosti pribl. 7. Da se spriječi denaturiranje složene strukture i otkidanje cinkovog kompleksa u hedgehog proteinu pH vrijednost farmaceutskog sastava mora biti prikladno viša od pH 4. Koncentracija pufera je ponajprije 1-500 mmolova/1, ponajprije 10-100 mmolova/1. Stoga se dakle u prikladnoj izvedbi kao pufer upotrebljava 20 mmolova/1 kalijevog fosfatnog pufera pH 7,2. In a preferred embodiment, the pharmaceutical composition additionally contains a pharmaceutically acceptable buffer which is biologically compatible preferably in the range between pH 4 and pH 10, especially preferably in the range between pH 6 and 9, and especially at a pH value of approx. 7. To prevent the denaturation of the complex structure and the breaking of the zinc complex in the hedgehog protein, the pH value of the pharmaceutical composition must be suitably higher than pH 4. The buffer concentration is preferably 1-500 mmol/1, preferably 10-100 mmol/1. Therefore, in a suitable embodiment, 20 mmol/l potassium phosphate buffer pH 7.2 is used as a buffer.
Za proizvodnju farmaceutskog sastava prednosno se nadalje dodaju pomoćne tvari kao šećer (manitol, saharoza, laktoza, glukoza, trehaloza, ponajprije 20-100 mg/ml) ili amino kiselina kao glicin ili arginin kao i antioksidanti kao EDTA, citrat, polietilen glikol (1 - 10% mas. %) , askorbinska kiselina, tokofenol, detergenti, ponajprije neionski detergenti (ponajprije 0,005 - 1 mas. %) kao polisorbati ili detergenti polioskietilenskog tipa (npr. Tween® 20, Tween® 80) ili polioksietilenski ili ionski detergenti kao natrijev holat, natrijev deoksiholat ili natrijev taurodeoksiholat, anti-upalna sredstva, lokalni anestetici, antibiotici i/ili stabilizatori kao lipidi, masne kiseline i glicerol. For the production of the pharmaceutical composition, auxiliary substances such as sugar (mannitol, sucrose, lactose, glucose, trehalose, preferably 20-100 mg/ml) or amino acids such as glycine or arginine, as well as antioxidants such as EDTA, citrate, polyethylene glycol (1 - 10% wt. %), ascorbic acid, tocophenol, detergents, preferably non-ionic detergents (preferably 0.005 - 1 wt. %) such as polysorbates or detergents of the polyoxyethylene type (e.g. Tween® 20, Tween® 80) or polyoxyethylene or ionic detergents such as sodium cholate, sodium deoxycholate or sodium taurodeoxycholate, anti-inflammatory agents, local anesthetics, antibiotics and/or stabilizers such as lipids, fatty acids and glycerol.
Konjugati prema izumu mogu se korisno upotrijebiti za induciranje ili za stimulaciju kondrocita ili osteocita u osteoinduktivnom farmaceutskom sastavu ili također za induciranje mišićnih i nervnih stanica. Osteoinduktivni farmaceutski sastavi poznati su, na primjer, iz US patenta 5,364,839, WO 97/35607 i WO 95/16035. The conjugates according to the invention can be advantageously used to induce or stimulate chondrocytes or osteocytes in an osteoinductive pharmaceutical composition or also to induce muscle and nerve cells. Osteoinductive pharmaceutical compositions are known, for example, from US Patent 5,364,839, WO 97/35607 and WO 95/16035.
Aktivnost hedgehog konjugata prema izumu može se ocijeniti in vivo u skladu s Glansbeek, H.L., et al., Laboratory Investigation 78 (1998) 133-142; US patent br. 5,270,300; Toriumi, D.M., et al., Arch. Otolaryngol. Head Neck Surg. 117 (1991) 1101-1112; Cook, S.D., et al., J. Bone and Joint Surgery 76-A (1994) 827-837; i Riley, E.H., et al., Clin. Orthopaed. and Realted Research 324 (1996) 39-46. The activity of hedgehog conjugates according to the invention can be evaluated in vivo according to Glansbeek, H.L., et al., Laboratory Investigation 78 (1998) 133-142; US patent no. 5,270,300; Toriumi, D.M., et al., Arch. Otolaryngol. Head Neck Surg. 117 (1991) 1101-1112; Cook, S.D., et al., J. Bone and Joint Surgery 76-A (1994) 827-837; and Riley, E.H., et al., Clin. Orthopaedist. and Realted Research 324 (1996) 39-46.
Kad se konjugat prema izumu daje lokalno prednosno ga je upotrijebiti u kombinaciji s prikladnom matricom kao nosačen i/ili s sekvestracijskim sredstvom. Takova matrica prikladna je za polagano oslobađanje proteina in vivo u aktivnom obliku, posebno u blizini kostiju ili tkiva kralježnice. Sekvestracijsko sredstvo je tvar koja olakšava davanje, na primjer, injekcijom i/ili sprečava ili barem usporava migraciju proteina prema izumu od strane davanje. When the conjugate according to the invention is administered locally, it is preferable to use it in combination with a suitable matrix as a carrier and/or with a sequestering agent. Such a matrix is suitable for the slow release of proteins in vivo in an active form, especially in the vicinity of bone or spinal tissue. A sequestering agent is a substance that facilitates administration, for example by injection, and/or prevents or at least slows down the migration of the protein according to the invention by the administration.
Farmaceutski sastav prema izumu sadrži ponajprije polimer (strukturna tvar) koji ima funkciju adhezije na stanice. Takova strukturna tvar je primjerice kolagen. The pharmaceutical composition according to the invention primarily contains a polymer (structural substance) that has the function of adhesion to cells. Such a structural substance is, for example, collagen.
Biološki kompatibilni/ razgradljivi materijali/ na primjer na osnovi kolagena ili drugih polimera na osnovi poli-mliječne kiseline, poliglikolne kiseline ili kopolimera mliječne kiseline i glikolne kiseline, posebno su prikladni kao matrični materijali. Takove polimerne matrice opisane su, na primjer, u W0 93/00050. Biocompatible/degradable materials/ for example based on collagen or other polymers based on poly-lactic acid, polyglycolic acid or copolymers of lactic acid and glycolic acid, are particularly suitable as matrix materials. Such polymer matrices are described, for example, in WO 93/00050.
Sekvestracijska sredstva jesu, na primjer, celuloza i materijali slični celulozi i, na primjer, alkil celuloza/ karboksimetil celuloza, hijaluronska kiselina, natrijev alginat, polietilen glikol i polivinil alkohol, od kojih je posebno prednosna hijaluronska kiselina, naročito u farmaceutskom sastavu također i bez noseće matrice. Sequestering agents are, for example, cellulose and cellulose-like materials and, for example, alkyl cellulose/carboxymethyl cellulose, hyaluronic acid, sodium alginate, polyethylene glycol and polyvinyl alcohol, of which hyaluronic acid is particularly preferred, especially in a pharmaceutical composition also without supporting matrices.
Slijedeći primjeri, publikacije, protokol sekvenci i slike dalje objašnjavaju izum, čiji smisao zaštite proizlazi iz patentnih zahtjeva. Opisane metode treba podrazumijevati kao primjere koji čak i nakon modifikacija samo opisuju glavni predmet izuma. The following examples, publications, sequence protocol and figures further explain the invention, the sense of protection of which derives from the patent claims. The described methods should be understood as examples which, even after modifications, only describe the main subject of the invention.
Opis slika Description of images
Slika 1 prikazuje aktivnost rekombinantnog humanog shh nakon derivatizacije s palmitoil CoA. Figure 1 shows the activity of recombinant human shh after derivatization with palmitoyl CoA.
Slika 2 prikazuje aktivnost rekombinantnog humanog shh nakon derivatizacije s tiokolesterolom. Figure 2 shows the activity of recombinant human shh after derivatization with thiocholesterol.
Primjer 1 Example 1
a) Kloniranje humanog sunčanog hedgehog proteina koji je pričvršćen za sidro His-6 kao i za stranu odcjepljenja enterokinaze; ekspresija u E. coli a) Cloning of the human sunny hedgehog protein that is attached to the His-6 anchor as well as to the cleavage site of enterokinase; expression in E. coli
Za pojačanje zrelog N-terminalnog dijela humanog sunčanog hedgehog proteina (aa 24 - Cys do 197 Gly) iz bilo kojeg željenog plazmida ili odgovarajućih cDNA koje sadrže sunčani hedgehog može se primijeniti slijedeći postupak: The following procedure can be used to amplify the mature N-terminal part of the human sun hedgehog protein (aa 24 - Cys to 197 Gly) from any desired plasmid or corresponding cDNAs containing sun hedgehog:
Komadić shh gena koji se širi od unutarnje RsrII strane odcjepljenja pa sve do sekvence koja kodira za amino kiselinu 198 pojačava se pomoću dva prajmera (344 i 345), a istovremeno nekoliko zaustavnih kodona kao i PstI strana odcjepljenja može biti učvršćena na C kraju. A piece of the shh gene extending from the internal RsrII cleavage site up to the sequence coding for amino acid 198 is amplified using two primers (344 and 345), and at the same time several stop codons as well as the PstI cleavage site can be fixed at the C end.
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Fragment DNA pojačan na taj način može se ponovno odcijepiti s RsrII i Pstl, a potreban je u slijedećim stupnjevima (fragment 344/345). The DNA fragment amplified in this way can be cleaved again with RsrII and PstI, and is needed in the following steps (fragment 344/345).
Linker je konstruiran aneliranjem dvaju daljnjih prajmera (346 1 347) pomoću kojih se na N kraju ugrađuje 6 histidinskih ostataka i strana odcjepljenja enterokinaze (EK) : The linker was constructed by annealing two further primers (346 1 347) by means of which 6 histidine residues and the cleavage site of enterokinase (EK) are incorporated at the N end:
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Za ekspresiju, PCR fragment 344/345 klonira se zajedno s adapterom 346/347 u vektor odcijepljen s EcoRI/PstI. To se može provesti izravno ili nakon intermedijarnog kloniranja fragmenta 344/345 u drugi vektor. Vektor odcijepljen s EcoRI/PstI mora sadržavati prikladan promotor za ekspresiju u E. coli na kraju EcoRI, ponajprije T5, tac, lac itd. shh ekspresijski plazmidi transfektiraju se u vrstu E. coli prikladnu za ekspresiju. Sistem ekspresije sadrži nadalje nukleinske kiseline koje kodiraju arginin-tRNA^GA/AGG sadržan u prokariotskim stanicama (Brinkmann et al.,Gene 85 (1989) 109-114). For expression, PCR fragment 344/345 is cloned together with adapter 346/347 into an EcoRI/PstI cleaved vector. This can be done directly or after intermediate cloning of the 344/345 fragment into another vector. The vector cleaved with EcoRI/PstI must contain a suitable promoter for expression in E. coli at the EcoRI end, preferably T5, tac, lac etc. The shh expression plasmids are transfected into an E. coli strain suitable for expression. The expression system further contains nucleic acids encoding arginine-tRNA^GA/AGG contained in prokaryotic cells (Brinkmann et al., Gene 85 (1989) 109-114).
b) Fermentacija b) Fermentation
10 1 fermentacija E. coli ekspresijskog klona za hedgehog: 10 1 E. coli fermentation of hedgehog expression clone:
Predkultura pripravljena je iz tipova kultura (premazana ploča ili ampule pohranjene pri -20°) koje se inkubiraju pri 30 - 37°C uz mućkanje. Inokulacijski volumen u slijedećoj višoj dimenziji je u svakom slučaju 1-10 volumnih %. U predkulturu dodan je ampicilin (50 - 100 mg/l), a glavna kultura je odabrana prema gubitku plazmida. The pre-culture is prepared from types of cultures (coated plate or ampoules stored at -20°) which are incubated at 30 - 37°C with shaking. The inoculation volume in the next higher dimension is in any case 1-10% by volume. Ampicillin (50 - 100 mg/l) was added to the pre-culture, and the main culture was selected according to plasmid loss.
Hranjive tvari koje se mogu upotrijebiti su enzimski probavljiv protein i/ili gljivični ekstrakt kao izvor dušika i ugljika, kao i glicerol i/ili glukoza kao dodatni izvor ugljika. Medij je puferiran na pH 7, a za stabilizaciju procesa fermentacije mogu se dodati metalne soli u fiziološki podnošljivim koncentracijama. Temperatura fermentacije je 25-37°C. Rast se određuje mjerenjem optičke gustoće pri 528 nm. Po isteku perioda fermentacije od pribl. 30 sati biomasu se skupi centrifugiranjem pri zastoju optičke gustoće. Nutrients that can be used are enzymatically digestible protein and/or fungal extract as a nitrogen and carbon source, as well as glycerol and/or glucose as an additional carbon source. The medium is buffered at pH 7, and metal salts can be added in physiologically tolerable concentrations to stabilize the fermentation process. Fermentation temperature is 25-37°C. Growth is determined by measuring the optical density at 528 nm. After the fermentation period of approx. For 30 hours, the biomass is collected by centrifugation when the optical density stops.
Primjer2 Example 2
a) Pripravljanje dimernog humanog shh a) Preparation of dimeric human shh
55 g biomase proizvedene u primjeru 1b lizirano je pomoću visokotlačne preše, centrifugirano i supernatant je apliciran u 50 ml helatinirajuće Sepharose (Pharmacia Biotech) koja je prethodno bila opterećena sa cinkom. shh fuzijski protein je ispran s gradijentom od 0 do 200 mM imidazola u 50 mM Hepesa; 250 mM NaCl; pH 7,4. Frakcije koje su sadržavale shh identificirane su pomoću SDS-PAGE, skupljene i razrijeđene s jednim volumenom 50 mM Hepesa; pH 7,4. Talozi nastali tijekom razrjeđivanja su centrifugirani i supernatant je dijaliziran pri 4°C nasuprot 50 mM Hepesa; pH 7,4. K 500 mg na taj način dobivenog shh fuzijskog proteina dodana je enterokinaza (1:500; masa/masa; Boehinger Mannheim GmbH) i P-ME (do 10 mM) i inkubirano je 16 sati u vodenoj kupelji pri 35°C. Zatim je dodan kruti DTT do koncentracije od 10 mM. Uzorak je apliciran u 166 ml SP-Sepharose (Pharmacia Biotech) i ispran s gradijentom od 0-800 mM NaCl u 20 mM HEPES-u, pH 7,4. Nakon analize vrsnih frakcija pomoću SDS-PAGE skupljene su glavne frakcije, podijeljene u alikvote i do daljnje upotrebe pohranjene su pri -80°C. Te glavne frakcije sadrže shh pod nereducirajućim uvjetima kao dimer, umrežen preko disulfidnog mosta, koji se može reducirati i ima prividnu molekulsku masu od pribl. 38 kDa. 55 g of the biomass produced in example 1b was lysed using a high-pressure press, centrifuged and the supernatant was applied to 50 ml of gelatinizing Sepharose (Pharmacia Biotech) which was previously loaded with zinc. The shh fusion protein was eluted with a gradient from 0 to 200 mM imidazole in 50 mM Hepes; 250 mM NaCl; pH 7.4. Fractions containing shh were identified by SDS-PAGE, pooled and diluted with one volume of 50 mM Hepes; pH 7.4. Precipitates formed during dilution were centrifuged and the supernatant dialyzed at 4°C against 50 mM Hepes; pH 7.4. Enterokinase (1:500; mass/mass; Boehinger Mannheim GmbH) and P-ME (up to 10 mM) were added to 500 mg of shh fusion protein thus obtained and incubated for 16 hours in a water bath at 35°C. Solid DTT was then added to a concentration of 10 mM. The sample was applied to 166 ml of SP-Sepharose (Pharmacia Biotech) and washed with a gradient of 0-800 mM NaCl in 20 mM HEPES, pH 7.4. After the analysis of the best fractions using SDS-PAGE, the main fractions were collected, divided into aliquots and stored at -80°C until further use. These major fractions contain shh under non-reducing conditions as a dimer, cross-linked by a disulfide bridge, which can be reduced and has an apparent molecular weight of approx. 38 kDa.
b) Modificiranje inkubacijom pod redukcijskim uvjetima b) Modification by incubation under reducing conditions
Dimerni shh (c = 1 mg/ml) reduciran je s 10 ml mM DTE (30 minuta, 25°C) i dijaliziriran je preko noći nasuprot PBS-u koji je sadržavao 0,5 mM DTT-a. Nakon odsoljavanja preko RP-HPLC maseni spektar dijalizata pokazao je da glavnina monomeriziranog shh pokazuje između ostalog (tj . dodatno uz ostale modifikacije koje dovode do proširenja pika u masenom spektru i koje dalje nisu specificirane) adukte od 32±4 Da (dvostruka oksidacija N-terminalnog cisteina) i 47±4 Da. Oblici shh modificirani na taj način ne mogu se ponovno dimerizirati ponovnom oksidacijom. To je pokazalo da je SH skupina N-terminalnog cisteina bila modificirana na postojan način. Posljedica toga je da ta SH skupina nije više sposobna za daljnje reakcije, npr. za tvorbu tioestera i iskorištenje derivatizacije s hidrofobnim spojevima je stoga jako reducirano. Zbog toga se kod aciliranja N-terminalnog cisteina in vitro periodi u kojima je u otopini prisutan reducirani shh moraju držati što je moguće kraćim ili u siučaju prikladne kemije povezivanja, reakcija se mora provoditi u prisutnosti Tris(2-karboksietil) fosfin hidroklorida (TCEP • HC1) jer se tada napadaju samo disuifodni spojeve, ali ne i (aktivirani) tioesteri. Dimeric shh (c = 1 mg/ml) was reduced with 10 ml mM DTE (30 min, 25°C) and dialyzed overnight against PBS containing 0.5 mM DTT. After desalting via RP-HPLC, the mass spectrum of the dialysate showed that the bulk of monomerized shh shows, among other things (i.e. in addition to other modifications that lead to the broadening of the peak in the mass spectrum and which are not further specified) adducts of 32±4 Da (double oxidation of N- terminal cysteine) and 47±4 Da. Forms of shh modified in this way cannot re-dimerize by re-oxidation. This indicated that the SH group of the N-terminal cysteine was modified in a persistent manner. The consequence of this is that this SH group is no longer capable of further reactions, eg for the formation of thioesters, and the use of derivatization with hydrophobic compounds is therefore greatly reduced. Therefore, when acylating the N-terminal cysteine in vitro, the periods in which reduced shh is present in the solution must be kept as short as possible or, in the case of suitable coupling chemistry, the reaction must be carried out in the presence of Tris(2-carboxyethyl) phosphine hydrochloride (TCEP • HC1) because then only disulfide compounds are attacked, but not (activated) thioesters.
c) Kinetika ponovne oksidacije shh za tvorbu dimera u odnosu prema pH vrijednosti otapala c) Kinetics of shh re-oxidation for dimer formation in relation to the pH value of the solvent
0,5 ml shh dimera (c = 2,7 mg/ml) monomerizirano je redukcijom intermolekularnog disulfidnog mosta dodatkom 2 mM TCEP-a (Tris(2-karboksietil)fosfin hidroklorid) tijekom 15 minuta pri 25°C. Zatim je uzorak ponovno puferiran pomoću stupca PD-10 (Pharmacia) u PBS-u, pH 7/0 ili PBS-u pH 5,0 i spontana dimerizacija (pri 25°C) analizirana je, nakon odstranjivanja redukcijskog sredstva, ovisno o periodu inkubacije pomoću RP-HPLC. 0.5 ml of shh dimer (c = 2.7 mg/ml) was monomerized by reduction of the intermolecular disulfide bridge by the addition of 2 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) for 15 minutes at 25°C. Then the sample was re-buffered using a PD-10 column (Pharmacia) in PBS, pH 7/0 or PBS, pH 5.0 and spontaneous dimerization (at 25°C) was analyzed, after removal of the reducing agent, depending on the period incubation by RP-HPLC.
Rezultati su zbirno prikazani u tablici 2: The results are summarized in table 2:
Tablica 2 Table 2
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Može se vidjeti da shh monomer spontano dimerizira. Dimerizacija je vrlo brza pri pH 7,0, dok se pri pH 5,0 ona odvija sa sporijom kinetikom. The shh monomer can be seen to dimerize spontaneously. Dimerization is very fast at pH 7.0, while at pH 5.0 it takes place with slower kinetics.
Primjer 3 Example 3
a) Pripravljanje reagenata povezivanje za selektivno aciliranje SH skupina u cisteinima a) Preparation of coupling reagents for selective acylation of SH groups in cysteines
(I) Imidazolidni postupak (I) Imidazolid procedure
Polazni materijali (imidazolidi) proizvedeni su općim postupkom A, B ili C analogno postupcima opisanim u literaturi (Leksyszyn, J. et al., Synthesis (1978) 478-479; Staab H.A., Angew. Chem. 74 (1962) 407-423; Fahrenholtz, K.E. et al., J. Med. Chem. 17 (1974) 337-342. The starting materials (imidazolides) were produced by the general procedure A, B or C analogous to the procedures described in the literature (Leksyszyn, J. et al., Synthesis (1978) 478-479; Staab H.A., Angew. Chem. 74 (1962) 407-423 , Fahrenholtz, KE et al., J. Med. Chem. 17 (1974) 337-342.
Ia) Opći postupak A Ia) General procedure A
10 mmolova odgovarajuće karboksilne kiseline i 10 mmolova 1,l'-karbonildiimidazola otopi se u apsolutnom tetrahidrofuranu i miješa se od 30 minuta do 24 sata pri sobnoj temperaturi. Smjesu se ispari u vakuumu i upotrebljava se bez daljnjeg čišćenja. 10 mmoles of the corresponding carboxylic acid and 10 mmoles of 1,1'-carbonyldiimidazole are dissolved in absolute tetrahydrofuran and stirred for 30 minutes to 24 hours at room temperature. The mixture is evaporated under vacuum and used without further cleaning.
Ib) Opći postupak B Ib) General procedure B
10 mmolova odgovarajućeg kiselinskog klorida i 20 mmolova imidazola grije se od 1 do 24 sata pod refluksom u apsolutnom toluenu uz isključenje vlage. Imidazolni hidroklorid, koji tako nastane, se odfiltrira i filtrat se ispari u vakuumu. Ostatak se očisti kromatografijom na silika gelu upotrebom etil acetat/heptana ili prekristalizacijom. 10 mmol of the corresponding acid chloride and 20 mmol of imidazole are heated from 1 to 24 hours under reflux in absolute toluene with exclusion of moisture. The resulting imidazole hydrochloride is filtered off and the filtrate is evaporated under vacuum. The residue is purified by chromatography on silica gel using ethyl acetate/heptane or by recrystallization.
Ic) Opći postupak C Ic) General procedure C
10 mmolova odgovarajuće karboksilne kiseline miješa se od 1 do 48 sati pri sobnoj temperaturi s 10 mmolova dicikloheksilkarbodiimida i 10 ml imidazola u diklormetanu. Kad se ohladi na 0°C tako nastalu ureu se odstrani filtracijom i filtrat se ekstrahira s vodom, osuši preko natrijevog sulfata, ispari u vakuumu i prekristalizira. 10 mmoles of the corresponding carboxylic acid are mixed from 1 to 48 hours at room temperature with 10 mmoles of dicyclohexylcarbodiimide and 10 ml of imidazole in dichloromethane. When it cools down to 0°C, the resulting urea is removed by filtration and the filtrate is extracted with water, dried over sodium sulfate, evaporated in a vacuum and recrystallized.
Dobiveni su slijedeći imidazolidni derivati masnih kiselina: The following imidazolide derivatives of fatty acids were obtained:
1-imidazol-l-il-heksan-l-on (bezbojno ulje; iskorištenje 66%), 1-imidazol-l-yl-hexan-l-one (colorless oil; yield 66%),
1-imidazol-1-il-oktan-1-on (bezbojni kristali; iskorištenje 59%), 1-imidazol-1-yl-octan-1-one (colorless crystals; yield 59%),
1-imidazol-1-il-dekan-1-on (bezbojni kristali; iskorištenje 79%) , 1-imidazol-1-yl-decan-1-one (colorless crystals; yield 79%),
1-imidazol-1-il-dodekan-1-on (bezbojni kristali; iskorištenje 86%), 1-imidazol-1-yl-dodecan-1-one (colorless crystals; yield 86%),
1-imidazol-1-il-tetradekan-1-on (bezbojni kristali; iskorištenje 52%) , 1-imidazol-1-yl-tetradecan-1-one (colorless crystals; yield 52%),
1-imidazol-1-il-heksadekan-1-on (bezbojni kristali; iskorištenje 70%) , 1-imidazol-1-yl-hexadecan-1-one (colorless crystals; yield 70%),
17-(4-imidazol-1-il-metil-4-okso-butil)-10,13-dimetil-dodekahidro-ciklopenta[a]fenantren-3,7,12-trion), (bezbojni kristali; iskorištenje 56%), 17-(4-imidazol-1-yl-methyl-4-oxo-butyl)-10,13-dimethyl-dodecahydro-cyclopenta[a]phenanthrene-3,7,12-trione), (colorless crystals; yield 56% ),
4-(10,13-dimetil-heksadekahidro-ciklopenta[a]fenantren-17-il)-1-imidazol-l-il-pentan, (bezbojni kristali; iskorištenje 71%). 4-(10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-1-imidazol-1-yl-pentane, (colorless crystals; yield 71%).
(II) Piridil sulfidni postupak (II) Pyridyl sulfide process
Polazni materijali (piridil disulfidi) proizvedeni su općim postupkom A u skladu s opisom iz literature (Lee, S. et al., Bull. Chem. Soc. Jpn. 64 (1991) 2019-2021). 10 mmolova odgovarajućeg merkaptana i 10 mmolova 2,2 ́-ditio-piridina otopi se u 30 ml apsolutnog diklormetana i miješa se 6 do 24 sata pri sobnoj temperaturi. Otapalo se odstrani u vakuumu i k ostatku se doda dietil eter i profiltrira se. Filtrat se ispari u vakuumu i očisti kromatografijom na silika gelu upotrebom etil acetat/heptana. Starting materials (pyridyl disulfides) were prepared by general procedure A according to the literature description (Lee, S. et al., Bull. Chem. Soc. Jpn. 64 (1991) 2019-2021). 10 mmol of the corresponding mercaptan and 10 mmol of 2,2 ́-dithio-pyridine are dissolved in 30 ml of absolute dichloromethane and stirred for 6 to 24 hours at room temperature. The solvent was removed in vacuo and diethyl ether was added to the residue and filtered. The filtrate was evaporated in vacuo and purified by silica gel chromatography using ethyl acetate/heptane.
Dobiveni su slijedeći spojevi: The following compounds were obtained:
2-decildisulfanil piridin (bezbojno ulje; iskorištenje 76%), 2-decyldisulfanyl pyridine (colorless oil; yield 76%),
2-heksadecildisulfanil piridin (bezbojni kristali; iskorištenje 76%), 2-hexadecylsulfanyl pyridine (colorless crystals; yield 76%),
2-{17-(1,5-dimetil-heksil)-10,13-dimetil-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradekahidro-1H-ciklopenta[a]-enantren-3-il-disulfanil)-piridin} (bezbojni kristali; iskorištenje 56%). 2-{17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro -1H-cyclopenta[a]-enanthren-3-yl-disulfanyl)-pyridine} (colorless crystals; yield 56%).
b) Pripravlajnje palmitoiliranog rekombinantnog humanog shh upotrebom palmitoil imidazolida b) Preparation of palmitoylated recombinant human shh using palmitoyl imidazolide
Kao edukt upotrijebljen je dimerni nemodificirani rekombinantni humani shh u skladu s primjerom 2. Protein je bio u PBS puferu (10 mM pufera natrijevog fosfata, 150 mM NaCl, pH 7,4) pri koncentraciji i od 3 mg/ml. U 2 ml tog uzorka dodan je detergent, prednosno 200 μl 10%-tne otopine Zwittergenta 3-14. Zatim je dodan tri (2-karboksietil)fosfin (TCEP) do koncentracije od 2mM. Nakon 15 minuta inkubacije pri sobnoj temperaturi dodano je 50 μl 60 mM otopine palmitoil imidazolida (1-imidazol-l-il-heksadekan-l-on) u dioksanu. Otopina je inkubirana 1-3 dana prednosno pri 4°C uz mućkanje. Dimeric unmodified recombinant human shh according to example 2 was used as educt. The protein was in PBS buffer (10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.4) at a concentration of 3 mg/ml. A detergent, preferably 200 μl of a 10% solution of Zwittergent 3-14, was added to 2 ml of that sample. Tri(2-carboxyethyl)phosphine (TCEP) was then added to a concentration of 2mM. After 15 minutes of incubation at room temperature, 50 μl of a 60 mM solution of palmitoyl imidazolide (1-imidazol-1-yl-hexadecan-1-one) in dioxane was added. The solution was incubated for 1-3 days preferably at 4°C with shaking.
Pod tim uvjetima nastao je dipalmitoilirani shh kao glavni proizvod (70 - 75%), koji je identificiran kao uzorak 6 pomoću RP-HPLC i masenom spektrometrijom. shh modificiran na taj način pokazao je mnogo višu biološku aktivnost nego nemodificirani shh u pokusu ispitivanja aktivnosti opisanom u primjeru 7. Polu-maksimalna aktivnost dobivena je pri 70 ng/ml. Under these conditions, dipalmitoylated shh was formed as the main product (70 - 75%), which was identified as sample 6 by RP-HPLC and mass spectrometry. shh modified in this way showed much higher biological activity than unmodified shh in the activity assay experiment described in Example 7. Half-maximal activity was obtained at 70 ng/ml.
Tip upotrijebljenog detergenta ima glavni utjecaj na dobitak dipalmitoiliranog shh i razinu biološke aktivnosti(tablica 1). The type of detergent used has the main influence on the gain of dipalmitoylated shh and the level of biological activity (table 1).
Tablica1 Utjecaj upotrijebljenog detergenta na udio dipalmitoiliranog shh u reakcijskoj smjesi (provedeno pri sobnoj temperaturi). Metoda za brojčano utvrđivanje data je u primjeru 6. Table 1 Effect of the used detergent on the proportion of dipalmitoylated shh in the reaction mixture (carried out at room temperature). The method for numerical determination is given in example 6.
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c) Pripravljanje alkiliranog rekombinantnog humanog shh upotrebom 2-heksadecildisulfanilpiridina c) Preparation of alkylated recombinant human shh using 2-hexadecyldisulfanylpyridine
Kao edukt je upotrijebljen dimerni nemodificirani rekombinantni humani shh u skladu s primjerom 2a. Protein je bio u PBS puferu (pufer 10 mM natrijevog fosfata, 150 mM NaCl, pH 7,4) pri koncentraciji od 3 mg/ml. Da bi se dobio mnomer nakon dodatka 2 mM TCEP-a inkubirano je 15 minuta. Zatim je TCEP odstranjen pomoću stupca PD-10 uravnoteženog u puferu 5 mM kalijevog fosfata, 150 mM NaCl, pH 5,0. Alikvoti od 200 μl shh, proizvedenog na taj način, upotrijebljeni su za provedbu reakcije s 2-heksadecil-disulfanil-piridinom primjenom dviju različitih metoda. Dimeric unmodified recombinant human shh according to example 2a was used as educt. The protein was in PBS buffer (10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.4) at a concentration of 3 mg/ml. In order to obtain the minomer after the addition of 2 mM TCEP, it was incubated for 15 minutes. TCEP was then eluted using a PD-10 column equilibrated in a buffer of 5 mM potassium phosphate, 150 mM NaCl, pH 5.0. Aliquots of 200 μl shh, produced in this way, were used to carry out the reaction with 2-hexadecyl-disulfanyl-pyridine using two different methods.
A) K shh monomeru doda se 20 μl 10%-tne otopine Zwittergenta 3-14; 20 μl 0,5 M HEPES-a, pH 7,0, i jednostruki do dvadeseterostruki molarni suvišak reagenta za povezivanje (otopljen u dioksanu, 60 mM) . A) 20 μl of 10% solution of Zwittergent 3-14 is added to shh monomer; 20 μl of 0.5 M HEPES, pH 7.0, and a one- to twenty-fold molar excess of coupling reagent (dissolved in dioxane, 60 mM).
B) K shh monomeru doda se 2 μl 10%-tnog natrijevog deoksiholata, 20 μl pufera 1M kalijevog fosfata, pH 7,6, i dvostruki do dvadeseterostruki molarni suvišak reagenta za povezivanje (otopljenog u metanolu, 60 mM). B) To the shh monomer is added 2 μl of 10% sodium deoxycholate, 20 μl of 1M potassium phosphate buffer, pH 7.6, and a 2- to 20-fold molar excess of coupling reagent (dissolved in methanol, 60 mM).
U obje inačice reakcija je bila gotova za 15 minuta. In both versions, the reaction was complete in 15 minutes.
Deseterostruki molarni suvišak reagenta za povezivanje u inačici A) imao je za posljedicu udio od 43%, a u inačici B) udio od 47% mono-alkiliranog shh u reakcijskoj smjesi. U inačici A biološka aktivnost bila je jako povišena u usporedbi s nemodificiranim shh, a polu-maksimalna aktivnost bila je 200 ng/ml shh. Jednostruki molarni suvišak 2-heksadecildisulfanilpiridina dao je samo malo niža iskorištenja. Inačica B također je dovela do povišenja aktivnosti u usporedbi s nemodificiranim shh, ali i do manjeg dometa: s jednakom količinom proteina postignuto je samo 30% biološke aktivnosti. Stoga se prednost daje inačici A. A tenfold molar excess of the coupling reagent in version A) resulted in a proportion of 43%, and in version B) a proportion of 47% of mono-alkylated shh in the reaction mixture. In variant A, the biological activity was greatly increased compared to unmodified shh, and the half-maximal activity was 200 ng/ml shh. A one-fold molar excess of 2-hexadecylsulfanylpyridine gave only slightly lower yields. Version B also led to an increase in activity compared to unmodified shh, but also to a smaller range: with the same amount of protein, only 30% biological activity was achieved. Therefore, preference is given to version A.
d) Čišćenje alkiliranih shh derivata d) Purification of alkylated shh derivatives
Proizvod povezivanja razrijeđen je 1:4 u 50 mM HEPES-u, 0,1% Tweena® 80, pH 7,4 i zatim je ispran pomoću SP-Sepharose HP (Pharmacia). The ligation product was diluted 1:4 in 50 mM HEPES, 0.1% Tween® 80, pH 7.4 and then washed with SP-Sepharose HP (Pharmacia).
Ravnotežni pufer: 50 mM HEPES, 0,2% Tweena® 80, pH 7,4; pufer za ispiranje: 50 mM HEPES, 0,2% Tweena® 80, pH 7,4 koji je sadržavao 0,8 M NaCl gradijent za ispiranje (volumen stupca 2 x 6) i diskontinuirano ispiranje. Equilibration buffer: 50 mM HEPES, 0.2% Tweena® 80, pH 7.4; elution buffer: 50 mM HEPES, 0.2% Tweena® 80, pH 7.4 containing a 0.8 M NaCl elution gradient (column volume 2 x 6) and discontinuous elution.
U slučaju dipalmitoiliranog shh bilo je moguće odvojiti suvišak palmitoil-imidazolida i djelomično odvojiti nemodificirani shh. U slučaju modifikacije s 2-heksadeksildisulfanilpiridinom dobiveno je značajno obogaćenje monopalmitiliranog shh od 75%. Modificirani shh mogao se je, na primjer, dalje očistiti pomoću heparin Sepharose. In the case of dipalmitoylated shh, it was possible to separate excess palmitoyl-imidazolide and partially separate unmodified shh. In the case of modification with 2-hexadecyldisulfanylpyridine, a significant enrichment of monopalmitylated shh of 75% was obtained. The modified shh could, for example, be further purified using heparin Sepharose.
Primjer 4 Example 4
a) Pripravljanje palmitoiliranog rekombinantnog humanog shh upotrebom plamitoil-CoA: a) Preparation of palmitoylated recombinant human shh using palmitoyl-CoA:
2 ml očišćenog dimernog shh uzorka s koncentracijom shh od 0,35 mg/ml pomiješano je s DTE na krajnju koncentraciju od 20 mM, inkubirano je 2 sata pri 37°C i zatim je dijalozirano nasuprot 2 ml of purified dimeric shh sample with shh concentration of 0.35 mg/ml was mixed with DTE to a final concentration of 20 mM, incubated for 2 hours at 37°C and then dialyzed against
a) 50 mM Tris/HCl, 1 mM DTE, 0,1 mM ZnCl;,, 0,5% Triton® X-100, pH 8,5, a) 50 mM Tris/HCl, 1 mM DTE, 0.1 mM ZnCl;,, 0.5% Triton® X-100, pH 8.5,
b) 100 mM MOPS, 1 mM DTE, 0,1% Triton® X-100, 0,1 mg/ml suramin, pH 7,4, b) 100 mM MOPS, 1 mM DTE, 0.1% Triton® X-100, 0.1 mg/ml suramin, pH 7.4,
c) 100 mM MOPS, 1 mM DTE, 0,1% Triton® X-100, pH 7,4. c) 100 mM MOPS, 1 mM DTE, 0.1% Triton® X-100, pH 7.4.
Zatim su u alikvote od 0,5 ml uzorka dodani različiti volumeni otopine palmitoil-CoA (10 mg/ml u 100 mM MOPS/ 1 Different volumes of palmitoyl-CoA solution (10 mg/ml in 100 mM MOPS/ 1
mM DTT, 0,2% Triton®X-100, pH 7,6), čime su u mješavini dobivene koncentracije palmitoil-CoA od mM DTT, 0.2% Triton®X-100, pH 7.6), which resulted in palmitoyl-CoA concentrations of
1) 0 μM, 1) 0 μM,
2) 5 0 μM, 2) 50 μM,
3) 500 μm, 3) 500 μm,
nakon čega su inkubirani 1 sat pri 37°C. Za stanično ispitivanje, prije filtracije i razrjeđivanja 1/200, uzorci su pomiješani s BSA (krajnja koncentracija 1 mg/ml) i suraminom (krajnja koncentracija 0,1 mg/ml). S gornjim uzorcima ispitivanje aktivnosti, kako je opisano u primjeru 7 za shh, pokazalo je da uzorci shh inkubirani u puferima b) i c) , koji sadrže 500 μM palmitoil-CoA, pokazuju značajno povišenje biološke aktivnosti (usporedi sliku 1) u usporedbi s mješavinama bez palmitoil-CoA. Na taj način palmitoilirani shh može se očistiti metodama koje su bile opisane za membranske proteine, npr. u ``A practical Guide to Membrane Protein Purification" (1994; izd. Jagow & Schlager; Academic Press) ili u Europskoj patentnoj prijavi br. 98 102 095.1. after which they were incubated for 1 hour at 37°C. For cellular assay, samples were mixed with BSA (final concentration 1 mg/ml) and suramin (final concentration 0.1 mg/ml) before filtration and 1/200 dilution. With the above samples, the activity test, as described in example 7 for shh, showed that shh samples incubated in buffers b) and c), containing 500 μM palmitoyl-CoA, show a significant increase in biological activity (compare Figure 1) compared to mixtures without palmitoyl-CoA. In this way, palmitoylated shh can be purified by methods that have been described for membrane proteins, for example in ``A practical Guide to Membrane Protein Purification'' (1994; ed. Jagow &Schlager; Academic Press) or in European Patent Application No. 98 102 095.1.
b) Ovisnost acilacije o shh koncentraciji i molarnom omjeru između shh i palmitoil-CoA (Pal-CoA) b) Dependence of acylation on shh concentration and molar ratio between shh and palmitoyl-CoA (Pal-CoA)
Dimerni shh (u PBS-u) razrijeđen je s PBS-om na c = 150 μM ili na 37,5 μM i dodan je Tween® 80 (krajnja koncentracija 0,05%), DTE (3 mM krajnja koncentracija) i TCEP (krajnja koncentracija 1mM). Zatim su dodane utvrdene količine Pal-CoA (20 mM u vodi) i smjesa je inkubirana preko noći pri 25°C. Uzorci su analizirani pomoću RP-HPLC ili različitih razrjeđenja u staničnom ispitivanju nakon razrjeđivanja u PBS-u, 1 mg/ml BSA, 0,05% Tveen® 80 pH 7,3 i sterilne filtracije. Rezultati su zbirno prikazani u tablici 3: Dimeric shh (in PBS) was diluted with PBS to c = 150 μM or to 37.5 μM and Tween® 80 (final concentration 0.05%), DTE (3 mM final concentration) and TCEP ( final concentration 1mM). Determined amounts of Pal-CoA (20 mM in water) were then added and the mixture was incubated overnight at 25°C. Samples were analyzed by RP-HPLC or different dilutions in the cell assay after dilution in PBS, 1 mg/ml BSA, 0.05% Tveen® 80 pH 7.3 and sterile filtration. The results are summarized in table 3:
Tablica 3 Table 3
[image] [image]
*EC50 je koncentracija shh koja se mora upotrijebiti da bi se u staničnom ispitivanju postiglo poli-maksimalnu indukciju alkalne fosfataze. Zbog razlika u broju prolaza stanica, i stoga promjena u staničnoj fiziologiji, ova vrijednost može biti malo različita između pokusa provedenih u različita vremena. ;Umjereni suvišak reagenta za povezivanje (< od pedeseterostrukog) uglavnom dovodi do tvorbe shh derivata dvostruko aciliranog na N-terminalnom cisteinu. S povećanjem omjera Pal-CoA prema shh, u povišenom opsegu nastao je shh koji je bio palmitiliran više nego dvostruko. Palmitiliranje s više od dva ostatka masne kiseline takoder povisuje aktivnost. S koncentracijom shh od 37,5 μM, što se tiče aktivnosti optimlan je pribl. deseterostruki suvišak Pal-CoA. Pri shh koncentracij i od 150 p,M trostruki suviša Pal-CoA već dosiže visoko iskorištenje dvostruko palmitiliranog shh i visoku aktivnost. ;c) Ovisnost aciliranja o tipu i koncentraciji detergenta. ;Dimerni shh u PBS-u pri pH 7,4 bio je namješten na koncentraciju od 0,75 mg/ml i pomiješan u utvrđenim količinama dotičnih detergenata, te u svakom slučaju s 500 ;pM Pal-CoA i 3 mM DTE-a ili 3 mM TCEP-a (krajnja koncentracija). Smjese za povezivanje inkubirane su preko noći pri 25°C i zatim su analizirane pomoću RP-HPLC ili staničnim pokusom upotrebom različitih razređenja, nakon razređenja u PBS-u, 1 mg/ml BSA, 0,05% Tween® 80, pH 7,3 i nakon sterilne filtracije. ;Rezultati su zbirno prikazani u tablici 4: ;Tablica 4 ;[image] ;pal. = palmitilirano ;*Reduciran s 3 mM TCEP-om umjesto s 3 mM DTE-om. *EC50 is the concentration of shh that must be used to achieve poly-maximal induction of alkaline phosphatase in a cellular assay. Due to differences in the number of cell passages, and therefore changes in cell physiology, this value may be slightly different between experiments performed at different times. ;A moderate excess of coupling reagent (< 50-fold) generally leads to the formation of shh derivatives doubly acylated at the N-terminal cysteine. As the ratio of Pal-CoA to shh increased, an increased amount of shh was produced that was more than twofold palmitylated. Palmytylation with more than two fatty acid residues also increases activity. With a shh concentration of 37.5 μM, in terms of activity the optimum is approx. a tenfold excess of Pal-CoA. At shh concentrations of even 150 p.M, triple excess Pal-CoA already reaches high utilization of doubly palmitylated shh and high activity. ;c) Dependence of acylation on the type and concentration of detergent. ;Dimeric shh in PBS at pH 7.4 was adjusted to a concentration of 0.75 mg/ml and mixed in determined amounts of respective detergents, and in each case with 500 ;pM Pal-CoA and 3 mM DTE or 3 mM TCEP (final concentration). Ligation mixtures were incubated overnight at 25°C and then analyzed by RP-HPLC or cell assay using different dilutions, after dilution in PBS, 1 mg/ml BSA, 0.05% Tween® 80, pH 7, 3 and after sterile filtration. ;The results are summarized in table 4: ;Table 4 ;[image] ;pal. = palmitylated ;*Reduced with 3 mM TCEP instead of 3 mM DTE.
Pokazalo se je da se upotrebom Tweena®80 ili 0,5%-tnog oktil glikozida mogu dobiti visoki omjeri palmitiliranja; također je i palmitiliranje u 0,05%-tnom Zwittergentu dovelo do visokog povećanja aktivnosti. Istovremena redukcija s TCEP-om dovela je do sličnih rezultata kao redukcija s DTE-om. It has been shown that using Tween®80 or 0.5% octyl glycoside can obtain high palmitylation ratios; also palmitylation in 0.05% Zwittergent led to a high increase in activity. Simultaneous reduction with TCEP led to similar results as reduction with DTE.
d) Aciliranje reduciranog, monomernog shh u odnosu prema pH vrijednosti smjese za povezivanje d) Acylation of reduced, monomeric shh in relation to the pH value of the coupling mixture
Dimerni shh (c = 0,7 mg/ml) reduciran je s 10 mM DTE i Dimeric shh (c = 0.7 mg/ml) was reduced by 10 mM DTE i
dijaliziran preko noći nasuprot PBS-u, 0,05 %-tnom Tweenu® 80, 0,5 mM DTE-u. Dijalizat je namješten na krajnju koncentraciju c = 0,25 mg/ml i na dotičnu pH vrijednost s PBS-om, 0,05%-tnim Tweenom® 80, 0,5 mM DTE i dodano je 125 dialyzed overnight against PBS, 0.05% Tween® 80, 0.5 mM DTE. The dialysate was adjusted to a final concentration of c = 0.25 mg/ml and to the respective pH value with PBS, 0.05% Tween® 80, 0.5 mM DTE and 125
μl Pal-CoA. Smjesa je inkubirana 2 sata pri 37°C i zatim je analizirana u razređenju od 1:500 u staničnom pokusu. Aktivnost uzoraka zbirno je prikazana u tablici 5 kao stimulacija AP aktivnosti u odnosu prema bazalnoj aktivnosti nestimuliranog C3H10T1/2 stanica (= 100%): μl Pal-CoA. The mixture was incubated for 2 hours at 37°C and then assayed at a 1:500 dilution in a cell assay. The activity of the samples is summarized in table 5 as the stimulation of AP activity in relation to the basal activity of unstimulated C3H10T1/2 cells (= 100%):
Tablica 5 Table 5
[image] [image]
Maksimalna aktivacija događa se prema tome pri neutralnoj do slabo lužnatoj pH vrijednosti. Maximum activation therefore occurs at a neutral to slightly alkaline pH value.
e) Povezivanje acil-CoA derivata koji sadrže acilne skupine koje imaju različite duljine ugljikovog lanca Dimerni shh namješten je na koncentraciju od 0,75 mg/ml u PBS-u, 0,05% Tweenu® 80, pH 7,4, 3 mM DTE i dodano je 0,5 mM dotičnog acil-CoA derivata i inkubirano preko noći pri 25°C. Uzorci su analizirani pomoću RP-HPLC ili primjenom staničnog ispitivanja pri različitim razređenjima, nakon razređenja u PBS-u, 1 mg/ml BSA, 0,05%-tnom Tweenu® 80 pH 7,3 i nakon sterilne filtracije. Pomak u zadržavanju na RP-HPLC kao i dobitak diaciliranog shh i aktivnost u staničnom ispitivanju zbirno su prikazani u tablici 6. e) Linking of acyl-CoA derivatives containing acyl groups having different carbon chain lengths Dimeric shh was adjusted to a concentration of 0.75 mg/ml in PBS, 0.05% Tween® 80, pH 7.4, 3 mM DTE and 0.5 mM of the respective acyl-CoA derivative was added and incubated overnight at 25°C. Samples were analyzed by RP-HPLC or by cell assay at different dilutions, after dilution in PBS, 1 mg/ml BSA, 0.05% Tween® 80 pH 7.3 and after sterile filtration. The shift in retention on RP-HPLC as well as the gain of diacylated shh and activity in the cellular assay are summarized in Table 6.
Tablica 6. Table 6.
[image] [image]
To pokazuje da se acilne skupine s različitim duljinama lanca mogu učinkovito prenijeti primjenom metode u kojoj se upotrebljavaju derivati Pal-CoA. Koncentracija detergenta, kao i tip upotrijebljenog detergenta zahtjeva fino optimiranje ovisno o duljini lanca prenesenog acilnog ostatka. Smanjenje duljine lanca acilnog ostatka ili smanjenje broja nezasićenih ugljikovih veza dovodi do smanjenja specifične aktivnosti aciliranih shh derivata. This shows that acyl groups with different chain lengths can be efficiently transferred using a method using Pal-CoA derivatives. The concentration of the detergent, as well as the type of detergent used, requires fine optimization depending on the chain length of the transferred acyl residue. A decrease in the chain length of the acyl residue or a decrease in the number of unsaturated carbon bonds leads to a decrease in the specific activity of acylated shh derivatives.
Primjer 5 Example 5
a) Derivatizacija rekombinantnog humanog shh s tiokolesterolom a) Derivatization of recombinant human shh with thiocholesterol
Da bi se shh modificirao pomoću tiokolesterola povezanog na aminoterminalni cistein preko disulfidnog mosta, reducirani monomerni shh pri koncentraciji od 0,66 mg/ml u 0,5 mM DTT oH 7,0 razrijeđen je u omjeru 1:3 u slijedećem reakcijskom puferu: To modify shh with thiocholesterol linked to the amino-terminal cysteine via a disulfide bridge, reduced monomeric shh at a concentration of 0.66 mg/ml in 0.5 mM DTT oH 7.0 was diluted 1:3 in the following reaction buffer:
100 mM etanolamina 100 mM ethanolamine
100 mM NaCl 100 mM NaCl
50 μM CuCl2 50 μM CuCl2
pH 9,5 pH 9.5
0,8 %-tni (masa/volumen) natrijev deoksiholat ili 0,4%-tni (masa/volumen) natrijev holat ili l,3%-tni (masa/volumen) n-oktil glikozid ili 0,3%-tni Triton®X-100. 0.8% (w/v) sodium deoxycholate or 0.4% (w/v) sodium cholate or 1.3% (w/v) n-octyl glycoside or 0.3% Triton®X-100.
Reakcija je započeta dodatkom (krajnje koncentracije): 5% (v/v) acetona ili 100 μM tiokolesterola (iz 10 mM otopine u acetonu) ili 500 μM tiokolesterola (iz 10 mM otopine u acetonu) i smjesa je mućkana 30 minuta pri sobnoj temperaturi. The reaction was started by the addition of (final concentration): 5% (v/v) acetone or 100 μM thiocholesterol (from a 10 mM solution in acetone) or 500 μM thiocholesterol (from a 10 mM solution in acetone) and the mixture was shaken for 30 minutes at room temperature .
Neposredno prije sterilne filtracije uzorci su razrijedeni s devet volumena 20 nM natrijevog fosfata, 0,9% NaCl, 0,05% Tveena® 80, 1 mg/ml suramina, pH 7,2 i analizirani su u staničnom pokusu u dodatnom razređenju 1/20. Immediately before sterile filtration, the samples were diluted with nine volumes of 20 nM sodium phosphate, 0.9% NaCl, 0.05% Tveena® 80, 1 mg/ml suramin, pH 7.2 and were analyzed in a cell experiment in an additional dilution of 1/ 20.
Kako je prikazano na slici 2, povećanje aktivnosti koje ovisi o koncentraciji tiokoleserola, koja je bila najučinkovitija, dobivena je ili stabilizirana u uzorcima koji su sadržavali anionske detergente. As shown in Figure 2, an increase in activity dependent on the concentration of thiocholesrol, which was the most effective, was obtained or stabilized in samples containing anionic detergents.
b) Derivatizacija rekombinantnog humanog shh s tiokolesterol piridil disulfodom b) Derivatization of recombinant human shh with thiocholesterol pyridyl disulfide
Da bi se shh modificirao s tiokolesterolom povezanim na aminoterminalni cistein preko disulfodnog mosta, shh dimer koji je bio kovalentno povezan između N-terminalnih cisteina preko disulfidne veze reduciran je pomoću 2 mN TCEP-a tijekom 15 minuta pri sobnoj temperaturi i zatim je ponovno puferiran da se odvoji redukcijsko sredstvo pomoću stupca PDIO (Pharmacia). Za povezivanje koncentracija shh namještena je na c = 1 mg/ml s PBS-om, pH 5. pH je bio namješten na neutralno do slabo lužnate pH vrijednosti (npr. pH 7,6) dodatkom koncentrirane otopine pufera (npr. 40 volumnih postotaka 0,4 M Na fosfata, pH 9,2) i neposredno zatim najprije je dodan detergent i zatim je dodan reagent za povezivanje (5 mM tiokoiesterol piridil disulfid) otopljen u organskom otapalu (npr. u metanolu, 40°C). Reakcijska smjesa je inkubirana nekoliko sati pri sobnoj temperaturi i zatim je analizirana pomoću HPLC i masenom spektrometrijom ili su različita razređenja bila upotrijebljena u staničnom pokusu nakon razređenja u PBS-u, 1 mg/ml PBS-a, 1 mg/ml BSA, 0,05% Tween® 80 pH 7,3 i nakon sterilne filtracije. To modify shh with thiocholesterol linked to the amino-terminal cysteine via a disulfide bridge, the shh dimer covalently linked between the N-terminal cysteines via a disulfide bond was reduced with 2 mN TCEP for 15 min at room temperature and then rebuffered to the reducing agent is separated using a PDIO column (Pharmacia). For binding, the concentration of shh was adjusted to c = 1 mg/ml with PBS, pH 5. The pH was adjusted to a neutral to slightly alkaline pH value (e.g. pH 7.6) by the addition of a concentrated buffer solution (e.g. 40% by volume 0.4 M Na phosphate, pH 9.2) and immediately afterwards detergent was first added and then a coupling reagent (5 mM thiocoesterol pyridyl disulfide) dissolved in an organic solvent (eg in methanol, 40°C) was added. The reaction mixture was incubated for several hours at room temperature and then analyzed by HPLC and mass spectrometry or different dilutions were used in the cell experiment after dilution in PBS, 1 mg/ml PBS, 1 mg/ml BSA, 0, 05% Tween® 80 pH 7.3 and after sterile filtration.
Rezultati za neke uzorke smjesa za povezivanje zbirno su prikazani u tablici 7. The results for some samples of bonding compounds are summarized in Table 7.
Tablica 7 Table 7
[image] [image]
Kako se vidi u tablici 7, pod prikladnim uvjetima i s iskorištenjem većim od 50% može se proizvesti disulfidno povezan tiokolesterolni derivat shh koji u staničnom pokusu ima najmanje 10% specifične aktivnosti dipalmitiliranog shh. As can be seen in Table 7, under appropriate conditions and with a yield greater than 50%, a disulfide-linked thiocholesterol derivative of shh can be produced, which in a cellular experiment has at least 10% of the specific activity of dipalmitylated shh.
Primjer 6 Example 6
Karakterizacija pripravka povezivanja shh s tiokoiesterol piridil disulfidom pomoću RP-HPLC i masenom spektrometrijom Characterization of shh binding preparation with thiocoesterol pyridyl disulfide by RP-HPLC and mass spectrometry
100 μl pripravka povezivanja dijaliziranog nasuprot PBS-u, 0,05%-tnom Tweenu 80, koji sadrži 0,7 mg/ml shh, 1% natrijevog holata, 250 μM tiokolesterol piridil disulfida, pH 7,6 stavljeno je na butilni stupac 1 x 150 mm (VydacTM 214TP5115) koji je bio uravnotežen u 18%-tnom acetonitrilu, 0,1% trifluoroctenoj kiselini (TFA). Razrijeđen je pri 25°C u gradijentu 18-19% acetonitrila u 0,1% TFA. Eluat je podijeljen i analiziran s jedne strane kontinuiranom detektcijom apsorbancije pri 220 nm i s druge strane kontinuiranom elektrosprejnom masenom spetrometrijom (API100, Sciex; namještani parametri: polazna masa (m/z) 900 jedinica atomske mase (amu), zaustavna masa (m/z) 1500 amu, stupanj 0,3 amu, vrijeme zadržavanja 0,5 ms, ulazni napon 30 V) . Reducirani monomerni shh i ponovno oksidirani dimerni shh s masom od 19560 Da i 39120 Da isprani su pri 18 min (42-44% acetonitrii) , disulfidno umreženi shh, koji sadrži tiokolesterol, s masom od 19962,7 Da ispran je pri 24,5 min (48,5% acetonitril). 100 μl of the binding preparation dialyzed against PBS, 0.05% Tween 80, containing 0.7 mg/ml shh, 1% sodium cholate, 250 μM thiocholesterol pyridyl disulfide, pH 7.6 was loaded onto a butyl column 1 x 150 mm (VydacTM 214TP5115) which was equilibrated in 18% acetonitrile, 0.1% trifluoroacetic acid (TFA). It was diluted at 25°C in a gradient of 18-19% acetonitrile in 0.1% TFA. The eluate was divided and analyzed on the one hand by continuous absorbance detection at 220 nm and on the other hand by continuous electrospray mass spectrometry (API100, Sciex; set parameters: starting mass (m/z) 900 atomic mass units (amu), stop mass (m/z ) 1500 amu, step 0.3 amu, dwell time 0.5 ms, input voltage 30 V). Reduced monomeric shh and re-oxidized dimeric shh with a mass of 19560 Da and 39120 Da were eluted at 18 min (42-44% acetonitrile), disulfide cross-linked shh, containing thiocholesterol, with a mass of 19962.7 Da was eluted at 24.5 min (48.5% acetonitrile).
Primjer 7 Example 7
Indukcija alkalne fosfataze u staničnom pokusu (određivanje aktivnosti alkalne fosfataze) Induction of alkaline phosphatase in a cell experiment (determination of alkaline phosphatase activity)
5000 stanica mišje mezenhimalne pluripotentne linije C3H10T1/2 (ATCC CCl-226) zasijano je u svaku jamicu 5000 cells of mouse mesenchymal pluripotent line C3H10T1/2 (ATCC CCl-226) were seeded in each well
mikrotitarske ploče s 96 jamica. Stanice su bile u 100 μl DMEM-a, 2 mM glutamina, 100 IU/ml penicilina, 100 μg/ml streptomicina i 10% fetalnog telećeg seruma, FCS. Slijedećeg dana dodane su ispitne aktivne tvari u prikladnim koncentracijama u volumenu od 100 μl, nakon razređvanja u mediju za kulturu. Ispitivanje je zaustavljeno nakon 5 dana. U tu svrhu supernatant je odbačen i stanice su isprane jednom s PBS-om. Stanice su lizirale u 50 μl 0,1% Tritona® X-100 i zamrznute pri -20°C. Nakon otapanja 25 μl je upotrijebljeno za određivanje proteina, a 25 μl je upotrijebljeno za određivanje aktivnosti alkalne fosfataze. 96-well microtiter plates. Cells were in 100 μl DMEM, 2 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum, FCS. The following day, the test active substances were added in suitable concentrations in a volume of 100 μl, after dilution in the culture medium. The trial was stopped after 5 days. For this purpose, the supernatant was discarded and the cells were washed once with PBS. Cells were lysed in 50 μl of 0.1% Triton® X-100 and frozen at -20°C. After dissolution, 25 μl was used for protein determination, and 25 μl was used for alkaline phosphatase activity determination.
Određivanje proteina prema uputama proizvodača Pierce: Determination of protein according to the instructions of the manufacturer Pierce:
75 μl redestilirane vode doda se k mješavini, zatim se doda 100 μl BCA proteinskog reagenta (Pierce Micro BCA, br. 23225). Nakon 60 minuta izmjeri se optičku gustoću (OD, e. optical density) pri 550 nm. 75 μl of redistilled water is added to the mixture, then 100 μl of BCA protein reagent (Pierce Micro BCA, no. 23225) is added. After 60 minutes, the optical density (OD, e. optical density) at 550 nm is measured.
Aktivnost alkalne fosfataze prema uputama proizvodača Sigma: Alkaline phosphatase activity according to Sigma manufacturer's instructions:
100 μl reakcijskog pufera (Sigma 221) doda se k pripravku. Kapsulu supstrata (Sigma 104-40) otopi se u 10 ml redestilirane vode i zatim se s pipetom u mješavinu doda 100 μl . OD se izmjeri pri 405 nm nakon žutog obojenja. U reakciji alkalna fosfataza pretvara p-nitrofenil fosfat u p-nitrofenol. 100 μl of reaction buffer (Sigma 221) is added to the preparation. The substrate capsule (Sigma 104-40) is dissolved in 10 ml of redistilled water and then 100 μl is added to the mixture with a pipette. OD is measured at 405 nm after yellow staining. In the reaction, alkaline phosphatase converts p-nitrophenyl phosphate into p-nitrophenol.
Svaka optička gustoća bila je preračunata pomoću standardnih krivulja u nmol ili u μg. Ocjena aktivnosti data je u skladu s formulom: Each optical density was converted using standard curves in nmol or in μg. The activity rating is given in accordance with the formula:
nmol PNP po (izmjerenoj) minuti po mg (staničnog) proteina. nmol PNP per (measured) minute per mg (cellular) protein.
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