Production Of Polyketides And Other Natural Products

• US 20090253732A1
• Filed: 08/11/2005
• Published: 10/08/2009
• Est. Priority Date: 08/11/2004
• Status: Active Grant

• Current Assignee: Biotica Technology Limited (Abliva AB), Wyeth (Pfizer Inc.)
• Original Assignee: Biotica Technology Limited (Abliva AB)
• Inventors: Martin, Christine Janet, Gregory, Matthew Alan
• Granted Patent: US 7,803,808 B2
• Time in Patent Office: Days
• US Class Current: 514/291
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 17/08 Antiseborrheics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/08 : Vasodilators for multiple indications
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/14 : Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07D 498/18 : Bridged systems

Abstract

The present invention relates to production of polyketides and other natural products and to libraries of compounds and individual novel compounds. Therefore in aspect the present invention provides 17-desmethylrapamycin and analogues thereof, methods for their production, including recombinant strains, and isolation and uses of the compounds of the invention. In a further aspect the present invention provides for the use of 17-desmethylrapamycin and analogues thereof in the induction or maintenance of immunosuppression, the stimulation of neuronal regeneration or the treatment of cancer, B-cell malignancies, fungal infections, transplantation rejection, graft vs. host disease, autoimmune disorders, diseases of inflammation vascular disease and fibrotic diseases, and in the regulation of wound healing.

Claims

1. 17-desmethylrapamycin and analogues thereof.

2. A compound according to claim 1 which has the following formula:

3. A compound according to claim 2, wherein R₄ represents a structural fragment selected from the group consisting of A, B, C, E and F, and wherein R₇ represents H, OH or OMe and R₈ represents H, OH, OMe, Cl or F.

4. A compound according to claim 3, wherein R₄ represents a structural fragment selected from the group consisting of A(i), C(i), E(i) and F(i), wherein A(i), C(i), E(i) and F(i) have the following structures:

5. A compound according to claim 2, wherein when R₅ represents OH, R₆ represents H and when R₆ represents OH, R₅ represents H.

6. A compound according to claim 2, wherein x represents a direct bond or CH₂.

7. A compound according to claim 2, wherein x represents CH₂.

8. A compound according to claim 2, wherein R₂ represents OH.

9. A compound according to claim 4 wherein R₁ represents (H, H), R₂ represents OH, R₃ represents H, R₄ represents the structural fragment C(i), R₇ and R₈ both represent OH, and x represents CH₂.

10. A compound according to claim 4 wherein R₁ represents (H, H), R₂ represents OH, R³ represents H, R₄ represents the structural fragment C(i), R₇ represents OH, R₈ represents H, and x represents CH₂.

11. A compound according to claim 4 wherein R₁ represents (H, H) or ═

O, R₂ represents OH or OMe, R₃ represents OH or OMe, R₄ represents the structural fragment C(i), R₇ represents OH, R₈ represents H, OH or OMe, and x represents CH₂.

12. A compound according to claim 4 wherein R₁ represents ═

O, R₂ represents OH or OMe, R₃ represents H, OH or OMe, R₄ represents the structural fragment C(i), R₇ represents OH, R₈ represents F or Cl, and x represents CH₂.

13. A compound according to claim 4 wherein R₁ represents ═

O, R₂ represents OH, R₃ represents OMe, R₄ represents the structural fragment C(i), R₇ represents OH, R₈ represents H, and x represents CH₂.

14. A compound according to claim 4 wherein R₁ represents ═

O, R₂ represents OH, R₃ represents OH, R₄ represents the structural fragment C(i), R₇ represents OH, R₈ represents H, and x represents CH₂.

15. A compound according to claim 4 wherein R₁ represents ═

O, R₂ represents OH, R₃ represents OMe, R₄ represents the structural fragment C(i), R₇ represents OH, R₈ represents OMe, and x represents CH₂.

17. A method for the induction or maintenance of immunosuppression, the stimulation of neuronal regeneration or the treatment of cancer, B-cell malignancies, fungal infections, transplantation rejection, graft vs. host disease, autoimmune disorders, diseases of inflammation or vascular disease in a subject in need thereof, said method comprising administering an effective amount of a compound according to claim 1 to said subject.

18. A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

19. A method for the production of 17-desmethylrapamycin or an analogue thereof according to claim 1 said method comprising:

(a) replacing the methylmalonyl-CoA specific AT domain of module 10 of the rapamycin PKS with malonyl-CoA-specific AT domain,

(b) expressing the engineered rapamycin PKS in a suitable host cell,

(c) culturing the host cell under conditions such that 17-desmethylrapamycin or an analogue thereof is produced including optionally supplying exogenous precursors, and

(d) optionally isolating the compound thus produced.

21. The method according to claim 19, wherein the methylmalonyl-CoA specific AT domain of module 10 is replaced with a malonyl-CoA specific AT domain from one of the following clusters:

rapamycin, monensin, FK506, erythromycin, FK520, amphotericin, angolamycin, tylosin, ‘

hyg’

, FK523, meridamycin, antascomicin, FK525 or tsukubamycin.

22. The method according to claim 21, wherein the malonyl-CoA specific AT domain is selected from one of the following clusters:

rapamycin, monensin or FK506.

23. The method according to claim 22, wherein the AT domain is selected from the group consisting of the AT domain from rapamycin module 2, the AT domain from monensin module 3, the AT domain from monensin module 6, the AT domain from monensin module 8, the AT domain from FK508 module 3 and the AT domain from FK506 module 7.

24. The method according to claim 23, wherein the malonyl-CoA specific AT domain is from module 2 of rapamycin.

25. The method according to claim 19, wherein the method comprises the additional steps of:

(a) isolating the AT to be introduced as a single DNA fragment with suitable flanking restriction sites,

(b) amplifying and isolating the regions of DNA sequence homologous to the flanking sequences of the target AT using appropriate restriction sites,

(c) ligating the three DNA fragments as described in (a) and (b) together to give an in-frame sequence of LHS homology followed by donor AT domain followed by RHS homology,

(d) introducing the complete sequence from (c) into a vector for introduction into the host strain to give the final plasmid, said plasmid comprising;

i) the oriT for conjugation,

ii) one or more resistance markers,

iii) a temperature sensitive origin of replication such that integrants can be selected by culturing at 37°

C., and

iv) an E coli origin of replications

(e) transforming the host strain by conjugation using the final plasmid as described in (d) above,

(f) selecting the transformants by resistance to the relevant antibiotic,

(g) generating the primary integrants by culturing at 37°

C. with antibiotic selection,

(h) screening the secondary recombinants by growth in the absence of antibiotic selection, also at 37°

C.,

(i) identifying the desired strain by its ability to produce the target product and

(j) optionally confirming the genetics of the strain by standard methods.

16. (canceled)

20. A method for generating a recombinant strain that contains a biosynthetic cluster that encodes an engineered rapamycin polyketide synthase where the methylmalonyl-CoA specific AT domain of module 10 is replaced with a malonyl-CoA specific AT domain.

26. A method for making a 17-desmethylrapamycin analogue, said method comprising:

(a) generating a recombinant strain according to claim 20, in which the methylmalonyl-CoA specific AT domain of module 10 of the rapamycin PKS has been replaced with a malonyl-CoA specific AT domain, and

(b) feeding non-natural starter acids to said recombinant strain under conditions suitable for polyketide production.

27. A method for generating a 17-desmethylrapamycin analogue, said method comprising:

(a) generating a recombinant strain according to claim 20, in which the methylmalonyl-CoA specific AT domain of module 10 of the rapamycin PKS has been replaced with a malonyl-CoA specific AT domain,

(b) additionally deleting or inactivating one or more rapamycin auxiliary genes selected from the group consisting of rapI, rapJ, rapK, rapL, rapM, rapN, rapO and rapQ, and

(c) culturing the strain thus obtained, optionally in the presence of an exogenous precursor if required, to produce the 17-desmethylrapamycin analogue.

28. A method for generating a 17-desmethylrapamycin analogue, said method comprising:

(a) generating a recombinant strain according to claim 20, in which the methylmalonyl-CoA specific AT domain of module 10 of the rapamycin PKS has been replaced with a malonyl-CoA specific AT domain,

(b) additionally deleting or inactivating one or more rapamycin auxiliary genes selected from the group consisting of rapI, rapJ, rapK, rapL, rapM, rapN, rapO and rapQ,

(c) re-introducing all or a subset of the rapamycin auxiliary genes in-trans to complement or partially complement the deletion, and

(d) culturing the strain thus obtained, optionally in the presence of an exogenous precursor if required, to produce the 17-desmethylrapamycin analogue.

29. A method for making a 17-desmethylrapamycin analogue, said method comprising:

(a) generating a recombinant strain according to claim 20, in which the methylmalonyl-CoA specific AT domain of module 10 of the rapamycin PKS has been replaced with a malonyl-CoA specific AT domain,

(b) additionally deleting or inactivating one or more rapamycin auxiliary genes selected from the group consisting of rapI rapJ, rapK, rapL, rapM, rapN, rapO and rapQ,

(c) optionally re-introducing all or a subset of the rapamycin auxiliary genes in-trans to complement or partially complement the deletion, and

(d) feeding non-natural starter acids to said recombinant strain under conditions suitable for polyketide production.