Regulation of biological events using novel compounds

• US 6,649,595 B2
• Filed: 02/12/2001
• Issued: 11/18/2003
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Ariad Pharmaceuticals Incorporated (Takeda Pharmaceutical Company Limited)
• Original Assignee: ARIAD Gene Therapeutics, Inc. (Takeda Pharmaceutical Company Limited)
• Inventors: Yang, Wu, Holt, Dennis A., Clackson, Timothy P., Keenan, Terence P., Rozamus, Leonard, Gilman, Michael Z.
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): Lambertson, David A.
• Application Number:
  US09/781,804
• Publication Number:
  US 20020107189A1
• Time in Patent Office: 1,009 Days
• Field of Search: 514/31, 514/44, 435/325, 435/355, 435/372, 435/372.3, 435/375, 536/6.5
• US Class Current: 514/31
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07D 498/18 Bridged systems
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 14/715 for cytokines; for lymphokines; for interferons
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2710/16622 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Abstract

Materials and methods are disclosed for regulation of biological events such as target gene transcription and growth, proliferation or differentiation of engineered cells.

Claims

1. A method for multimerizing chimeric proteins in cells which comprises:

(a) providing cells which contain;

(i) a first recombinant nucleic acid encoding a first chimeric protein which binds to rapamycin or an analog thereof and which comprises at least one FK506-binding protein (FKBP) domain and at least one protein domain heterologous thereto, wherein the FKBP domain comprises a naturally occurring FKBP or a variant thereof in which up to 10 amino acid residues have been deleted, inserted, or replaced with substitute amino acids;

(ii) a second recombinant nucleic acid encoding a second chimeric protein which forms a complex with both (a) rapamycin or a rapamycin analog and (b) the first chimeric protein, and which comprises at least one FKBP;

rapamycin binding (FRB) domain and at least one domain heterologous thereto, wherein the FRB domain comprises a peptide sequence selected from a naturally occurring FRB domain, or a variant thereof in which up to 10 amino acid residues have been deleted, inserted, or replaced with substitute amino acids; and

,

(b) contacting the cells with a rapalog which forms a complex containing itself and at least one molecule of each of the first and second chimeric proteins, where the rapalog has an immunosup-pressive effect less than 0.01 times that of rapamycin and comprises the substructure of formula I;

bearing one or more optional substituents, optionally unsaturated at one or more carbon—

carbon bonds spanning carbons 1 through 8, as a substantially pure stereoisomer or mixture of stereoisomers, or a pharmaceutically acceptable derivative thereof.

5. The method of any of claims 1-4 wherein at least one of R^C7a and R^C7b is a moiety other than —

OMe.

6. The method of any of claims 1-4 wherein the chimeric protein encoded by the first recombinant nucleic acid comprises at least one FKBP domain whose peptide sequence contains up to three amino acid replacements relative to a naturally occurring FKBP peptide sequence.

7. The method of any of claims 1-4 wherein the chimeric protein encoded by the first recombinant nucleic acid comprises at least one FKBP domain whose peptide sequence contains one amino acid replacement relative to a naturally occurring FKBP peptide sequence.

8. The method of any of claims 1-4 wherein the chimeric protein encoded by the first recombinant nucleic acid comprises at least one FKBP domain whose peptide sequence contains a replacement amino acid for Phenylalanine-36 of a naturally occurring FKBP peptide sequence.

9. The method of any of claims 1-4 wherein the chimeric protein encoded by the second recombinant nucleic acid comprises at least one FRB whose peptide sequence contains up to three amino acid replacements relative to a naturally occurring FRB peptide sequence.

10. The method of any of claims 1-4 wherein the chimeric protein encoded by the second recombinant nucleic acid comprises at least one FRB whose peptide sequence contains one amino acid replacement relative to a naturally occurring FRB peptide sequence.

11. The method of any of claims 1-4 wherein the chimeric protein encoded by the second recombinant nucleic acid comprises at least one FRB whose peptide sequence contains a replacement amino acid for one or more of Tyr2038, Phe2039, Thr2098, Gln2099, Trp2101 or Asp2102 in a naturally occurring FRB peptide sequence.

12. The method of any of claims 1-4 wherein at least one of the chimeric proteins comprises a heterologous domain which is a DNA-binding domain, transcription activation domain or a cellular signaling domain for triggering growth, proliferation, differentiation or apoptosis upon dimerization with another protein containing at least one such signaling domain.

13. The method of any of claims 1-4 wherein the cells are grown in a culture medium and the contacting with a rapalog is effected by adding the rapalog to the culture medium.

14. The method of any of claims 1-4 wherein the cells are present in a whole organism and the contacting with a rapalog is effected by administering the rapalog to the organism.

20. The method of claim 5 wherein one of R^C7a and R^C7b is H and the other is phenyl, di- or tri-substituted phenyl or a mono- or di-substituted heterocyclic moiety.

21. The method of claim 5 wherein one of R^C7a and R^C7b is H and the other is o,p-dialkoxyphenyl or trialkoxyphenyl.

22. The method of claim 5 wherein one of R^C7a and R^C7b is H and the other is o,p-dimethoxyphenyl, o-methoxy-p-ethoxyphenyl, o-ethoxy-p-methoxyphenyl, o,p-diethoxyphenyl, trimethoxyphenyl or triethoxyphenyl.

23. The method of claim 5 wherein the chimeric protein encoded by the first recombinant nucleic acid comprises at least one FKBP domain whose peptide sequence contains a replacement amino acid for Phenylalanine-36 of a naturally occurring FKBP peptide sequence.

24. The method of claim 5 wherein the cells are present in a whole organism and the contacting with a rapalog is effected by administering the rapalog to the organism.

25. The method of claim 6 wherein the chimeric protein encoded by the second recombinant nucleic acid comprises at least one FRB whose peptide sequence contains up to three amino acid replacements relative to a naturally occurring FRB peptide sequence.

26. The method of claim 6 wherein the chimeric protein encoded by the second recombinant nucleic acid comprises at least one FRB whose peptide sequence contains a replacement amino acid for one or more of Tyr2038, Phe2039, Thr2098, Gln2099, Trp2101 or Asp2102 in a naturally occurring FRB peptide sequence.

27. The method of claim 6 wherein at least one of the chimeric proteins comprises a heterologous domain which is a DNA-binding domain, transcription activation domain or a cellular signaling domain for triggering growth, proliferation, differentiation or apoptosis upon dimerization with another protein containing at least one such signaling domain.

28. The method of claim 6 wherein the cells are present in a whole organism and the contacting with a rapalog is effected by administering the rapalog to the organism.

29. The method of claim 25 wherein at least one of the chimeric proteins comprises a heterologous domain which is a DNA-binding domain, transcription activation domain or a cellular signaling domain for triggering growth, proliferation, differentiation or apoptosis upon dimerization with another protein containing at least one such signaling domain.

30. The method of claim 25 wherein the cells are present in a whole organism and the contacting with a rapalog is effected by administering the rapalog to the organism.

31. The method of claim 30 wherein the cells are of primate origin and the organism is a primate.

32. The method of claim 31 wherein the primate is a human.

33. The method of claim 9 wherein at least one of the chimeric proteins comprises a heterologous domain which is a DNA-binding domain, transcription activation domain or a cellular signaling domain for triggering growth, proliferation, differentiation or apoptosis upon dimerization with another protein containing at least one such signaling domain.

34. The method of claim 9 wherein the cells are present in a whole organism and the contacting with a rapalog is effected by administering the rapalog to the organism.

35. The method of claim 14 wherein the cells are mammalian and the organism is a mammal.

36. The method of claim 35 wherein the rapalog is administered orally.

2. A method for multimerizing chimeric proteins in cells which comprises:

(a) providing cells which contain;

(i) a first recombinant nucleic acid encoding a first chimeric protein which binds to rapamycin or an analog thereof and which comprises at least one FKBP domain and at least one protein domain heterologous thereto, wherein the FKBP domain comprises a naturally occurring FKBP or a variant thereof in which up to 10 amino acid residues have been deleted, inserted, or replaced with substitute amino acids;

(ii) a second recombinant nucleic acid encoding a second chimeric protein which forms a complex with both (a) rapamycin or a rapamycin analog and (b) the first chimeric protein, and which comprises at least one FRB domain and at least one domain heterologous thereto, wherein the FRB domain comprises a naturally occurring FRB domain or a variant thereof in which up to 10 amino acid residues have been deleted, inserted, or replaced with substitute amino acids; and

(b) contacting the cells with a rapalog which forms a complex containing itself and at least one molecule of each of the first and second chimeric proteins, where the rapalog is of the formula;

wherein

one of R^C7a and R^C7b is H and the other is —

H, halo, —

R², —

OR¹, —

SR¹, —

OC(O)R¹, —

OC(O)NHR¹, —

NHR¹, —

NHR¹R², —

NHC(O)R¹, or —

NH—

SO₂—

R¹, where R²=aliphatic, heteroaliphatic, aryl, heteroaryl or alkylaryl,

R^C30 is halo, —

OR³ or (═

O),

R^C24 is ═

O, ═

NR⁴, ═

NOR⁴, ═

NNHR⁴, —

NHOR⁴, —

NHNHR⁴, —

OR⁴, —

OC(O)R⁴, —

OC(O)NR⁴, halo or —

H,

R^C14 is ═

O, —

OR⁶, —

NR⁶, —

H, —

NC(O)R⁶, —

OC(O)R⁶ or —

OC(O)NR⁶

R³ is H, —

R⁷, —

C(O)R⁷ or —

C(O)NHR⁷ or a cyclic moiety bridging C28 and C30

R^C28 is halo or —

OR³

R^C29 is H, OH or OMe

where each substituent is present in either stereochemical orientation unless otherwise indicated, and where R¹, R⁴, R⁵, R⁶, R⁷, R⁹, and R¹⁰ are independently selected from H, aliphatic, heteroaliphatic, aryl or heteroaryl;

R⁸ is H, halo, —

CN, ═

O, —

OH, —

NR⁹R¹⁰, OSO₂CF₃, OSO₂F, OSO₂R^4′

, OCOR^4′

, OCONR^4′

R^5′

, or OCON(OR4′

)R^5′

;

in which one or both of R^C13 and R^C28 is a halo substituent;

both R^C24 and R^C30 are other than ═

O;

one of R^C7a and R^C7b is H and the other is phenyl, di- or tri-substituted phenyl or a mono- or di-substituted heterocyclic moiety;

n is 1; and

/or moiety

as a substantially pure stereoisomer or mixture of stereoisomers, or a pharmaceutically acceptable derivative thereof.

3. The method of claim 2, wherein both R^C24 and R^C30 are moieties other than (═

O).

4. The method of claim 3 wherein one or both of R^C24 and R^C30 are —

OH, —

OR1 or halo.

15. The method of any of claims 2-4 wherein the rapalog has an immunosuppressive effect less than 0.01 times that of rapamycin.

16. The method of claim 15 wherein the cells are present in a whole organism and the contacting with a rapalog is effected by administering the rapalog to the organism.

17. The method of claim 16 wherein the cells are mammalian and the organism is a mammal.

18. The method claim 17 wherein the cells are of primate origin and the organism is a primate.

19. The method of claim 17 wherein the rapalog is administered orally.