Claims
- 1. A composition comprising a purified farnesyl:protein transferase enzyme, characterized as follows:
(a) capable of catalyzing the transfer of farnesol to a protein or peptide having a farnesyl acceptor moiety; (b) capable of binding to an affinity chromatography medium comprised of TKCVIM coupled to a suitable matrix; (c) exhibiting a molecular weight of between about 70,000 kDa and about 100,000 kDa upon gel filtration chromatography, and comprised of two different subunits, each exhibiting a molecular weight of approximately 45,000 kDa to 50,000 kDa upon SDS-PAGE; and (d) having a farnesyl transferase activity that is capable of being inhibited by TKCVIM; CVIM; or KKSKTKCVIM.
- 2. The composition of claim 1, further defined as exhibiting a farnesyl transferase specific activity of between about 5 and about 600,000 units/mg protein.
- 3. The composition of claim 2, further defined as exhibiting a farnesyl transferase specific activity of between about 500 and about 600,000 units/mg protein.
- 4. The composition of claim 1, wherein said farnesyl transferase enzyme is purified by a process which includes the steps of
(a) preparing a cellular extract which includes the enzyme; (b) subjecting the extract to affinity chromatography on an affinity chromatography medium to bind the enzyme thereto, the medium comprised of a farnesyl transferase binding peptide coupled to a suitable matrix; (c) washing the medium to remove impurities; and (d) eluting the enzyme from the washed medium.
- 5. The composition of claim 4, wherein the farnesyl transferase binding peptide comprises a peptide of at least 4 amino acids in length and including a carboxy terminal sequence of —C-A-A-X, wherein:
C=cysteine; A=an aliphatic or hydroxy amino acid; and X=any amino acid
- 6. The composition of claim 5, wherein the farnesyl transferase binding peptide includes a carboxy terminal sequence of —C—V—I-M, —C—S—I-M or —C-A-I-M.
- 7. The composition of claim 6, wherein the farnesyl transferase binding peptide comprises T-K—C—V—I-M.
- 8. The composition of claim 1, wherein the farnesyl transferase enzyme is prepared by recombinant mans.
- 9. A method of preparing a farnesyl transferase enzyme, comprising the steps of:
(a) preparing a cellular extract which includes the enzyme; (b) subjecting the extract to affinity chromatography on an affinity chromatography medium to bind the enzyme thereto, the medium comprised of a farnesyl transferase binding peptide coupled to a suitable matrix; (c) washing the medium to remove impurities; and (d) eluting the enzyme from the washed medium.
- 10. The method of claim 9, wherein the farnesyl transferase binding peptide comprises a peptide of at least 4 amino acids in length and including a carboxy terminal sequence of —C-A-A-X, wherein:
C=cysteine; A=an aliphatic or hydroxy amino acid; and X=any amino acid
- 11. The method of claim 10, wherein the farnesyl transferase binding peptide includes a carboxy terminal sequence of —C—V—I-M, —C—S—I-M or —C-A-I-M.
- 12. The method of claim 10, wherein the farnesyl transferase binding peptide is biotinylated.
- 13. The method of claim 11, wherein the farnesyl transferase binding peptide comprises T-K—C—V—I-M.
- 14. A method for assaying for the presence of farnesyl transferase activity in a composition comprising determining the ability of said composition to catalyze the transfer of farnesol to a farnesyl acceptor protein or peptide.
- 15. The method of claim 14, wherein said farnesol is transferred from farnesyl pyrophosphate.
- 16. The method of claim 15, wherein said farnesyl pyrophosphate contains a label on the farnesyl moiety.
- 17. The method of claim 14, wherein said farnesyl acceptor protein or peptide comprises a carboxy terminal sequence of —C-A-A-X, wherein:
C=cysteine; A=an aliphatic or hydroxy amino acid; and X=any amino acid
- 18. The method of claim 17, wherein said farnesyl acceptor protein or peptide comprises a p21ras protein
- 19. The method of claim 17, wherein said farnesyl acceptor protein or peptide comprises a peptide of at least 4 amino acids in length.
- 20. The method of claim 19, wherein the farnesyl acceptor protein or peptide comprises CVIM; KKSKTKCVIN; TKCVIM; RASNRSCAIM; TQSPQNCSIM; CIIM; CVVN; or CVLS.
- 21. A farnesyl transferase inhibitor comprising a peptide, or protein other than a p21ras protein, lamin a, lamin b, or yeast mating factor a, said peptide or protein having a farnesyl acceptor or inhibitor sequence within its structure and capable of inhibiting the farnesylation of p21ras by farnesyl transferase.
- 22. The inhibitor claim 21, wherein the farnesyl acceptor or inhibitor sequence is further defined as a farnesyl acceptor amino acid sequence which includes the amino acids CAAX, wherein:
C=cysteine; A=an aliphatic or hydroxy amino acid; and X=any amino acid
- 23. The inhibitor of claim 22, wherein the farnesyl acceptor or inhibitor amino acid sequence is positioned at the carboxy terminus of the protein or peptide.
- 24. The inhibitor of claim 23, further defined as peptide of from four to 10 amino acids in length.
- 25. The inhibitor of claim 24, further defined as a peptide incorporating one of the following peptide sequences at its carboxy terminus: CVIM; KKSKTKCVIM; TKCVIM; RASNRSCAIM; TQSPQNCSIM; CIIM; CVVM; CVLS; CVLM; CAIM; CSIM; CCVQ; CIIC; CIIS; CVIS; CVLS; CVIA; CVIL; CLIL; CLLL; CTVA; CVAM; DKIM; CLIM; CVLM; CFIM; CVFM; CVIF; CEIM; CGIM; CPIM; CVYM; CVTM; CVPM; CVSM; CVIF; CVIV; CVIP; or CVII.
- 26. The inhibitor of claim 24, further defined as a tetrapeptide.
- 27. The inhibitor of claim 25, further defined as one of the following peptides. CVIM; CIIM; CVVM; CVLS; CVLM; CAIM; CSIM; CCVQ; CIIC; CIIS; CVIS; CVLS; CVIA; CVIL; CLIL; CLLL; CTVA; CVAM; CKIM; CLIM; CVLM; CFIM; CVFM; CVIF; CEIM; CGIM; CPIM; CVYM; CVTM; CVPM; CVSM; CVIF; CVIV; CVIP; or CVII.
- 28. The inhibitor of calim 25, further defined as a peptide having a sequence which consistws essentially of one of the specified peptide sequences.
- 29. The inhibitor of claim 24 wherein the peptide is modified by biotinylation, esterification, acylation, or alkylation.
- 30. The inhibitor of claim 23, further defined as a pure inhibitor.
- 31. The inhibitor of claim 30, further defined as a peptide comprising the structure—C-A1-A2-X, wherein C=cysteine, A1—any aliphatic, aromatic or hydroxy amino acid; A2=any aromatic amino acid or amino acid modified to incorporate one or more aromatic moieties; and X=any amino acid.
- 32. The inhibitor of claim 31, further defined as the tetrapetide CVFM.
- 33. The inhibitor of claim 31, wherein the aromatic moiety of the A2 amino acid is modified to include a fluoro, chloro, or nitro group.
- 34. The inhibitor of claim 33, wherein the A2 amino acid comprises parachlorophenylalanine.
- 35. The inhibitor of claim 31, wherein the A2 amino acid comprises a naphthyl ring.
- 36. The method of claim 31, wherein the A2 amino acid comprises phenylalanine, tyrosine or tryptophan.
- 37. A method for determining the ability of a candidate substance to inhibit a farnesyl transferase enzyme, comprising the steps of:
(a) obtaining an enzyme composition comprising a farnesyl transferase enzyme that is capable of transferring a farnesyl moiety to a farnesyl acceptor substance; (b) admixing a candidate substance with the enzyme composition; and (c) determining the ability of the farnesyl transferase enzyme to transfer a farnesyl moiety to a farnesyl acceptor substrate in the presence of the candidate substance.
- 38. The method of claim 37, wherein the farnesyl transferase composition comprises the composition of claim 1.
- 39. The method of claim 37, wherein the farnesyl acceptor substrate comprises a p21ras, or any peptide containing a cysteine at the fourth position from the carboxyl terminus.
- 40. The method of claim 37, wherein step (c) comprises determining the ability of the candidate substance to inhibit the transfer of farnesyl from farnesyl pyrophosphate to the acceptor substrate.
- 41. The method of claim 37, wherein the farnesyl moiety is labeled.
- 42. The method of claim 41, wherein the farnesyl moiety is radiolabeled.
- 43. A method of inhibiting a farnesyl transferase enzyme comprising subjecting the enzyme to an effective concentration of a farnesyl transferase inhibitor in accordance with claim 21, or a candidate substance identified in accordance with the method of claim 29 to be such an inhibitor.
- 44. A method of inhibiting the attachment of a farnesyl moiety to a ras protein in malignant cells comprising subjecting said cells to an effective concentration of a farnesyl transferase inhibitor in accordance with claim 21, or a candidate substance identified in accordance with the method of claim 29 to be such an inhibitor.
- 45. A DNA segment encoding the α or β subunit of farnesyl:protein transferase.
- 46. The DNA segment of claim 45, further defined as encoding the α subunit.
- 47. The DNA segment of claim 45, further defined as encoding the β subunit.
- 48. A recombinant vector comprising the DNA segment of claim 45.
- 49. The recombinant vector of claim 48, further defined as comprising a DNA segment encoding the α subunit of farnesyl protein transferase.
- 50. The recombinant vector of claim 48, further defined as comprising a DNA segment encoding the β subunit of farnesyl protein transferase.
Parent Case Info
[0001] This is a continuation of co-pending application Ser. No. 07/937,893, filed Dec. 22, 1992, which is US nationalization of PCT application PCT/US91/02650, filed Apr. 18, 1991, which PCT application is a continuation-in-part of application Ser. No. 07/615,715, filed Nov. 20, 1990, now U.S. Pat. No. 5,141,851, which is a continuation-in-part of application Ser. No. 07/510,706, filed Apr. 18, 1990, abandoned. The government may own certain rights in the present invention pursuant to NIH grant number 5-PO1-HL20948.
Continuations (1)
|
Number |
Date |
Country |
Parent |
07937893 |
Dec 1992 |
US |
Child |
10083894 |
Feb 2002 |
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
07615715 |
Nov 1990 |
US |
Child |
PCT/US91/02650 |
Apr 1991 |
US |
Parent |
07510706 |
Apr 1990 |
US |
Child |
07615715 |
Nov 1990 |
US |