Methods for the identification of farnesyltransferase inhibitors

• US 5,962,243 A
• Filed: 04/27/1995
• Issued: 10/05/1999
• Est. Priority Date: 04/18/1990
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: James, Guy L., Brown, Michael S., Goldstein, Joseph L.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Slobodyansky, Elizabeth
• Application Number:
  US08/429,964
• Time in Patent Office: 1,622 Days
• Field of Search: 435/15, 435/193, 435/69.1, 435/240.2, 435/252.3, 435/320.1, 514/2, 530/329, 530/330, 536/23.2
• US Class Current: 435/15
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C07K 5/1013 the side chain containing O or S as heteroatoms, e.g. Cys, Ser
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 9/1085 transferring alkyl or aryl groups other than methyl groups (2.5)
• CPC Class Codes: C12Q 1/48 involving transferase

Abstract

Disclosed are methods and compositions for the identification of inhibitors of farnesyltransferase enzymes involved in the prenylation of various cellular proteins, including cancer related ras proteins, such as p21^ras, particularly, K-rasB. Enclosed are procedures for using purified farnesyltransferase enzymes and K-rasB proteins in screening protocols for the identification of possible anticancer agents that inhibit the enzyme and thereby prevent prenylation of proteins such as K-RasB.

Claims

1. A method for assaying for the presence of farnesyl protein transferase activity in an enzyme composition, comprising determining the ability of said enzyme composition to catalyze the transfer of a farnesyl moiety to a K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity.

2. The method of claim 1, wherein the ability of said enzyme composition to catalyze the transfer of a farnesyl moiety to a K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity is determined by a method comprising the steps of:

(a) admixing said enzyme composition with a substrate composition comprising a K-RasB protein, or K-RasB peptide fragment having farnesyl acceptor activity, and a farnesyl pyrophosphate compound having a labeled farnesyl moiety; and

(b) evaluating the ability of said enzyme composition to transfer said labeled farnesyl moiety to said K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity.

3. The method of claim 2, wherein said substrate composition comprises a K-RasB protein.

4. The method of claim 2, wherein said substrate composition comprises a K-RasB peptide fragment having farnesyl acceptor activity.

5. The method of claim 2, wherein said farnesyl pyrophosphate compound has a radiolabeled farnesyl moiety.

6. A method for determining whether a candidate substance has the ability to inhibit a farnesyl protein transferase enzyme, the method comprising the steps of:

(a) obtaining an enzyme composition comprising a farnesyl protein transferase enzyme;

(b) obtaining a substrate composition comprising a K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity;

(c) admixing said enzyme composition with said substrate composition, a farnesyl pyrophosphate and a candidate substance; and

(d) determining the ability of the farnesyl protein transferase enzyme to transfer a farnesyl moiety to the K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity in the presence of the candidate substance and in the absence of the candidate substance.

7. The method of claim 6, wherein said enzyme composition is a native enzyme composition obtained by purification from natural sources.

8. The method of claim 6, wherein said enzyme composition is a recombinant enzyme composition obtained by recombinant expression.

9. The method of claim 6, wherein said substrate composition comprises a K-RasB protein.

10. The method of claim 6, wherein said substrate composition comprises a K-RasB peptide fragment having farnesyl acceptor activity.

11. The method of claim 6, wherein said substrate composition is obtained by purification from natural sources.

12. The method of claim 6, wherein said substrate composition is obtained by recombinant expression.

13. The method of claim 6, wherein the farnesyl moiety of said farnesyl pyrophosphate is labeled.

14. The method of claim 13, wherein the farnesyl moiety of said farnesyl pyrophosphate is radiolabeled.

15. A method for inhibiting a farnesyl protein transferase enzyme, comprising contacting the enzyme with an effective amount of a farnesyl protein transferase inhibitor substance identified by the method of claim 6.

16. A method of inhibiting the attachment of a farnesyl moiety to a K-RasB protein in a cell, comprising transferring into said cell an effective amount of a farnesyl protein transferase enzyme inhibitor, the inhibitor identified by a method comprising the steps of:

(a) admixing a farnesyl protein transferase enzyme composition with a K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity substrate composition, a farnesyl pyrophosphate and a candidate inhibitory substance; and

(b) determining the ability of the farnesyl protein transferase enzyme to transfer a farnesyl moiety to the K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity in the presence of the candidate inhibitory substance and in the absence of the candidate inhibitory substance.

17. A method for inhibiting a farnesyl protein transferase enzyme, comprising contacting the enzyme with an effective amount of a farnesyl protein transferase inhibitor identified by a process comprising the steps of:

(a) obtaining an enzyme composition comprising a farnesyl protein transferase enzyme that is capable of transferring a farnesyl moiety to a farnesyl acceptor substance comprising a K-RasB protein or K-RasB peptide fragment having farnesyl acceptor activity;

(b) admixing a candidate substance with the enzyme composition and farnesyl pyrophosphate; and

(c) identifying a farnesyl protein transferase inhibitor by selecting a candidate inhibitor substance that inhibits the ability of the farnesyl protein transferase enzyme to transfer a farnesyl moiety to the farnesyl acceptor substance.

18. A method of providing a farnesyl protein transferase inhibitor comprising:

(a) obtaining a test composition comprising a compound suspected of being a farnesyl protein transferase inhibitor;

(b) obtaining an enzyme composition comprising a farnesyl protein transferase enzyme that is capable of transferring a farnesyl moiety to a farnesyl acceptor substance wherein the farnesyl protein transferase enzyme comprises a recombinant farnesyl protein transferase enzyme obtained by recombinant expression;

(c) admixing farnesyl pyrophosphate, the enzyme composition and the farnesyl acceptor substance with the test composition;

(d) determining the ability of the enzyme composition to transfer a farnesyl moiety to the farnesyl acceptor substance in the presence of the test composition, wherein the ability of the test composition to inhibit the transfer of the farnesyl moiety to the farnesyl acceptor substance confirms the identity of the test composition to be a farnesyl protein transferase inhibitor; and

(e) providing the identified farnesyl protein transferase inhibitor.

19. The method of claim 18, wherein the farnesyl acceptor substance comprises a peptide of at least 4 amino acids in length that includes a carboxy terminal amino acid sequence of --C--A--A--X, wherein:

C=cysteine;

A=an aliphatic or hydroxy amino acid; and

X=any amino acid.

20. The method of claim 19, wherein the farnesyl acceptor substance comprises a peptide of at least 6 amino acids in length that includes a carboxy terminal amino acid sequence of Thr Lys Cys Val Ile Met.

21. The method of claim 18, wherein the farnesyl acceptor substance comprises a p21^ras protein.

22. The method of claim 21, wherein the p21^ras protein comprises a K-RasB protein.

23. The method of claim 18, wherein the farnesyl pyrophosphate comprises a labeled farnesyl moiety.

24. The method of claim 23, wherein the farnesyl pyrophosphate comprises a radiolabeled farnesyl moiety.

25. The method of claim 18, wherein the farnesyl protein transferase enzyme comprises a farnesyl protein transferase enzyme characterized as:

(a) catalyzing the transfer of all-trans farnesol to a protein or peptide having a carboxy terminal farnesyl acceptor moiety;

(b) binding to an affinity chromatography medium that comprises a peptide of the amino acid sequence Thr Lys Cys Val Ile Met coupled to a matrix;

(c) exhibiting a molecular weight of between about 70,000 Da and about 100,000 Da upon gel filtration chromatography, and comprising to two different subunits, each exhibiting a molecular weight of approximately 45,000 Da to 50,000 Da upon SDS-PAGE;

(d) having a farnesyl protein transferase activity that is capable of being inhibited by a peptide having the sequence Thr Lys Cys Val Ile Met;

Cys Val Ile Met;

or Lys Lys Ser Lys Thr Lys Cys Val Ile Met; and

(e) exhibiting a farnesyl protein transferase specific activity of between about 5 and about 600,000 units/mg protein.

26. The method of claim 25, wherein the farnesyl protein transferase enzyme exhibits a farnesyl protein transferase specific activity of between about 500 and about 600,000 units/mg protein.

27. The method of claim 18, wherein the enzyme composition comprises a farnesyl protein transferase enzyme purified by a process that includes the steps of:

(a) preparing a cellular extract that 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 protein transferase binding peptide coupled to a suitable matrix;

(c) washing the medium to remove impurities; and

(d) eluting the enzyme from the washed medium.

28. The method of claim 27, wherein said farnesyl protein transferase binding peptide comprises a peptide of at least 4 amino acids in length that includes a carboxy terminal sequence of --C--A--A--X, wherein:

C=cysteine;

A=an aliphatic or hydroxy amino acid; and

X=any amino acid.

29. The method of claim 28, wherein said farnesyl protein transferase binding peptide comprises the sequence Thr Lys Cys Val Ile Met.

30. A method of providing a farnesyl protein transferase inhibitor comprising:

(a) obtaining an enzyme composition comprising an enzyme suspected of having farnesyl protein transferase inhibitor activity;

(b) admixing the enzyme composition, farnesyl pyrophosphate, a farnesyl protein transferase enzyme that is capable of transferring a farnesyl moiety to a farnesyl acceptor substance, and the farnesyl acceptor substance wherein the farnesyl protein transferase enzyme is further defined as exhibiting a farnesyl transferase specific activity of between about 500 and about 600,000 units/mg protein;

(c) determining the ability of the farnesyl protein transferase enzyme to transfer a farnesyl moiety to the farnesyl acceptor substance in the presence and absence of the enzyme composition, wherein the ability of the enzyme composition to inhibit the transfer of the farnesyl moiety to the farnesyl acceptor substance confirms the identity of the enzyme composition to be a farnesyl protein transferase inhibitor; and

(d) providing the identified farnesyl protein transferase inhibitor.

31. A method of providing a candidate substance having the ability to inhibit a farnesyl protein transferase enzyme comprising:

(a) obtaining an enzyme composition comprising a farnesyl protein transferase enzyme that is capable of a farnesyl moiety to a farnesyl acceptor substance;

(b) admixing a candidate substance with the enzyme composition and farnesyl pyrophosphate, wherein the enzyme composition comprises an enzyme characterized as follows;

(i) catalyzing the transfer of all-trans farnesol to a protein or peptide having a carboxy terminal farnesyl acceptor moiety;

(ii) binding to an affinity chromatography medium that comprises peptide of the amino acid sequence Thr Lys Cys Val Ile Met coupled to a matrix;

(iii) exhibiting a molecular weight of between about 70,000 Da and about 100,000 Da upon gel filtration chromatography, and comprising two different subunits, each exhibiting a molecular weight of approximately 45,000 Da to 50,000 Da upon SDS-PAGE;

(iv) having a farnesyl protein transferase activity that is capable of being inhibited by a peptide having the sequence Thr Lys Cys Val Ile Met;

Cys Val Ile Met;

or Lys Lys Ser Lys Thr Lys Cys Val Ile Met; and

(v) exhibiting a farnesyl protein transferase specific activity of between about 5 and about 600,000 units/mg protein;

(c) comparing the ability of the farnesyl protein transferase enzyme to transfer a farnesyl moiety to a farnesyl acceptor substance in the presence of the candidate substance to the ability of the farnesyl protein transferase enzyme to transfer a farnesyl moiety to the substrate in the absence of the candidate substance; and

(d) the candidate substance that is determined to have the ability to inhibit the farnesyl protein transferase enzyme.

32. The method of claim 31, wherein the enzyme is further defined as exhibiting a farnesyl transferase specific activity of between about 500 and about 600,000 units/mg protein.

33. The method of claim 32, wherein the farnesyl protein transferase enzyme is prepared by recombinant means.

34. The method of claim 32, wherein the farnesyl protein transferase enzyme comprises a native farnesyl protein transferase enzyme obtained by purification from natural sources.