Fluorescence assay of Ras farnesyl protein transferase

• US 5,525,479 A
• Filed: 02/17/1994
• Issued: 06/11/1996
• Est. Priority Date: 06/17/1992
• Status: Expired due to Fees

• Current Assignee: Merck & Co., Inc.
• Original Assignee: Merck & Co., Inc.
• Inventors: Pompliano, David L., Anthony, Neville J.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/199,778
• Time in Patent Office: 845 Days
• Field of Search: 435/15, 435/4, 435/24, 435/18, 514/305, 514/2
• US Class Current: 435/15
• CPC Class Codes: C12Q 1/48 involving transferase

Abstract

This invention is directed to a continuous fluorescence assay of Ras farnesyl:protein transferase. This assay can be used to screen for inhibitors of farnesyl:protein transferase. The assay may also be modified to screen for inhibitors of other protein transferases.

Claims

1. An assay for determining prenyl:

protein transferase activity, comprising;

(a) reacting an acceptor peptide substrate, wherein the acceptor peptide substrate has a fluorescence probe positioned proximally to the reaction center, with a prenyl diphosphate in the presence of a substance suspected of having a corresponding prenyl;

protein transferase activity; and

(b) detecting whether the prenyl residue is incorporated into the peptide substrate, in which incorporation of the prenyl residue into the peptide substrate indicates prenyl;

protein transferase activity and wherein the incorporation of the prenyl residue is detected by an enhancement of the fluorescence of the reaction.

2. The assay according to claim 1 wherein the prenyl:

protein transferase activity is selected from farnesyl;

protein transferase activity and geranylgeranyl;

protein transferase activity.

3. An assay for determining farnesyl:

protein transferase activity, comprising;

(a) reacting an acceptor peptide substrate, wherein the acceptor peptide substrate has a fluorescence probe positioned proximally to the reaction center, with farnesyl diphosphate in the presence of a substance suspected of having farnesyl;

protein transferase activity; and

(b) detecting whether the farnesyl residue is incorporated into the peptide substrate, in which incorporation of the farnesyl residue into the peptide substrate indicates farnesyl;

protein transferase activity and wherein the incorporation of the farnesyl residue is detected by an enhancement of the fluorescence of the reaction.

4. The assay according to claim 3 wherein the substance suspected of having farnesyl:

protein activity is human FPTase.

5. The assay according to claim 3 for determining farnesyl:

protein transferase activity, wherein the acceptor peptide substrate is selected from Ds-GlyCysValLeuSer (SEQ.ID.NO.;

     1), Ds-CysValIleMet (SEQ.ID.NO.;

     3), Ds-CysValLeuSer (SEQ.ID.NO.;

     4), Ds-LysCysValLeuSer (SEQ.ID.NO.;

     5), Ds-GlyLysCysValLeuSer (SEQ.ID.NO.;

     6) and Ds-SerLysCysValLeuSer (SEQ.ID.NO.;

     7).

6. An assay for identifying compounds that inhibit prenyl:

protein transferase activity which comprises

(a) reacting an acceptor peptide substrate, wherein the acceptor peptide substrate has a fluorescence probe positioned proximally to the reaction center, with a prenyl diphosphate in the presence of a substance having a corresponding prenyl;

protein transferase activity in the presence of a test compound; and

(b) detecting whether the prenyl residue is incorporated into the peptide substrate, in which the ability of the test compound to inhibit prenyl;

protein transferase activity is indicated by a decrease of the incorporation of the prenyl residue into the peptide substrate as compared to the amount of the prenyl residue incorporated into the peptide substrate in the absence of the test compound and wherein the decrease of the incorporation of the prenyl residue in the presence of the test compound is detected by a reduction in the enhancement of the fluorescence of the reaction when compared to the enhancement of the fluorescence of the reaction in the absence of the test compound.

7. The assay according to claim 6 wherein the prenyl:

protein transferase activity is selected from farnesyl;

protein transferase activity and geranylgeranyl;

protein transferase activity.

8. An assay for identifying compounds that inhibit farnesyl:

protein transferase activity which comprises

(a) reacting an acceptor peptide substrate, wherein the acceptor peptide substrate has a fluorescence probe positioned proximally to the reaction center, with farnesyl diphosphate in the presence of a substance having farnesyl;

protein transferase activity in the presence of a test compound; and

(b) detecting whether the farnesyl residue is incorporated into the peptide substrate, in which the ability of the test compound to inhibit farnesyl;

protein transferase activity is indicated by a decrease of the incorporation of the farnesyl residue into the peptide substrate as compared to the amount of the farnesyl residue incorporated into the peptide substrate in the absence of the test compound and wherein the decrease of the incorporation of the prenyl residue in the presence of the test compound is detected by a reduction in the enhancement of the fluorescence of the reaction when compared to the enhancement of the fluorescence of the reaction in the absence, of the test compound.

9. An acceptor peptide substrate which is selected from Ds-GlyCysValLeuSer (SEQ.ID.NO.:

1 ), Ds-CysValIleMet (SEQ.ID.NO.;

     3), Ds-CysValLeuSer (SEQ.ID.NO.;

     4), Ds-LysCysValLeuSer (SEQ.ID.NO.;

     5), Ds-GlyLysCysValLeuSer (SEQ.ID.NO;

     6) and Ds-SerLysCysValLeuSer (SEQ.ID.NO.;

     7).