Abstract
A method for identifying compounds that inhibit HIV protease is disclosed. A substrate that comprises an HIV protease cleavage site is combined with HIV protease and test compounds. Cleavage of the substrate indicates protease activity and can be detected using antibodies against a cleavage product which do not cross react with uncleaved substrate. A method of detecting the presence of anti-HIV protease antibodies in a sample is also disclosed. A substrate is combined with the sample and HIV protease. Detection of substrate cleavage indicates that the protease is active and that there is an absence of neutralizing anti-HIV protease antibodies.
Filing date: Apr 4, 1991
Issue date: Dec 15, 1992
Inventor: Satish K. Sharma
Assignee: The Upjohn Company
Primary Examiner: Chris Dubrule
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What is claimed is:
1. A method for identifying compounds which inhibit HIV protease activity comprising the steps of:
- a) combining a substrate, said HIV protease, anti-Ang I antibodies and a compound that is an HIV protease inhibitor candidate, said substrate comprising an HIV protease cleavage site, wherein said substrate does not bind with anti-Ang I antibodies and cleavage of said substrate by said HIV protease generates at least one reactive cleavage product which binds with anti-Ang I antibodies; and
- b) detecting the presence of said anti-Ang I antibodies that are bound with said reactive cleavage product, binding of anti-Ang I antibodies with a reactive cleavage product indicates that said HIV protease cleaved said substrate.
2. A method according to claim 1 wherein said HIV protease is HIV-1 protease.
3. A method according to claim 2 wherein said HIV protease cleavage site is selected from the group consisting of Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, Pro-Phe-His-Leu-Leu-Glu-Ile-Ser and Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.
4. A method according to claim 2 wherein said substrate is selected from the group consisting of: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Ile-Ser; and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.
5. A method according to claim 1 wherein said anti-Ang I antibodies are immobilized upon a solid phase.
6. A method according to claim 5 wherein said anti-Ang I antibodies are immobilized upon the inner surface of a container such that said anti-Ang I antibodies will come into contact with material put into said container, and wherein said substrate, said HIV protease and said compound that is an HIV protease inhibitor candidate are combined in said container.
7. A method according to claim 6 comprising the additional steps of:
- a) adding a radiolabeled peptide to said container, wherein said radiolabeled peptide competes with said reactive cleavage product to bind with said anti-Ang I antibodies;
- b) removing unbound reactive cleavage product and unbound radiolabeled peptide from said container; and
- c) measuring amount of radiolabeled material is bound to said anti-Ang I antibodies.
8. A kit for identifying compounds that inhibit HIV protease activity, wherein said kit comprises a carton comprising:
- a) a container having anti-Ang I antibodies immobilized on its inner surface;
- b) a container comprising a substrate comprising an HIV protease cleavage site wherein said substrate does not bind to said anti-Ang I antibodies and wherein cleavage of said substrate by said HIV protease generates at least one reactive cleavage product which binds with said anti-Ang I antibodies; and,
- c) a container comprising HIV protease, said container comprising HIV protease being a different container than said container comprising said substrate.
9. A kit according to claim 8 wherein said HIV protease is HIV-1.
10. A kit according to claim 9 wherein said HIV protease cleavage site is an amino acid sequence is selected from the group consisting of Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, Pro-Phe-His-Leu-Leu-Glu-Ile-Ser and Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.
11. A kit according to claim 9 wherein said substrate is selected from the group consisting of: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Ile-Ser; and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.
12. A method of detecting the presence of anti-HIV protease antibodies in a sample that is suspected of containing antibodies to HIV protease comprising the steps of:
- a) combining a substrate, HIV protease, anti-Ang I antibodies and a sample that is suspected of containing antibodies to HIV protease, said substrate comprising an HIV protease cleavage site wherein said substrate does not bind with anti-Ang I antibodies and cleavage of said substrate by said HIV protease generates at least one reactive cleavage product which binds with anti-Ang I antibodies; and
- b) detecting the presence of said anti-Ang I antibodies that are bound with said reactive cleavage product.
13. A method according to claim 12 wherein said anti-HIV protease antibodies are anti-HIV-1 antibodies and said HIV protease is HIV-1 protease.
14. A method according to claim 13 wherein said HIV protease cleavage site is selected from the group consisting of Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, Pro-Phe-His-Leu-Leu-Glu-Ile-Ser and Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.
15. A method according to claim 13 wherein said substrate is selected from the group consisting of: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Ile-Ser; and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.
16. A method according to claim 12 wherein said anti-Ang I antibodies are immobilized upon a solid phase.
17. A method according to claim 16 wherein said anti-Ang I antibodies are immobilized upon the inner surface of a container such that said anti-Ang I antibodies will come into contact with material put into said container, and wherein said substrate, said HIV protease and said sample that is suspected of containing antibodies to HIV protease are combined in said container.
18. A method according to claim 17 comprising the additional steps of:
- a) adding a radiolabeled peptide to said container, wherein said radiolabeled peptide competes with said reactive cleavage product to bind with said anti-Ang I antibodies;
- b) removing unbound reactive cleavage product and unbound radiolabeled peptide from said container; and
- c) measuring amount of radiolabeled material is bound to said anti-Ang I antibodies.
19. A kit for detecting the presence of anti-HIV protease antibodies in a sample that is suspected of containing antibodies to HIV protease, wherein said kit comprises a carton comprising:
- a) a container having anti-Ang I antibodies immobilized on its inner surface;
- b) a container comprising a substrate comprising an HIV protease cleavage site wherein said substrate does not bind to said anti-Ang I antibodies and wherein cleavage of said substrate by said HIV protease generates at least one reactive cleavage product which binds with said anti-Ang I antibodies; and,
- c) a container comprising HIV protease, said container comprising HIV protease being a different container than said container comprising said substrate.
20. A kit according to claim 19 wherein said anti-HIV protease antibodies are anti-HIV-1 protease antibodies and said HIV protease is HIV-1 protease.
21. A kit according to claim 20 wherein said HIV protease cleavage site is selected from the group consisting of Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, Pro-Phe-His-Leu-Leu-Glu-Ile-Ser and Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.
22. A kit according to claim 19 wherein said substrate is selected from the group consisting of: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Ile-Ser; and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser.