Methods for identifying HCV protease inhibitors

1. A method comprising the steps of:

(a) contacting a test compound with an NS3-containing protein;

(b) measuring NS3 self-cleaving activity by measuring at least one of:

(a) a level of NS3; and

(b) a level of a NS3 self-cleavage product; and

(c) classifying the test compound as a reversible or irreversible inhibitor of NS3;

wherein:

an irreversible inhibitor of NS3 is characterized by providing, when at a 5 uM concentration, at least 10% inhibition of NS3 self-cleaving activity four hours after removing the test compound; and

a reversible inhibitor of NS3 is characterized by providing, when at a 5 uM concentration, less than 10% inhibition of NS3 self-cleaving activity four hours after removing the test compound.

2. A method comprising the steps of:

(a) contacting a test compound with an HCV NS3-containing protein;

(b) measuring HCV NS3 self-cleaving activity by measuring at least one of:

(a) a level of HCV NS3; and

(b) a level of a HCV NS3 self-cleavage product; and

(c) classifying the test compound as a reversible or irreversible inhibitor of HCV NS3;

wherein:

an irreversible inhibitor of HCV NS3 is characterized by providing, when at a 5 uM concentration, at least 10% inhibition of HCV NS 3 self-cleaving activity four hours after removing the test compound; and

a reversible inhibitor of HCV NS3 is characterized by providing, when at a 5 uM concentration, less than 10% inhibition of HCV NS3 self-cleaving activity four hours after removing the test compound.

3. The method according claim 2 , wherein said method is used to identify one or more reversible inhibitors of HCV NS3.

4. The method according to claim 2 , wherein said method is used to identify one or more covalent irreversible inhibitors of HCV NS3.

5. The method according to claim 4 , wherein said method is used to determine duration of action of the covalent irreversible inhibitor.

6. The method according to claim 2 , wherein the HCV NS3 is selected from any one or more genotypes and subtypes.

7. The method according to claim 2 , wherein the HCV NS3 is a variant.

8. The method according to claim 7 , wherein the HCV NS3 is a variant selected from A156T, A156S, D168V, D168A, and R155K.

9. The method according to claim 4 , wherein said irreversible inhibitor covalently modifies Cys159 of HCV protease subtype 1b, or a variant thereof

10. The method according to claim 2 , further comprising the step of performing an additional method of determining inhibitory activity of the test compound.

11. The method according to claim 10 , wherein the additional method is a method for confirming that the test compound is a covalent irreversible inhibitor.

12. The method according to claim 11 , wherein the additional method is a washout experiment or mass spectrometric analysis.

13. A method comprising the steps of:

(a) contacting a cell with a test compound;

(b) washing said cells at one or more time intervals;

(c) measuring HCV NS3 self-cleaving activity by measuring at least one of:

(a) a level of HCV NS3; and

(b) a level of a HCV NS3 self-cleavage product; and

(d) classifying the test compound as a reversible or irreversible inhibitor of HCV NS3;

wherein:

an irreversible inhibitor of HCV NS3 is characterized by providing, when at a 5 uM concentration, at least 10% inhibition of HCV NSself-cleaving activity four hours after removing the test compound; and

a reversible inhibitor of HCV NS3 is characterized by providing, when at a 5 uM concentration, less than 10% inhibition of HCV NS3 self-cleaving activity four hours after removing the test compound.

14. The method according to claim 13 , wherein the cell is any cell line expressing both NS3 and NS4A.

15. The method according to claim 14 , wherein the cell line is selected from Huh-7-Luc-Neo-ET cells, Huh-9- 13 cells, Huh-5-15 cells, or Huh-11-7 cells.

16. The method according to claim 14 , wherein the NS3 is a variant.

17. The method according to claim 15 , wherein the Huh-7-Luc-Neo-ET cells comprise NS3 mutations A156T, A156S, D168V, D168A, and R155K.

18. The method according to claim 13 , wherein the cells are washed at any time point between about 1 and 48 hours.

19. The method according to claim 2 , wherein the method is repeated or performed with a plurality of different concentrations of the test compound.

20. The method according to claim 2 , wherein the quantifying step involves direct measurement of HCV NS3 internal self-cleavage.

21. The method according to claim 13 , wherein the quantifying step involves direct measurement of HCV NS3 internal self-cleavage.

22. The method according to claim 1 , wherein the NS3-containing protein comprises NS3 and NS4A.

23. The method according to claim 2 , wherein the HCV NS 3 -containing protein comprises NS3 and NS4A.