Method for identifying protease inhibitors

Abstract

The invention describes a method for assaying HCV NS3 protease activity using an NS3•4A protease molecule. The invention also provides a method for screening and identifying modulators of NS3 protease.

1 . A method for measuring the activity of the Hepatitis C NS3 protease comprising:
 a) providing a sample comprising an isolated protein comprising SEQ ID NO:1;   b) adding a peptide substrate based on the NS5A/NS5B cleavage site for Hepatitis C genotype 1a;   c) separating the substrate and products on a reverse phase HPLC column; and   d) quantifying the product peaks.   
     
     
         2 . The method of  claim 1  further comprising adding to the sample an NS4A cofactor peptide comprising SEQ ID NO:4. 
     
     
         3 . The method of  claim 1  or  2  wherein the peptide substrate is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6. 
     
     
         4 . The method of  claim 3 , wherein the peptide substrate is SEQ Id NO:2. 
     
     
         5 . The method of  claim 3 , wherein the peptide substrate is SEQ ID NO:5. 
     
     
         6 . The method of  claim 3 , wherein the peptide substrate is SEQ ID NO:6. 
     
     
         7 . The method of  claim 4 , wherein the C-terminal product peak is quantified using absorbance data collected at about 210 ηm. 
     
     
         8 . The method of  claim 5 , wherein the C-terminal product peak is quantified using fluorescence data collected at about 350 ηm excitation/about 490 ηm emission. 
     
     
         9 . The method of  claim 6 , wherein the N-terminal product peak is quantified using fluorescent data collected at about 440 ηm excitation/about 520 ηm emission. 
     
     
         10 . A method for identifying modulators of the Hepatitis C NS3 protease comprising
 a) providing a sample comprising a protein comprising SEQ ID NO:1;   b) introducing a test compound to the sample;   c) adding a peptide substrate based on the NS5A/NS5B cleavage site for Hepatitis C genotype 1a;   d) separating the substrate and products on a reverse phase HPLC column; and   e) quantifying the product peaks.   
     
     
         11 . The method of  claim 10  further comprising adding to the sample and test compound an NS4A cofactor peptide comprising SEQ ID NO:4. 
     
     
         12 . The method of  claim 10  or  11 , wherein the peptide substrate is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6. 
     
     
         13 . A method for determining whether a test compound modifies the activity of the NS3 protease or the activity of the NS4A cofactor comprising:
 a) providing a sample comprising a protein comprising SEQ ID NO:1;   b) introducing a test compound to the sample;   c) adding an NS4A cofactor peptide;   d) adding a peptide substrate based on the NS5A/NS5B cleavage site for Hepatitis C genotype 1a;   e) measuring first amounts of products by separating the substrate and products on a reverse phase HPLC column; and quantifying the product peaks; and   f) comparing first amounts of products to second amounts of products, wherein the second amounts of products are measured in the absence of a NS4A cofactor peptide.   
     
     
         14 . The method of  claim 13 , wherein the NS4A cofactor peptide comprises SEQ ID NO:4. 
     
     
         15 . The method of  claim 13  or  14 , wherein the peptide substrate is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6. 
     
     
         16 . A kit comprising
 a) a sample comprising a protein containing SEQ ID NO:1 and   b) a peptide substrate based on the NS5A/NS5B cleavage site for Hepatitis C genotype 1a.   
     
     
         17 . The kit according to  claim 16  further comprising an NS4A cofactor peptide comprising SEQ ID NO:4. 
     
     
         18 . The kit according to  claim 16  or  17 , wherein the peptide substrate is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6, or a combination thereof. 
     
     
         19 . The kit according to  claim 16 ,  17  or  18 , further comprising one or more known modulators of NS3 protease. 
     
     
         20 . The kit according to  claim 17 ,  18  or  19 , further comprising one or more known modulators of NS4A cofactor.