Background:  Hepatitis C virus (HCV) NS3 protease is essential to the viral lifecycle by cleaving at least 4 sites along the viral polyprotein, and for this reason, has been viewed as an attractive therapeutic target. Although several protease inhibitors have shown promise in clinical trials, drug resistance has been documented in both replicon studies and patient populations. From our previous studies of the balance of substrate recognition with the occurrence of drug resistance in HIV-1 protease, we found that most primary active site mutations do not extensively contact substrates, but are critical to inhibitor binding. We are extending this investigation to study drug resistance in HCV NS3 protease in the context of substrate recognition.

Methods:  Peptides corresponding to each of the 4 HCV NS3 substrates were modeled in the active site of full-length single-chain NS3 structure (1CU1). Crystal structures (from the database) of NS3 protease in complex with inhibitors were then superposed to determine areas where the inhibitor van der Waals surface extended beyond that of the bound substrates.

Results:  The molecular modeling of substrates in HCV NS3 provides a rationale that begins to explain a basis for drug resistance mutations to NS3 inhibitors at R155 and A156. In particular, substrate recognition is preserved when resistance occurs, as these residues appear to be making more extensive contacts with the inhibitors than they are making with the substrates. 

Conclusions:  Similar to our findings for HIV-1 protease, resistance to current NS3 protease inhibitors appear to occur in a manner that maintains substrate recognition. This implies that future NS3 protease inhibitors that fit better within the substrate binding region should be less susceptible to drug resistant mutations. We believe that drug design strategies can be utilized in the development of NS3 protease inhibitors, which are less susceptible to resistance and therefore more robust for HCV treatment.