18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

ANALYSING DRUG RESISTANCE IN TERMS OF SUBSTRATE RECOGNITION IN HCV NS3/4A PROTEASE

Antivir Ther 2009; 14 Suppl 1:A36 (abstract no. 34)

K Romano and C Schiffer
University of Massachusetts Medical School, Worcester, MA, USA

BACKGROUND: Hepatitis C virus (HCV) infects an estimated 180 million people worldwide causing over half of all liver cancer cases. The bifunctional NS3/4A protein is a prime therapeutic target as both protease and helicase functions are required for viral infection and replication. The first series of NS3/4A protease inhibitors in clinical trials are the result of recent structure-based drug design efforts. Although several of these inhibitors show promise, such as SCH503034 (Schering–Plough), drug resistance is being reported both in replicon studies and in clinical trials. Alarmingly, single site mutations at A156 and R155 have been shown to confer high-level multidrug resistance. A more detailed understanding of the molecular basis behind these observed resistance profiles is crucial to the development of future drugs that are less susceptible to drug resistance.

METHODS: In this study, we investigated the balance between substrate recognition by NS3/4A protease and the occurrence of drug resistance. Peptides corresponding to post-cleavage products (4A4B and 5A5B) were crystallized in complex with the inactive protease domain of NS3/4A. Structural alignments were performed to compare these structures with those of full-length NS3/4A (1CU1) and NS3/4A protease in complex with SCH503034 (2OC8). A structural analysis was then carried out to determine regions where the inhibitor bound relative to NS3/4A products.

RESULTS: Structural analysis reveals relative shifts in the region that contacts peptide products and SCH503034; however, bound ligands are shifted by the same degree in this area such that a common binding modality is preserved. When NS3/4A products are superposed, primary sites of drug resistance, such as A156 and R155, do not extensively contact substrates but are crucial to inhibitor binding.

CONCLUSIONS: These results imply that drug resistance is occurring in a manner that selectively weakens inhibitor binding, but maintains substrate recognition and cleavage. Therefore, future NS3/4A protease inhibitors that fit better within the substrate binding region should be less susceptible to drug resistant mutations. As we have observed previously in the system of HIV-1 protease, drug design strategies that incorporate this constraint can lead to the development novel protease inhibitors less susceptible to resistance and more robust in HCV treatment.

2009-06-09
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