Author:

Abstract:

The last decade great progress has been made in the development of direct-acting antivirals (DAA) against HCV. The RNA-dependent RNA polymerase of HCV is essential for viral RNA replication and is thus an excellent target for DAA. Non-nucleoside polymerase inhibitors based on a benzimidazole or indole scaffold have been reported. Compounds of this class can inhibit HCV replication by interacting with thumb domain 1 of the HCV polymerase. Escape mutants that confer resistance to these inhibitors in vitro map to amino acids P495, P496 or V499. We report a novel resistance mutation (T389S/A) that was identified following resistance selection with the benzimidazole non-nucleoside polymerase inhibitor JT-16 in a HCV genotype 1b subgenomic replicon. Clonal sequencing analysis of the JT-16res replicon revealed that the T389S mutation was present in 60% of all clones sequenced, whereas the P495A mutation was only identified in 2 clones (= 13%). Introduction of mutations T389A or T389S into a wild-type backbone induced moderate levels of resistance to JT-16 (7- and 13-fold, respectively). In contrast, P495A is associated with high level resistance (44-fold). Furthermore, the replication fitness of the T389S mutant was significantly higher than that of P495A. By means of molecular modelling a structural hypothesis was formulated to explain the emergence of the T389S/A mutation in the JT-16 resistant replicon. A ligplot interaction map of residue T389 showed that T389 makes H-bonding interactions with C488, D387 and K491. Mutation of threonine 389 into an alanine or serine would disrupt this H-bonding network and possibly induce a change of rotameric state of the side chain of K491, thereby interfering with the binding of the JT-16 inhibitor. In conclusion, we demonstrated that less resistant, but fitter variants can develop during in vitro resistance selection with the benzimidazole inhibitor JT-16. Surprisingly, the previously published signature mutation for benzimidazole resistance, P495A, was only detected in 13% of the resistant population. Our data show that structural modifications to inhibitors with a similar scaffold can affect the (pattern of) resistance mutations that emerge during resistance selection.