Interestingly, effective analogues were not affected by the L20F mutation, despite adamantyl moieties interacting identically with the Ama/Rim binding pocket. However, extended analogue side chains formed additional interactions with A41 and G46, which presumably overcame disruption caused by L20F. We next designed nonadamantane molecules using the “Draw” function in Maestro with a high predicted affinity for the J4 and JFH-1 binding sites. These were screened in a subgenomic replicon for effects on HCV RNA replication and cell viability

(data not shown).21 Compound CD (Fig. 5A) both inhibited GT1b p7 activity in vitro and showed an equivalent antiviral effect to Rim, to which L20F virus was resistant (Fig. 5B,C). To our knowledge, CD is the first molecule designed entirely against a de novo molecular model to display an antiviral effect in learn more culture. GT3a 452 isolate p7 displays resistance to NN-DNJ in vitro and in culture.21 This provided an excellent basis to investigate whether IS targeted oligomerization and to identify resistance polymorphisms. DHPC induces oligomerization of IS-sensitive J4 p7 in vitro, inducing heptameric complexes equivalent to liposomes.31 We therefore assessed

whether IS or Rim blocked oligomerization SCH727965 of J4 and 452 p7. NN-DNJ abrogated J4 p7 oligomerization and channel activity, yet 452 p7 activity was insensitive to this drug and oligomerization was not affected (Fig. 6A). Rim did not affect oligomerization, but it inhibited channel activity in both cases, confirming separate modes of action for these inhibitor classes. Comparing NN-DNJ binding sites revealed variation between J4 and 452 (Fig. 1C), however alignment with other p7 sequences revealed an F25A polymorphism to be covariant with IS resistance. F25 is located on a predicted bulge in the p7 N-terminal helix, which may link with adjacent protomers, but is also predicted to interact with IS head groups (Fig. 1B). We previously showed that J4 F(22, 25, 26)/A p7 formed hyperactive channels

in vitro that retained Ama sensitivity.31 We therefore tested whether this mutant or F25A in isolation could rescue p7 oligomerization from NN-DNJ. Both J4 mutant proteins and JFH-1 F25A p7 were insensitive to NN-DNJ check in vitro and displayed hyperactive channel phenotypes, consistent with a more open-form channel structure (Fig. 6B). Native PAGE again correlated IS resistance with the formation of drug-resistant oligomeric complexes (Fig. 6C). Interestingly, the major species formed by JFH-1 F25A p7 oligomer migrated more rapidly than other proteins, yet was stable in the presence of NN-DNJ; some heptameric JFH-1 F25A protein was also apparent. All mutant proteins remained sensitive to Rim in vitro (data not shown). We next tested F25A in cell culture and, despite a modest decrease in particle production, the mutant was resistant to both NN-DNJ and N-nonyl deoxygalactonojirimycin (NN-DGJ), but not Rim (Fig. 6D).