These experiments demonstrate the subtlety and complexity inherent to p7/inhibitor interactions and explain why variations in protein sequence or inhibitor structure can result in different experimental outcomes. Such studies will, however, inform the future development of more potent compounds through iterative refinement and improvement of rational drug MG-132 in vitro design. From a therapeutics perspective, alkylated IS p7 inhibitors

acted through a mechanism distinct from that of adamantanes, providing scope for the development of parallel yet complimentary p7 inhibitor series. In agreement with previous studies,15, 22 docking programs predicted that nonylated IS bound p7 protomers >10-fold more avidly than those with butyl side chains, occluded more of the p7 protomer interface and so disrupted channel oligomerization. IS compounds disrupted J4 p7 oligomerization and channel activity, but not that of resistant 452 protein.21 F residues have been purported to stabilize p7 oligomerization through hydrophobic interactions48 and F25 is predicted to interact with IS head groups in GT1b p7. In 452 p7, F25 is changed to A, and this polymorphism was shown to mediate IS resistance both in vitro and in culture while remaining Rim sensitive. F25A mutants also formed hyperactive channel complexes in

vitro which, in the Lumacaftor case of JFH-1, appeared to be less stable and migrated differently by native PAGE. Nevertheless, F25A HCV genomes were viable in culture, again showing a low fitness cost for the development of p7 inhibitor resistance. Resistance to p7 inhibitors mediated by single amino acid changes with little consequence for virus fitness readily explains their ineffectiveness

in clinical trials combined with IFN/Rib. Virus rebound has been noted during amantadine mono-26 and triple therapy.27 In addition, relatively high IC50 values compared with other STAT-C molecules Cyclooxygenase (COX) and a maximal reduction in virus production of ∼2log10 even for combinations of p7 inhibitors understandably generates skepticism over their usefulness. However, Rim and IS IC50 values in HCV culture are similar to those in influenza A virus and HIV in vitro/culture systems, where they progressed to clinical and trial-stage use in humans, respectively. Given the relatively high degree (∼30% of patients) of breakthrough in trials combining NS3 inhibitors with IFN/Rib,49 the recent success of STAT-C combinations,50 and lessons from HIV, expanding the STAT-C repertoire should be an immediate and ongoing priority. The availability of prototype p7 inhibitors could rapidly expedite this process, and future p7 inhibitors could complement STAT-C therapies as these are implemented over the next decade as an understanding of the molecular basis of resistance assists in the design of novel, more potent compounds.