Characterizations of HCV NS5A replication complex inhibitors

Abstract

The hepatitis C virus NS5A protein is an established and clinically validated target for antiviral intervention by small molecules. Characterizations are presented of compounds identified as potent inhibitors of HCV replication to provide insight into structural elements that interact with the NS5A protein. UV-activated cross linking and affinity isolation was performed with one series to probe the physical interaction between the inhibitors and the NS5A protein expressed in HCV replicon cells. Resistance mapping with the second series was used to determine the functional impact of specific inhibitor subdomains on the interaction with NS5A. The data provide evidence for a direct high-affinity interaction between these inhibitors and the NS5A protein, with the interaction dependent on inhibitor stereochemistry. The functional data supports a model of inhibition that implicates inhibitor binding by covalently combining distinct pharmacophores across an NS5A dimer interface to achieve maximal inhibition of HCV replication.

Synergistic Activity of Combined NS5A Inhibitors

“…We hypothesize that DCV binds to resistant NS5A dimers (a conformation different from the wild type [WT]) without disrupting NS5A function(s); however, DCV binding causes a conformational change that accommodates the binding of a 2nd inhibitor (synergist [Syn]) on adjacent NS5A dimers to disrupt function(s) of the oligomer. This model is further supported by studies indicating that NS5A compounds can bind to resistant mutants with no detectable consequence (6).…”

“…Yet, the synergistic functional interaction suggests the NS5A-Syn binding promotes perturbations outside the binding pocket(s). Cross-linking studies with NS5A inhibitors also indicate the presence of NS5A multimers inside cells (6). The formation of higher-order structures might endow NS5A with the ability to usurp cellular factors for viral advantage, similar to adenovirus ORF3, but it also reveals an Achilles' heel amenable to small-molecule targeting.…”

“…NS5A-Syn binding is proposed to be similar to DCV binding, since previous compounds related to this series have been shown to bind directly, although indirect effects cannot be ruled out (6,22). Located in a polymeric structure consisting of alternating NS5A dimers of differing conformations, one dimer form is postulated to interact with NS5A synergistic compounds, while another dimer form interacts with DCV (8,22).…”

“…The binding site(s) for NS5A RCIs has been modeled based on biochemical and crystal structure data, implicating at least one site that spans a region between NS5A proteins that associate as dimers (6). Available evidence for NS5A inhibitor binding suggests that NS5A RCIs, such as DCV, bind tightly and specifically to NS5A protein dimers present in infected cells (6).…”

“…Available evidence for NS5A inhibitor binding suggests that NS5A RCIs, such as DCV, bind tightly and specifically to NS5A protein dimers present in infected cells (6).…”