The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors

Abstract

New direct acting antivirals (DAAs) such as daclatasvir (DCV; BMS-790052), which target NS5A function with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however, major resistance mutations appear in the N-terminal region of NS5A that include the amphipathic helix and domain 1. The dimeric symmetry of these compounds suggests that they act on a dimer of NS5A, which is also consistent with the presence of dimers in crystals of NS5A domain 1 from genotype 1b. Genotype 1a HCV is less potently affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A homologue and intriguingly, our X-ray crystal structure reveals two new dimeric forms of this domain. Furthermore, the high solvent content (75%) makes it ideal for ligand-soaking. Daclatasvir (DCV) shows twofold symmetry suggesting NS5A dimers may be of physiological importance and serve as potential binding sites for DCV. These dimers also allow for new conformations of a NS5A expansive network which could explain its operation on the membranous web. Additionally, sulfates bound in the crystal structure may provide evidence for the previously proposed RNA binding groove, or explain regulation of NS5A domain 2 and 3 function and phosphorylation, by domain 1.

Synergistic Activity of Combined NS5A Inhibitors

“…The picomolar potency of DCV toward diverse HCV genotypes coupled with the abundance of the NS5A protein in infected cells supports a polymer-based interaction model of NS5A dimers, in which the binding of DCV to one NS5A dimer influences proximal and distal NS5A dimers that are arranged in a polymeric structure. The polymer-based interaction model (18,21) is consistent with our recently reported observations with NS5A synergists (22). 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.…”

“…A recent report of two additional crystal forms of NS5A domain 1 (gt 1a), together with the previously reported structures (17)(18)(19), suggests that NS5A domain 1 has…”

“…A recent report of two additional crystal forms of NS5A domain 1 (gt 1a), together with the previously reported structures (17)(18)(19), suggests that NS5A domain 1 has multiple conformations and a flexibility that may be important for an association of NS5A function and dimers. The ability to model the binding of NS5A compounds in a nonsymmetrical manner may also reflect this property (20).…”

Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity In Vitro

“…The protein is phosphorylated and organized into three domains (5,(54)(55)(56)(57)(58)(59). The structure of full-length NS5A has not yet been solved; however, three different crystal structures of N-terminal domain I of NS5A have been published (60)(61)(62). The structure published by Tellinghuisen et al (60) shows domain I as a homodimer with a positively charged cleft that can serve as a potential RNA binding groove.…”

“…In contrast, the cleft is absent in the second published structure (61), and the proposed RNA binding surfaces of monomers that formed the cleft in the first structure are exposed to the solvent and face away from each other. In the most recent crystal structure of NS5A domain I, two new dimeric forms of this domain were observed (62). The biological significance of these configurations of NS5A dimeric structure is unknown.…”