DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

Abstract

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

“…1D). The common binding region corresponds to the most conserved residues of NS5A-D2 over all genotypes, with five residues (Pro-310, Trp-312, Ala-313, Pro-315, and Pro-319) that are strictly conserved (see Hanoulle et al (53) and further corresponds to the location where CsA-resistance mutations were identified (37,70). To further probe this common binding motif in NS5A-D2, we prepared a new NMR sample with the three different proteins in a 1:1:1 molecular ratio (i.e.…”

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

“…Cyclophilins affect the folding of proteins through their peptidyl-prolyl cis-trans isomerase activity, which catalyzes the cis-trans isomerisation of the prolyl peptide bond preceding proline residues. Due to the resistance mutation, HCV NS5A adopted a conformation that was no longer dependent on the cis-trans isomerisation activity of the cyclophilins for its function [61]. These examples of mutant HCV and CVB3 demonstrate that viruses can acquire resistance even to compounds that target crucial host factors.…”

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…NS5A is a nonstructural protein that has no defined role in the virus life cycle but is absolutely required for RNA replication (8 -10) and particle assembly (11)(12)(13)(14). It has also been shown to interact with a large number of host proteins including CypA (cyclophilin A) (15)(16)(17)(18)(19)(20)(21)(22). NS5A is peripherally anchored to membranes by an N-terminal amphipathic helix (23)(24)(25)(26)(27).…”

“…RNA was purified by centrifugation for 30 min at 13,000 ϫ g, washed with 70% ethanol, and suspended in 5 mM Tris, pH 8.0. Quantitative real time PCR was carried out as we described (18).…”