Synergistic Activity of Combined NS5A Inhibitors

Abstract

). To extend the characterization of NS5 A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV-NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV-NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function. Hepatitis C virus (HCV) infections can be effectively cured using combinations of small-molecule direct-acting antivirals (DAAs) with different mechanisms of action. The nonstructural 5A replication complex inhibitor (NS5A RCI) class of compounds represented by daclatasvir (DCV) (BMS-790052, Daklinza; Bristol-Myers Squibb) has activity against genotypes (gts) 1a, 1b, 2a, 3a, 4a, 5a, and 6a and is the most potent class of HCV inhibitor yet described (1-3). NS3 protease inhibitors and NS5B polymerase inhibitors have thus far been the preferred DAA partners for DCV combination therapies (1). Combinations of small-molecule DAAs are required for effective curative oral therapies, because resistant variants existing in the quasispecies population in untreated HCV-infected patients are enriched during monotherapy. To prevent viral breakthrough or relapse and selection of resistant variants, multiple highly effective pangenotypic DAA combination therapies, with NS5A RCIs as the backbone, have been approved or are currently being developed (1, 4, 5).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).A recent report (7) of DCV binding to Escherichia coli-expressed NS5A protein provided the first direct evidence that DCV binds to NS5A. The authors demonstrated that DCV binding can reduce the affinity of NS5A protein for viral RNA, supporting one potential mechanism of inhibition. An additional report of direct binding to NS5A protein of DCV and the related compound ledipasvir also supports the direct associatio...

Design and Synthesis of Novel Symmetric Fluorene-2,7-Diamine Derivatives as Potent Hepatitis C Virus Inhibitors

“…This is supported by a previous manuscript that shows the synergistic effect between the NS5A inhibitor daclatasvir and other NS5A inhibitors [ 25 ].…”

The Discovery and Development of Daclatasvir: An Inhibitor of the Hepatitis C Virus NS5A Replication Complex

“…This result represented a 2,600-fold increase in the sensitivity of the Tyr93Asn replicon to 1 in the presence of 52. The synergistic relationship between 1 and 52 was confirmed in a reciprocal experiment where 52 was titrated in the presence of suboptimal amount of 1 affording a similar outcome [60,61].…”

“…These studies have suggested that the binding of inhibitors to NS5A interferes with the association of viral RNA with the protein, with the binding of compounds competed out by other NS5A inhibitors and demonstrating diminished affinity for the Tyr93His mutant protein [58,59]. However, profiling of inhibitors in cell-based assays has indicated that disruption of RNA binding to NS5A does not appear to occur and that the introduction of key resistant mutations leads to only a modest reduction in the binding of inhibitors [60,61]. Studies with 50 in resistant GT-1b replicons indicated that while the Tyr93His-resistant mutation reduced inhibitory potency by 220-fold, an estimate of the amount of NS5A protein pulled down by the chemical probe, as determined by an analysis of Western blots, suggested similar levels of protein-drug association for the resistant and wild-type strains [60,61].…”

“…However, profiling of inhibitors in cell-based assays has indicated that disruption of RNA binding to NS5A does not appear to occur and that the introduction of key resistant mutations leads to only a modest reduction in the binding of inhibitors [60,61]. Studies with 50 in resistant GT-1b replicons indicated that while the Tyr93His-resistant mutation reduced inhibitory potency by 220-fold, an estimate of the amount of NS5A protein pulled down by the chemical probe, as determined by an analysis of Western blots, suggested similar levels of protein-drug association for the resistant and wild-type strains [60,61]. Adding further to the complexity of the biochemistry was the observation that in a protein pull-down experiment, an attempt to outcompete the biotinylated tool compound 50 with a non-biotinylated analogue in a GT-1b Tyr93His-containing mutant (EC 50 ¼ 290 nM for stated analogue vs >7 μM for 50) not only failed but, at low concentration, appeared to have incrementally enhanced the amount of NS5A pulled down.…”

HA1077 displays synergistic activity with daclatasvir against hepatitis C virus and suppresses the emergence of NS5A resistance-associated substitutions in mice

“…Therefore, emergence of DAA RAVs is yet a major drawback of DAAs. To overcome this, combination therapies using DAAs become now representative approaches for HCV treatment 10 – 15 . Nevertheless, new combination therapies still need to be developed for patients with RAVs who do not respond to current DAAs and for those with difficult-to-treat HCV genotypes such that alternative or advanced treatment options can be provided for better management of HCV infection 4 .…”