Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A

Sun, Jin-Hua; O’Boyle, Donald R.; Fridell, Robert A.; Langley, David R.; Wang, Chunfu; Roberts, Susan B.; Nower, Peter T.; Johnson, Benjamin M.; Moulin, Frederic; Nophsker, Michelle; Wang, Ying-Kai; Liu, Mengping; Rigat, Karen; Tu, Yong; Hewawasam, Piyasena; Kadow, John F.; Meanwell, Nicholas A.; Cockett, Mark I.; Lemm, Julie A.; Kramer, Melissa; Belema, Makonen; Gao, Min

doi:10.1038/nature15711

Cited by 47 publications

(77 citation statements)

References 24 publications

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“…It has been reported that the NS5A synergistic compound Syn-395, in a triple combination containing DCV, is as effective against gt 1a as either the NS3 or the NS5B inhibitor that it replaced (22). The initial observations reported for Syn-395 are confirmed and extended in this study of gts 1a, 1b, 2a, 3a, 4a, 5a, and 6a using Syn-690 and Syn-535 ( Fig.…”

Section: Resultssupporting

confidence: 76%

“…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.…”

supporting

confidence: 72%

“…Assays were conducted as previously described, with minor modifications (22)(23)(24). Briefly, virus stocks harvested from transfected cell medium were used to infect fresh Huh-7.5 cells in T175 flasks and were monitored for luciferase activity.…”

Section: Methodsmentioning

confidence: 99%

“…NS5A synergists were discovered during the characterization of NS5A RCI specificity, when pulldown experiments in NS5A replicon cells revealed compounds that competed for binding even though they had little inhibitory activity (22). In addition, some weakly active compounds in combination with DCV were found to enhance inhibitory potency (22).…”

mentioning

confidence: 99%

“…In addition, some weakly active compounds in combination with DCV were found to enhance inhibitory potency (22). The observed synergistic effect led us to investigate the structure-activity relationship (SAR) of this chemotype on DCV-resistant variants.…”

mentioning

confidence: 99%

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Synergistic Activity of Combined NS5A Inhibitors

O’Boyle

Nower

Gao

et al. 2016

Antimicrob Agents Chemother

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). 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...

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Section: Resultssupporting

confidence: 76%

supporting

confidence: 72%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Synergistic Activity of Combined NS5A Inhibitors

O’Boyle

Nower

Gao

et al. 2016

Antimicrob Agents Chemother

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Impact of HCV genotype on treatment regimens and drug resistance: a snapshot in time

Cuypers

Ceccherini‐Silberstein

Laethem

et al. 2016

Reviews in Medical Virology

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The introduction of highly potent direct-acting antivirals (DAAs) has revolutionized hepatitis C virus treatment. Nevertheless, viral eradication worldwide remains a challenge also in the era of DAA treatment, because of the high associated costs, high numbers of undiagnosed patients, high re-infection rates in some risk groups and suboptimal drug efficacies associated with host and viral factors as well as advanced stages of liver disease. A correct determination of the HCV genotype allows administration of the most appropriate antiviral regimen. Additionally, HCV genetic sequencing improves our understanding of resistance-associated variants, either naturally occurring before treatment, acquired by transmission at HCV infection, or emerging after virological failure. Because treatment response rates, and the prevalence and development of drug resistance variants differ for each DAA regimen and HCV genotype, this review summarizes treatment opportunities per HCV genotype, and focuses on viral genetic sequencing to guide clinical decision making. Although approval of the first pan-genotypic DAA-only regimen is expected soon, HCV genetic sequencing will remain important because when DAA therapies fail, genotyping and resistance testing to select a new active DAA combination will be essential. Copyright © 2016 John Wiley & Sons, Ltd.

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NS5A as a Target for HCV Drug Discovery

O'boyleii

Gao²

2019

Topics in Medicinal Chemistry

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Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A

Cited by 47 publications

References 24 publications

Synergistic Activity of Combined NS5A Inhibitors

Synergistic Activity of Combined NS5A Inhibitors

Impact of HCV genotype on treatment regimens and drug resistance: a snapshot in time

NS5A as a Target for HCV Drug Discovery

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