Showing papers by "Min Gao published in 2013"
TL;DR: It is suggested that during the 14-day monotherapy, most wild-type virus was eradicated by DCV, and viral fitness, rather than DCV resistance, probably determines which viral variants emerge as dominant in populations after the end of DCV treatment.
Abstract: Daclatasvir (DCV; BMS-790052) is a hepatitis C virus (HCV) NS5A replication complex inhibitor (RCI) with picomolar to low nanomolar potency and broad genotypic coverage in vitro. Viral RNA declines have been observed in the clinic for both alpha interferon-ribavirin (IFN-α-RBV) and IFN-RBV-free regimens that include DCV. Follow-up specimens (up to 6 months) from selected subjects treated with DCV in 14-day monotherapy studies were analyzed for genotype and phenotype. Variants were detected by clonal sequencing in specimens from baseline and were readily detected by population sequencing following viral RNA breakthrough and posttreatment. The major amino acid substitutions generating resistance in vivo were at residues M28, Q30, L31, and Y93 for genotype 1a (GT-1a) and L31 and Y93 for GT-1b, similar to the resistance substitutions observed with the in vitro replicon system. The primary difference in the resistance patterns observed in vitro and in vivo was the increased complexity of linked variant combinations observed in clinical specimens. Changes in the percentage of individual variants were observed during follow-up; however, the overall percentage of variants in the total population persisted up to 6 months. Our results suggest that during the 14-day monotherapy, most wild-type virus was eradicated by DCV. After the end of DCV treatment, viral fitness, rather than DCV resistance, probably determines which viral variants emerge as dominant in populations.
89 citations
TL;DR: The results support the potential use of DCV as a component in combination therapies for HCV3a chronic infection and identify NS5A residues 31 and 93 as sites for DCV-selected resistance.
Abstract: The NS5A replication complex inhibitor daclatasvir (DCV; BMS-790052) inhibits hybrid replicons containing hepatitis C virus (HCV) genotype 3a (HCV3a) NS5A genes with 50% effective concentrations (EC50s) ranging from 120 to 870 pM. Selection studies with a hybrid HCV3a replicon identified NS5A residues 31 and 93 as sites for DCV-selected resistance. Our results support the potential use of DCV as a component in combination therapies for HCV3a chronic infection.
51 citations
TL;DR: 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.
47 citations
TL;DR: A mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A found that specificSerine residues were important for efficient hyperphosphorylation of JFH2NS5A, and intragenic complementation of replication-defective NS5a alleles was demonstrated.
Abstract: Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypo- and hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans-complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of replication-defective NS5A mutations in trans-complementation assays. While some behaved similarly to the S232I replicon, others displayed a unique trans-complementation phenotype, suggesting that NS5A trans-complementation can occur by two distinct modes. Moreover, we were able, for the first time, to demonstrate intragenic complementation of replication-defective NS5A alleles. Our results identified three complementation groups: group A, comprising mutations within NS5A domain I; group B, comprising mutations affecting serine residues important for hyperphosphorylation and a subset of the domain I mutations; and group C, comprising a single mutation within the C-terminal region of domain II. We postulate that these complementation groups define three distinct and genetically separable functions of NS5A in RNA replication.
40 citations
TL;DR: The contribution of the core region to the overall topology of the pharmacophore, primarily vector orientation and planarity, was determined, with a set of analogs exhibiting <10 nM EC(50) in a genotype 1b replicon assay.
28 citations
TL;DR: Structural-activity relationship studies that uncovered an alternate phenylglycine-based cap series that exhibit further improvements in virology profile, along with some insights into the pharmacophoric elements associated with the GT-1a potency, are described.
17 citations
TL;DR: A 96-well based replicon elimination and colony formation assay is presented for comparing the resistance barrier of the hepatitis C virus NS5A replication complex inhibitor daclatasvir on three HCV genotypes in a proof of concept experimental protocol.
4 citations