Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ϳ5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.Hepatitis C virus (HCV) is the major causative agent for non-A, non-B hepatitis worldwide, which affects more than 3% of the world population. HCV establishes chronic infections in a large percentage of infected individuals, increasing the risk for developing liver cirrhosis and, in some cases, hepatocellular carcinoma. Although the current standard of care for HCV infection involves the use of PEGylated interferon and ribavirin, a large proportion of patients fail to respond to this therapy, and treatment is associated with frequent and sometimes serious side effects (9). Given the limited efficacy of the current therapy, the development of safer and more effective therapies is of tremendous importance.HCV is a positive-strand RNA virus belonging to the family Flaviviridae. The HCV genome consists of a ϳ9.6-kb RNA with a large open reading frame encoding a polyprotein of ϳ3,010 amino acids. The polyprotein is cleaved co-and posttranslationally by both cellular and viral proteases into at least 10 different products (10, 11). The viral proteins required for RNA replication include NS3, NS4A, NS4B, NS5A, and NS5B (4, 19). NS3 consists of an amino-terminal protease domain required for the cleavage of the remaining nonstructural proteins and a carboxyl-terminal helicase/NTPase domain (8,11,30). NS4A serves as a cofactor for NS3 protease and helicase activities (8). NS4B is a...
Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.
BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC 50 ) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC 50 s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC 50 , 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC 50 s of >4 M) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC 50 . Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6-to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were >10-fold above the inhibitor EC 50 s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.
Three hepatitis C virus (HCV) inhibitors, asunaprevir (ASV; BMS-650032), daclatasvir (DCV; BMS-790052), and BMS-791325, each targeting a different nonstructural protein of the virus (NS3, NS5A, and NS5B, respectively), have independently demonstrated encouraging preclinical profiles and are currently undergoing clinical evaluation. Since drug-resistant variants have rapidly developed in response to monotherapy with almost all direct-acting antiviral agents (DAAs) for HCV, the need for combination therapies to effectively eradicate the virus from infected patients is clear. These studies demonstrated the additivesynergistic effects on replicon inhibition and clearance of combining NS3 protease or NS5B RNA polymerase inhibitors with the first-in-class, NS5A replication complex inhibitor daclatasvir (DCV) and reveal new resistance pathways for combinations of two small-molecule inhibitors that differ from those that develop during monotherapy. The results suggest that under a specific selective pressure, a balance must be reached in the fitness costs of substitutions in one target gene when substitutions are also present in another target gene. Further synergies and additional novel resistance substitutions were observed during triple-combination treatment relative to dual-drug therapy, indicating that, in combination, HCV inhibitors can exert cross-target influences on resistance development. Enhanced synergies in replicon inhibition and a reduced frequency of resistance together lend strong support to the utility of combinations of DAAs for the treatment of HCV, and the identification of altered resistance profiles during combination treatment provides useful information for monitoring resistance in the clinic. H epatitis C virus (HCV) is a positive-stranded RNA virus in theFlaviviridae family of enveloped virions which affects an estimated 170 million people worldwide and is the major cause of chronic hepatitis. Currently, approximately 50% of patients infected with genotype 1 (gt 1), the most prevalent form of the virus, fail to achieve a sustained reduction in viral load with therapy employing pegylated alpha interferon (IFN-␣) plus ribavirin (alfa/RBV) (52,54,56). A substantial fraction (20%) of chronically infected patients develop serious progressive liver disease, including cirrhosis or hepatocellular carcinoma. alfa/RBV treatment is associated with a high incidence (Ͼ30%) of adverse effects, some of which are of sufficient severity to cause patients to discontinue therapy (56). Despite the recent approval of two new direct-acting antiviral agents (DAAs), boceprevir and telaprevir, for use in combination with alfa/RBV (18, 47), their use may be limited by poor efficacy in some patient populations, inconvenient 3-times-daily dosing of the DAA, and association with side effects, including anemia, rash, and gastrointestinal effects, in addition to the well-documented spectrum of adverse effects associated with alfa/RBV. Although addition of these DAAs to the standard of care for HCV represents a significant i...
The hepatitis C virus (HCV) NS5B replicase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Inspired by the overlay of bound structures of three structurally distinct NS5B palm site allosteric inhibitors, the high-throughput screening hit anthranilic acid 4, the known benzofuran analogue 5, and the benzothiadiazine derivative 6, an optimization process utilizing the simple benzofuran template 7 as a starting point for a fragment growing approach was pursued. A delicate balance of molecular properties achieved via disciplined lipophilicity changes was essential to achieve both high affinity binding and a stringent targeted absorption, distribution, metabolism, and excretion profile. These efforts led to the discovery of BMS-929075 (37), which maintained ligand efficiency relative to early leads, demonstrated efficacy in a triple combination regimen in HCV replicon cells, and exhibited consistently high oral bioavailability and pharmacokinetic parameters across preclinical animal species. The human PK properties from the Phase I clinical studies of 37 were better than anticipated and suggest promising potential for QD administration.
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