BMS NS5A HCV Inhibitor - chemical genetics discovery process in Nature

BMS NS5A HCV Inhibitor - chemical genetics
discovery process in Nature jnl - pdf attached


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"Individuals infected with HCV are at considerable risk of developing liver cirrhosis, end-stage liver disease and hepatocellular carcinoma.....We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication.....we screened over one million compounds from the Bristol-Myers Squibb proprietary collection in high throughput mode.....The results with BMS-790052 provide clinical validation for the first in a new class of HCV inhibitors that target a viral protein with no known enzymatic function and an as yet poorly understood role in viral replication16, 22. The strategy used to identify a lead HCV NS5A inhibitor and to optimize this molecule into a clinical candidate offers a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics......In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered to subjects with genotype 1 chronic HCV at doses of 1, 10 and 100mg as an oral solution. All subjects were infected with HCV genotype 1a except for two subjects at 10mg and three subjects at 100mg who were infected with genotype 1b. BMS-790052 was safe and well tolerated in HCV-infected subjects after single oral doses up to 100mg.....BMS-790052 had a mean plasma elimination half-life ranging from 10 to 14h, and plasma drug levels were similar to those in non-HCV-infected subjects. After single oral doses of 10-100mg BMS-790052, all subjects had 24-h plasma concentrations above the tenfold protein binding-adjusted EC90 for HCV genotypes 1a and 1b, suggesting the possibility for once daily administration.....A single milligram dose of BMS-790052 produced a mean 1.8log10 reduction (range 0.2-3.0log10) in HCV viral load measured 24h after drug administration. Both the 10 and 100mg doses produced a greater antiviral effect, with mean plasma viral RNA falling by 3.2 log10 (range 2.9-4.0log10) and 3.3log10 (range 2.7-3.6log10), respectively, at 24h post-dose. Moreover, the 100mg dose resulted in a mean maximal HCV RNA decline of 3.6 log10 (range 3.0-4.1log10) and a prolonged antiviral response was observed in two subjects infected with genotype 1b virus, with an HCV RNA measurement that reached the lower limit of quantification (less than 25IUml-1) in one subject"

BMS - Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect - pdf of full article attached

Letters to Nature
Nature , | doi:10.1038/nature08960; Received 29 October 2009; Accepted 26 February 2010; Published online 21 April 2010

Min Gao1, Richard E. Nettles2, Makonen Belema3, Lawrence B. Snyder3, Van N. Nguyen3, Robert A. Fridell1, Michael H. Serrano-Wu3, David R. Langley4, Jin-Hua Sun1, Donald R. O'Boyle II1, Julie A. Lemm1, Chunfu Wang1, Jay O. Knipe5, Caly Chien2, Richard J. Colonno1, Dennis M. Grasela2, Nicholas A. Meanwell3 & Lawrence G. Hamann3

1. Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, USA
2. Department of Discovery Medicine and Clinical Pharmacology, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, USA
3. Department of Discovery Chemistry,
4. Department of Computer-Aided Drug Design,
5. Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, USA

Correspondence to: Nicholas A. Meanwell3 Correspondence and requests for materials should be addressed to N.A.M. (Email: Nicholas.Meanwell@bms.com).

Abstract

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200million people1. Current therapy relies upon a combination of pegylated interferon-α and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus2, 3. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B4. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC50) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3log10 reduction in mean viral load measured 24h post-dose that was sustained for an additional 120h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.

We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication. Our differentiating strategy centred on the identification of compounds functionally distinct from those acting on the traditional targets of antiviral research in this field, the NS3 protease and the NS5B RNA-dependent RNA polymerase5, 6, 7. Using a Con-1 genotype 1b replicon replicating in Huh-7 liver cells8, we screened over one million compounds from the Bristol-Myers Squibb proprietary collection in high throughput mode. We used a dual assay format that simultaneously evaluated replication of the related flavivirus bovine viral diarrhoea virus (BVDV) and host cell cytotoxicity as an efficient means of preliminarily eliminating compounds without specificity for HCV9. Active inhibitors were further triaged by evaluation in biochemical assays for NS3 protease, NS3 helicase and NS5B polymerase in addition to screening against a panel of unrelated viruses. The iminothiazolidinone BMS-858 (Fig. 1) emerged as a weak but, more importantly, specific inhibitor of HCV RNA replication (EC50 = 0.57µM, half-maximum cytotoxic concentration (CC50) = >50µM) for which resistance was mapped to a tyrosine to histidine substitution at residue 93 in the NS5A protein (Y93H or Y2065H in the HCV polyprotein)10. BMS-858 formed the basis of an extensive series of chemical refinements that focused on improving antiviral potency, broadening inhibitory activity to encompass the HCV 1a genotype, and optimizing for oral bioavailability and sustained pharmacokinetic properties. After defining symmetry as an important contributor to antiviral activity10, a discovery that preceded the disclosure of structural information (see below), we subsequently identified BMS-790052 (Fig. 1) as a development candidate for advancement into clinical trials11. (The studies leading to the identification of BMS-790052 and its preclinical profiling will be the subject of a series of separate publications.) This compound is the most potent HCV replication inhibitor reported so far, with mean EC50 values of 50 and 9pM against genotype 1a and 1b replicons, respectively (data summarized in Table 1). BMS-790052 displays a therapeutic index (CC50/EC50) of at least 100,000 in vitro and is inactive towards a panel of 10 RNA and DNA viruses, with EC50 values greater than 10µM, which confirms specificity for HCV (Supplementary Tables 1 and 2).


Upon further analysis, we determined that the inhibitory activity of BMS-790052 maps to the first 100 amino acids of HCV NS5A (see below). The antiviral activity of the compound towards additional genotypes was assessed by using replication-competent 1a12 or 1b replicons to construct hybrids in which the entire NS5A coding region or the first 100 amino acids of NS5A from genotypes 2a, 3a, 4a and 5a replaced the corresponding sequence of the parent replicon13. This approach has been validated using genotype-specific inhibitors of HCV NS5A10. As summarized in Table 1, BMS-790052 demonstrates potent inhibitory activity towards all genotypes tested, with EC50 values ranging from 9 to 146pM. In combination studies, BMS-790052 displayed additive-to-synergistic effects with interferon-α/ribavirin, an inhibitor of NS3 protease (ITMN-191), and both nucleoside and allosteric inhibitors of NS5B polymerase, which is indicative of the potential of this molecule as a candidate for combination therapy with other HCV therapeutic agents (data summarized in Table 2) (Supplementary Tables 3-7). More significantly, BMS-790052 is a potent inhibitor of the JFH-1 genotype 2a infectious virus that replicates in cell culture (EC50 = 28pM), an assay considered to be a more biologically relevant in vitro cell culture system14. In addition, BMS-790052 displayed similar potency in Huh-7, HeLa and HEK293T cells (Supplementary Table 8), demonstrating that the function(s) of NS5A inhibited by BMS-790052 is (are) highly conserved in different cellular environments.

To confirm that NS5A inhibitors bind to the viral protein, we prepared the biotin-tagged derivative 1, with the natural (S)-configuration at both proline stereocentres, and its diastereomer 2 (Fig. 1). Compound 1 inhibited subgenomic viral RNA replication in the Con-1 genotype 1b replicon with an EC50 of 33nM but was inactive towards a Y93H replicon (EC50>10µM) whereas the diastereomer 2, used as a control, was inactive in vitro (EC50>10µM). After exposure to active inhibitor 1, the replicon lysate was passed over streptavidin agarose beads, separated by SDS-polyacrylamide gel electrophoresis and probed with an antibody specific for HCV NS5A, with a parallel experiment using the inactive diastereomer 2 serving as a control. NS5A could only be pulled down efficiently with active inhibitor 1 but not by the inactive compound 2 (Fig. 2); NS3 and NS5B were not detected in pull-down experiments with 1, suggesting selective binding to NS5A. In a similar experiment conducted with replicons expressing BVDV RNA, compound 1 failed to bind to BVDV NS5A.

Table 3: Resistance profile of genotype 1a and 1b replicons exposed to BMS-790052

HCV NS5A is a 447 amino-acid, zinc-binding phosphoprotein that plays a critically important but enigmatic role in the virus life cycle16. The substitutions conferring resistance to BMS-790052 map to domain I of the protein, which incorporates an amphipathic amino (N)-terminal α-helix (residues 5-25) thought to anchor the protein to the membrane16. The solid-state structure of fragments of domain I (residues 36-198 and 33-191) of HCV NS5A reveal dimeric complexes with patterns of interfacial recognition that anticipate an oligomeric form of the protein17, 18, postulated to sequester RNA within the replication complex19. The unique palindromic topology of BMS-790052 complements the dimeric structure of the NS5A protein. The location of the resistant substitutions suggests that the compound binds across the dimer interface, proximal to the N terminus of domain I between the protein and the membrane, and on the face opposite that of the putative RNA binding domain. We speculate that BMS-790052 may interfere with the precision of dimer association, effecting subtle structural distortions that directly or allosterically interfere with protein function. Under these circumstances, the exquisite inhibitory potency exhibited by BMS-790052 in vitro may be related to the disruption of the function of a limited number of NS5A dimers that compromise the activity of an oligomeric complex. However, although this hypothesis allows for an amplification of inhibitory effect, an alternative explanation relies on the inhibition of two or more activities of NS5A that interact synergistically. Further studies with BMS-790052 will be required to define its exact mode of action more fully and, conversely, the compound is a useful tool with which to study aspects of HCV NS5A function in viral replication. These investigations may be aided by compounds with an in vitro profile similar to 1, which is restricted to genotype 1b inhibition10.

BMS-790052 exhibited no significant effects in an extensive battery of in vitro receptor binding and enzymatic assays designed to assess promiscuity. Despite a molecular mass of over 700daltons, the compound is orally bioavailable in four preclinical species, with plasma levels readily achieved that surpass the in vitro EC50, and it distributes effectively to the liver.

In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered at six dose levels to healthy, non-HCV-infected subjects over a range of 1 to 200mg as an oral solution. The compound was safe and well tolerated up to 200mg with no clinically relevant adverse effects. After oral administration, BMS-790052 was readily absorbed, with dose-proportional exposures over the studied dose range, and all subjects had drug concentrations greater than the protein-binding-adjusted EC90 for genotypes 1a and 1b, as measured in the replicon assay, at and beyond 24h post-dose (Fig. 3). (The protein binding-adjusted EC90 figures were derived from an analysis of the effect of the addition of human serum on antiviral activity in replicons. In the presence of 40% human serum, the EC90 for BMS-790052 is 383pM (0.28ngml-1) for the genotype 1a replicon and 49pM (0.04ngml-1) for the genotyope 1b replicon.)

In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered to subjects with genotype 1 chronic HCV at doses of 1, 10 and 100mg as an oral solution. All subjects were infected with HCV genotype 1a except for two subjects at 10mg and three subjects at 100mg who were infected with genotype 1b. BMS-790052 was safe and well tolerated in HCV-infected subjects after single oral doses up to 100mg. Specifically, there were no deaths, serious adverse events, discontinuations due to adverse events or clinically relevant adverse effects. Headache was the most frequent adverse event, reported by four subjects after administration of BMS-790052. In HCV-infected subjects, BMS-790052 had a mean plasma elimination half-life ranging from 10 to 14h, and plasma drug levels were similar to those in non-HCV-infected subjects. After single oral doses of 10-100mg BMS-790052, all subjects had 24-h plasma concentrations above the tenfold protein binding-adjusted EC90 for HCV genotypes 1a and 1b, suggesting the possibility for once daily administration. The plasma HCV RNA levels were measured for up to 6days after administration; the mean decline from the time of administration to 144h post-dose is shown in Fig. 4. A single milligram dose of BMS-790052 produced a mean 1.8log10 reduction (range 0.2-3.0log10) in HCV viral load measured 24h after drug administration. Both the 10 and 100mg doses produced a greater antiviral effect, with mean plasma viral RNA falling by 3.2 log10 (range 2.9-4.0log10) and 3.3log10 (range 2.7-3.6log10), respectively, at 24h post-dose. Moreover, the 100mg dose resulted in a mean maximal HCV RNA decline of 3.6 log10 (range 3.0-4.1log10) and a prolonged antiviral response was observed in two subjects infected with genotype 1b virus, with an HCV RNA measurement that reached the lower limit of quantification (less than 25IUml-1) in one subject and 35IUml-1 in the other measured at hour 144. Genotypic analysis of samples taken at baseline (T0), 24 (T24) and 144 (T144) hours post-dose revealed that, in general, a marked reduction in viral load was required to detect major HCV variants. Substitutions were observed at amino-acid positions identified using the in vitro replicon system (Supplementary Tables 12-14): M28T, Q30H/R and L31M for genotype 1a, and Y93H for genotype 1b, results that suggest the usefulness of the replicon system for assessing resistance in response to inhibitor pressure in vivo. Follow-up samples were available for only one of these subjects, which revealed that HCV RNA had returned to near baseline; however, genotype analysis was not performed on this sample. As would be anticipated based on the in vitro replicon potency of BMS-790052, a greater and more sustained decline in HCV RNA was observed for subjects infected with genotype 1b (mean 3.6log10 reduction (range 3.1-4.0log10) and mean 3.1log10 reduction (range 2.7-3.4log10) in HCV viral load measured 24h after a 10 and 100mg dose, respectively) than for subjects infected with genotype 1a (mean 1.8log10 reduction (range 0.2-3.0log10), mean 2.9log10 reduction (range 2.9-3.0log10) and mean 3.6log10 reduction (range 3.5-3.6log10) in HCV viral load measured 24h after a 1, 10 and 100mg dose, respectively). The mean rates of decline for subjects who received 10 and 100mg doses of BMS-790052 were similar up to 36h after dosing, after which the mean decline was greater and more sustained in the subjects who received 100mg. Subjects who received 1mg of BMS-790052 had a lower mean decline in HCV RNA than subjects treated with 10 and 100mg (Fig. 4). However, multiple-dose studies are needed to define the optimal dose range for maximal antiviral effect beyond the first phase of viral decline. The relationships between maximum decline from baseline in HCV RNA and drug pharmacokinetics exposures were explored using Pearson's correlation coefficients. All estimated Pearson's correlation coefficients were above 0.65, suggesting that the maximum declines in log10 HCV RNA and log pharmacokinetics exposures (BMS-790052 Cmax, AUC(0-T), AUC(INF), C12 and C24) were positively correlated; that is, that the maximum declines in log10 HCV RNA increase with the exposure to BMS-790052.

Individuals infected with HCV are at considerable risk of developing liver cirrhosis, end-stage liver disease and hepatocellular carcinoma20. The current standard of care for the treatment of HCV infection is a combination of weekly subcutaneous injections of pegylated interferon-α in conjunction with ribavirin, administered orally twice a day, for periods ranging from 24 to 72 weeks, depending on genotype2, 3. However, the side effects associated with this therapeutic regimen impose a significant physiological and psychological burden on the patient. Moreover, sustained virological response rates in those infected with genotype 1 are typically less than 50% (refs 2, 3). The direct-acting HCV antiviral drug candidates currently reported to be in late-stage clinical development are inhibitors of NS3 protease and NS5B polymerase, more traditional antiviral targets. Although these compounds are currently being developed as an add-on therapy to the standard of care, there is considerable anticipation for combinations of direct-acting agents, which will require at least two mechanistically distinct inhibitors to suppress the emergence of resistant virus4, 21. The preliminary in vitro and clinical profile of NS5A inhibitors described in this paper makes them a potentially valuable component of any interferon-free treatment regimen.

The results with BMS-790052 provide clinical validation for the first in a new class of HCV inhibitors that target a viral protein with no known enzymatic function and an as yet poorly understood role in viral replication16, 22. The strategy used to identify a lead HCV NS5A inhibitor and to optimize this molecule into a clinical candidate offers a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics6. Indeed, this methodology is uniquely applicable to targets similar to HCV NS5A for which precise function is enigmatic and the development of biochemical assays could neither be anticipated nor are feasible. As a specific inhibitor of HCV, BMS-790052 exhibits a spectrum of genotype inhibition, in vitro potency, and efficacy and potency after single oral doses to chronically HCV-infected subjects that is collectively and individually unprecedented. Achieving high potency and selectivity against a non-mammalian target, the traditional goal of antiviral medicinal chemistry, has in the past translated into a wider therapeutic index in the clinic. Although preliminary, these data indicate that inhibitors of HCV NS5A offer considerable promise for the treatment of HCV infection. The in vitro data demonstrating additive to synergistic interactions with known HCV inhibitors suggest that combinations of BMS-790052, with either current standard of care or, ultimately, emerging inhibitors of HCV NS3 and NS5B as part of a cocktail of direct-acting antiviral agents, may lead to therapeutic regimens with better tolerability and improved clinical outcomes21.