Read an Excerpt
Successful Strategies for the Discovery of Antiviral Drugs
By Manoj C. Desai, Nicholas A. Meanwell
The Royal Society of ChemistryCopyright © 2013 The Royal Society of Chemistry
All rights reserved.
Discovery and Clinical Validation of HCV Inhibitors Targeting the NS5A Protein
MAKONEN BELEMA, NICHOLAS A. MEANWELL, JOHN A. BENDER, OMAR D. LOPEZ, PIYASENA HEWAWASAM AND DAVID R. LANGLEY
Significant effort has been invested in elucidating the exact role and function of the NS5A protein in the hepatitis C virus (HCV) replication cycle. Although, unlike the NS3 and NS5B proteins, no enzymatic function has been identified thus far for NS5A, it has become apparent that this protein plays a diverse and critical set of roles both in the replication of the virus and in the mediation of host–virus interactions. Despite its multifunctional role, the lack of a well- characterized function coupled with the limited availability of structural information, compared with the NS3 protease and NS5B polymerase, initially made the NS5A protein a less compelling target for therapeutic intervention. That changed, however, with the validation of NS5A as a clinically relevant target by daclatasvir (1), where single doses effected pronounced and rapid declines in viral RNA in HCV-infected subjects (Figure 1.1). Highlights of the biochemical pharmacology of the NS5A protein, along with the discovery, the mode of action and the clinical characterization of a potent class of NS5A inhibitors, are discussed in this chapter.
1.2 The HCV NS5A Protein
The HCV RNA genome encodes a ~3000 amino acid polypeptide that is processed by both viral and cellular proteases into structural proteins (Core, E1 and E2), an ion channel (p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). The non-structural proteins are responsible for replication of the viral genome and for the assembly of the viral particle from the structural proteins, with the assistance of host factors. HCV NS5A is a 447 residue peptide that is comprised of three domains, which are interlinked with short fragments designated as low-complexity sequences (LCSs) (Figure 1.2). Various studies have demonstrated that NS5A is an RNA-binding protein, although the specific elements of the protein that are establishing the biologically relevant interactions with ribonucleic acid still need to be identified. For example, one study has indicated that all three domains of NS5A exhibit RNA-binding properties, albeit with differential affinities, whereas a different study showed that the Domain I/LCS I peptide fragment exhibited RNA-binding affinity that is comparable to that of the full-length NS5A protein, supported by the observation that the RNA-binding property of NS5A is abolished if Domain I is deleted. Whatever their specific RNA-binding properties may be, all three domains contribute to genome replication, while Domain III plays a key role in viral particle assembly. In addition, all three domains play a diverse set of regulatory roles in modulating host–virus interactions so as to facilitate the establishment of an environment conducive to successful viral replication.
Domain I of NS5A is a Zn2+-binding moiety with an amphipathic α-helix at its N-terminal that is believed to anchor the protein to cellular membranes. X-ray structural studies by two independent groups on similar amino acid constructs of Domain I, both lacking the amphipathic α-helix motif, revealed that the protein crystallizes as a homodimer (Figure 1.3). Interestingly, although the monomeric units in the two X-ray studies were highly structurally conserved, their modes of dimerization were different and involved non-overlapping contact surfaces. A positively charged groove created by the dimeric interface of the X-ray structure reported by Rice's group, which had the appropriate dimensions to support the hypothesis that it could be an RNA-binding site, is fully exposed in Love's X-ray structure. The reason for the differing modes of dimerization and how well either one may reflect a biochemically relevant structure of the NS5A protein, especially since about half of the protein is missing from the structural analyses, is not apparent at this stage. Some have postulated that the two dimeric modes may represent snapshots of an oligomeric state, the functional significance of which has yet to be revealed. In general agreement with the X-ray structural findings, a glutathione-S-transferase (GST)-tagged NS5A Domain I was able to pull down a His-tagged NS5A protein, presumably through a dimeric interaction, whereas this was not possible with GST alone. This interaction does not appear to be mediated by the presence of nucleic acids and yet, interestingly, there is a similarity between the minimal NS5A fragment required to effect this pull-down and the minimal fragment required to maintain the RNA-binding affinity of the full-length NS5A protein. It is also noteworthy that the minimal peptide fragment required to effect the dimerization in the pull-down study was longer than the peptide constructs used in the X-ray studies (amino acids 1–240 versus 25–215 for the Rice dimer and 33–202 for the Love dimer), and although the reason for this disparity is not apparent, it could be a result of the distinct physical states that the two studies are dealing with and of differences in experimental parameters, such as protein concentration.
In another study, a glutaraldehyde cross-linking experiment demonstrated that either Domain I or the full-length version of NS5A (but not Domain II–III) dimerize in solution, that the dimer is in equilibrium with the monomer and that the presence of uracil-rich RNA, which is known to bind to NS5A, shifts the equilibrium in favor of the dimer. Interestingly, in the same study, NS5A–RNA cross-linking followed by the mapping of the amino acids involved in the cross-linking on to either of the two X-ray structures indicated that, for the Rice dimer, the amino acids decorate the positively charged groove of the protein and not the similarly charged back side of the dimer, whereas for the Love dimer, a ribbon pattern surrounding the structure is observed. It was claimed that this cross-linking result is more consistent with Rice's dimer. Moreover, others have hypothesized that the groove in the Rice dimer may serve as an 'RNA railway system' that connects different functional states that the RNA has to traverse, along with providing a role of protecting the viral RNA from cellular factors that degrade exogenous RNA. Whatever the case may be, the fact that highly potent NS5A inhibitors with resistance mutations that map to Domain I constitute a dimeric pharmacophore that complements the symmetrical features revealed by the X-ray studies and supported by biochemical studies, is unlikely to be coincidental (see below).
Unlike Domain I, Domains II and III are disordered proteins that lack secondary structural elements. It is hypothesized that disordered proteins have an extended surface area that promotes simultaneous interactions with multiple proteins and/or an interaction with RNA, which could be a reflection of the multifunctional nature of these domains, the details of which still need to be delineated.
1.3 The Discovery of HCV NS5A Replication Complex Inhibitors
HCV replicons, cell-based assay systems that support the autonomous replication of the subgenomic and genomic HCV, have played a central role in the HCV drug discovery field since the introduction of the first genotype 1b (G-1b) system in 1999, most notably in creating opportunities to exploit the potential of viral targets devoid of enzymatic functions. As part of a campaign directed at identifying inhibitors of HCV that act by novel mechanisms to disrupt replication, scientists at Bristol-Myers Squibb (BMS) devised a unique, dual-replicon assay system that was used to conduct a phenotype-based, high- throughput screening (HTS) campaign. Specifically, this assay system utilized a mixture of a G-1b HCV replicon and a replicon of a closely related virus, bovine viral diarrhea virus (BVDV), in the same well. The two replicons had the same Huh-7 cellular background but orthogonal activity reporters – a FRET assay based on NS3 protease activity for HCV and a luciferase expression assay for BDVD. In addition, cell toxicity was assessed in the same well using a standard Alamar Blue assay. It is noteworthy that since a luciferase enzyme assay is more sensitive than a FRET assay, this reporter combination placed a stringent criterion for the identification of HCV-specific inhibitors. The BMS compound collection was screened with this dual replicon assay system and initial hits that had either cytotoxic properties or poor HCV specificity, as reflected by a <10-fold potency spread between HCV and BVDV inhibitory activities, were discarded. Counter-screening of the resultant hit set with NS3 protease, NS3 helicase and NS5B polymerase enzymatic assays afforded a thiazolidinone chemotype, exemplified by carbamate 2, as a novel class of HCV inhibitor (Figure 1.4). It is noteworthy that over one million compounds were assayed in the HTS effort and only this single chemotype class met the stringent screening criteria to qualify as a suitable lead structure. Carbamate 2 exhibited moderate HCV potency (G-1b EC50 1/4 0.58 μM), very good HCV specificity (BVDV EC50 450 μM) and a CC50 of 4100 μM.
In order to identify the HCV protein that carbamate 2 might be targeting, passage of a G-1b replicon system through increasing concentrations of the compound resulted in a resistant phenotype that was 410-fold less sensitive to the inhibitor. After confirming that the mutation that caused the resistance phenotype was associated with viral RNA and not cellular RNA, sequence analysis of viral RNA from resistant cell lines was conducted and two dominant mutations were identified in the Domain I region of NS5A (Y93H and Y93C). Either mutation, when introduced individually into a G-1b replicon, was sufficient to confer the observed resistant phenotype and no cross-resistance was observed with inhibitors targeting alternative HCV mechanisms. This resistance analysis was the first indication that the thiazolidinone chemotype might be engaging the NS5A protein.
Preliminary structure–activity relationship (SAR) studies directed at establishing the fidelity of the lead revealed that there was a preference for the S stereochemistry at the amino acid moiety and that changing the benzyl carbamate to phenylacetamide, as in amide 3, effected a ~100-fold potency enhancement, an SAR observation that was recapitulated in the analogous proline series (see amides 4 and 5). The SAR survey of the iminothiazolidinone region of the lead molecule revealed that variation of the substituent pattern also modulated potency; a 410-fold dynamic potency range that was dependent on structure was noted. However, the patterns of SAR associated with this region were less discrete than that of the amino acid moiety. Resistance selection with amide 3 yielded additional mutations in NS5A Domain I (L31V and Q54L) that resulted in a 9–60-fold potency loss and were cross-resistant to amide 2, suggesting commonality of the inhibitory mechanism between these two molecules, despite the difference in their resistance mutations.
At this juncture, it became apparent that this thiazolidinone chemotype was exhibiting chemical instability in certain organic solvents and in the replicon medium. Careful analysis of degradation products revealed that when 3 is stored in dimethyl sulfoxide (DMSO) under ambient conditions, it undergoes an oxidative rearrangement to afford the thiohydantoin 8, which was inactive in the replicon assay, EC50 >20 μM (Scheme 1.1). Incubation of 3 in the replicon assay medium initially afforded thiohydantoin 8, which degraded to thiourea 9, which also lacked replicon inhibitory activity. A critical and enlightening experiment in which 3 was pre-incubated in the assay medium until complete degradation had occurred, followed by assessment in the replicon, revealed that the HCV inhibitory activity was maintained, clearly indicating that some chemical entity other than the parental analog was likely responsible for the observed effect. A careful HPLC biofractionation study conducted on 3 after incubation in assay medium coupled with detailed spectroscopic analyses of the degradation products revealed the presence of two dimeric derivatives (see 14 in Scheme 1.2), both of which demonstrated inhibitory activity in the G-1b replicon with EC50s of 0.6 and 43 nM. Although the precise stereochemical relationship between these two dimers has not been established, the more potent dimer converts to the weaker dimer when heated at 55 °C in CD3CN, which is suggestive of either a rotameric or a stereoisomeric relationship. It is hypothesized that the formation of the dimeric species from 3 arises from its susceptibility to form a captodative radical (11) in the presence of a radical initiator such as molecular oxygen, which has a diradical ground state. Since 11 is a stabilized radical, it persists such that it can undergo either dimerization to afford 14 or combine with molecular oxygen to afford the peroxy intermediate 12, which would be susceptible to reduction to alcohol 6 followed by rearrangement to afford thiohydantoin 8 via ketoamide 7. Transfer of the ketoacyl moiety of intermediate 7 to a nucleophilic species in the replicon medium would afford thiourea 9. Although a simple hydrolysis of ketoamide 7 in assay medium is also possible, the byproduct of such a hydrolytic process, keto acid 10, was not identified. It is noteworthy that acetate 13, which can be prepared from 3 in 78% yield by oxidation with Mn(OAc)3–Cu(OAc)2–AcOH, afforded thiohydantoin 8 when treated with MeOH–K2CO3, providing supportive evidence for a key step of the proposed mechanism.
Although the identification of the dimeric derivatives represented marked progress for the medicinal chemistry effort, optimizing these architecturally complex leads to a drug candidate appeared to be a challenging task, given that their physical properties fall far outside conventional drug space. However, based on insights gleaned from the preliminary SAR investigation, a significant simplification of the dimeric species was achieved when the key pharmacophoric elements were successfully captured in the bibenzyl 15, which exhibited a G-1b EC50 of 30 nM, potency that was improved further with the structurally more rigid stilbene analog 16, which displayed an EC50 of 0.086 nM in the G-1b replicon assay. This new lead molecule was relatively stable when incubated in replicon medium for the length of the assay period and exhibited a resistance profile that mirrored that of 3, supportive of a similar mode of inhibitory effect and confirming that this molecule contains the key pharmacophore within dimers 14. With its impressive potency and simplified structure compared with 14, stilbene 16 served as the starting point for the next phase of the medicinal chemistry campaign. This enterprise focused on expanding genotype coverage, since the EC50 of 16 in a G-1a replicon was >10 μM, and optimizing ADME properties. The effort involved significant chemotype evolution based on the application of bioisostere concepts and ultimately culminated in the discovery of the highly potent, first-in-class NS5A replication complex inhibitor daclatasvir (1). Daclatasvir inhibits G-1b and G-1a replicons with EC50s of 0.009 and 0.05 nM, respectively. In addition, it inhibits G-2a to G-5a replicons with EC50s ranging from 0.033 to 0.146 nM. This unprecedented in vitro potency spectrum established a new benchmark for the HCV field.
A similar cell-based screening of compound libraries conducted by scientists at Arrow Pharmaceuticals (subsequently acquired by AstraZeneca) led to the identification of two distinct hits (17 and 19) with HCV inhibitory activity that also appeared to target the NS5A protein and which were optimized to the two clinical candidates AZD-2836 (18) and AZD-7295 (20) (Figure 1.5). Interestingly, although resistance associated with the quinazoline series mapped to the NS5A protein, along with some accompanying mutations in the NS4B and NS5B regions, reverse genetic engineering of the mutations into a G-1b replicon, either alone or in combination, failed to recapitulate the resistant phenotype. On the other hand, the biphenyl carboxamide series afforded mutations in Domain I, the Y93H/C change being the hallmark and in the C-terminal region of the NS5A protein, for which additional details were not provided.
Excerpted from Successful Strategies for the Discovery of Antiviral Drugs by Manoj C. Desai, Nicholas A. Meanwell. Copyright © 2013 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.