Repurposing the HCV NS3-4A protease drug boceprevir as COVID-19 therapeutics.
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ABSTRACT:
SUBMITTER: Oerlemans R
PROVIDER: S-EPMC8130630 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Asunaprevir (ASV), an isoquinoline-based competitive inhibitor targeting the hepatitis C virus (HCV) NS3/4A protease, is very potent in vivo. However, the potency is significantly compromised by the drug resistance mutations R155K and D168A. In this study three crystal structures of ASV and an analogue were determined to analyze the structural basis of drug resistance susceptibility. These structures revealed that ASV makes extensive contacts with Arg155 outside the substrate envelope. Arg155 in turn is stabilized by Asp168, and thus when either residue is mutated, the enzyme's interaction with ASV's P2* isoquinoline is disrupted. Adding a P1-P3 macrocycle to ASV enhances the inhibitor's resistance barrier, likely due to poising the inhibitor to its bound conformation. Macrocyclic inhibitors with P2* extension moieties avoiding interaction with the protease S2 residues including Arg155 must be chosen for future design of more robust protease inhibitors.
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Project description:A series of linear HCV NS3/4A protease inhibitors was designed by eliminating the P2-P4 macrocyclic linker in grazoprevir, which, in addition to conferring conformational flexibility, allowed structure-activity relationship (SAR) exploration of diverse quinoxalines at the P2 position. Biochemical and replicon data indicated preference for small hydrophobic groups at the 3-position of P2 quinoxaline for maintaining potency against resistant variants R155K, A156T, and D168A/V. The linear inhibitors, though generally less potent than the corresponding macrocyclic analogues, were relatively easier to synthesize and less susceptible to drug resistance. Three inhibitor cocrystal structures bound to wild-type NS3/4A protease revealed a conformation with subtle changes in the binding of P2 quinoxaline, depending on the 3-position substituent, likely impacting both inhibitor potency and resistance profile. The SAR and structural analysis highlight inhibitor features that strengthen interactions of the P2 moiety with the catalytic triad residues, providing valuable insights to improve potency against resistant variants.
| S-EPMC6047049 | biostudies-literature
Project description:Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve >95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.
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Project description:A variety of amino acid substitutions in the NS3-4A protease of the hepatitis C virus lead to protease inhibitor (PI) resistance. Many of these significantly impair the replication fitness of the resistant variants in a genotype- and subtype-dependent manner, a critical factor in determining the probability with which resistant variants will persist. However, the underlying molecular mechanisms are unknown. Here, we present a novel residue-interaction network approach to determine how near-neighbor interactions of PI resistance mutations in NS3-4A can impact protease functional sites dependent on their genomic background. We constructed subtype-specific consensus residue networks for subtypes 1a and 1b from protease structure ensembles combined with biological properties of protein residues and evolutionary amino acid conservation. By applying local and global network topology analysis and visual exploration, we characterize PI resistance-associated sites and outline differences in near-neighbor interactions. We find local residue-interaction patterns and features at protease functional sites that are subtype specific. The noncovalent bonding patterns indicate higher fitness costs conferred by PI resistance mutations in a subtype 1b genomic background and explain the prevalence of Q80K and R155K in subtype 1a. Based on local residue interactions, we predict a subtype-specific role for the protease residue NS3-Q80 in molecular mechanisms related to the assembly of infectious virus particles that is supported by experimental data on the capacity of Q80K variants to replicate and produce infectious virus in subtype 1a and 1b cell culture.
| S-EPMC6554042 | biostudies-literature
Project description:NS3/4A protease is an important emerging target for the cure of hepatitis C. There are many inhibitors of HCV NS3/4A protease that are passing through the clinical improvement indicating momentous reduction in the viral infection rate of patients. In this study molecular docking via MOE-Dock program was used to evaluate binding interactions of ligands with HCV NS3/4A protease. The docking and experimental results were found in good correlation. The best conformations of ligands were analyzed for binding interactions with the residues of binding cavity of NS3/4A protease. The valuable binding interactions and docking scores were observed for compounds 01, 05, 06, 07, 08 and 09.
| S-EPMC3607190 | biostudies-literature
Project description:Hepatitis C virus, causative agent of chronic viral hepatitis, infects 71 million people worldwide and is divided into seven genotypes and multiple subtypes with sequence identities between 68 to 82%. While older generation direct-acting antivirals had varying effectiveness against different genotypes, the newest NS3/4A protease inhibitors including glecaprevir (GLE) have pan-genotypic activity. The structural basis for pan-genotypic inhibition and effects of polymorphisms on inhibitor potency were not well-known due to lack of crystal structures of GLE-bound NS3/4A or genotypes other than 1. In this study, we determined the crystal structures of NS3/4A from genotypes 1a, 3a, 4a, and 5a in complex with GLE. Comparison with the highly similar grazoprevir indicated the mechanism of GLE's drastic improvement in potency. We found that, while GLE is highly potent against wild-type NS3/4A of all genotypes, specific resistance-associated substitutions (RASs) confer orders of magnitude loss in inhibition. Our crystal structures reveal molecular mechanisms behind pan-genotypic activity of GLE, including potency loss due to RASs at D168. Our structures permit for the first time analysis of changes due to polymorphisms among genotypes, providing insights into design principles that can aid future drug development and potentially can be extended to other proteins.
| S-EPMC7747061 | biostudies-literature
Project description:A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins.
| S-EPMC4999588 | biostudies-literature
Project description:We have previously reported the discovery of our P2-P4 macrocyclic HCV NS3/4a protease inhibitor MK-5172, which in combination with the NS5a inhibitor MK-8742 recently received a breakthrough therapy designation from the US FDA for treatment of chronic HCV infection. Our goal for the next generation NS3/4a inhibitor was to achieve pan-genotypic activity while retaining the pharmacokinetic profile of MK-5172. One of the areas for follow-up investigation involved replacement of the quinoxaline moiety in MK-5172 with a quinoline and studying the effect of substitution at 4-position of the quinoline. The rationale for this effort was based on molecular modeling, which indicated that such modifications would improve interactions with the S2 subsite, in particular with D79. We wish to report herein the discovery of highly potent inhibitors with pan-genotypic activity and an improved profile over MK-5172, especially against gt-3a and A156 mutants.
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Project description:Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168 To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.
| S-EPMC7535904 | biostudies-literature
Project description:During the current COVID-19 pandemic more than 160,000 people have died worldwide as of mid-April 2020, and the global economy has been crippled. Effective control of the SARS-CoV2 virus that causes the COVID-19 pandemic requires both vaccines and antivirals. Antivirals are particularly crucial to treat infected people during the period of time that an effective vaccine is being developed and deployed. Because the development of specific antiviral drugs can take a considerable length of time, an important approach is to identify existing drugs already approved for use in humans which could be repurposed as COVID-19 therapeutics. Here we focus on antivirals directed against the SARS-CoV2 Mpro protease, which is required for virus replication. A structural similarity search showed that the Hepatitis C virus (HCV) NS3/4A protease has a striking three-dimensional structural similarity to the SARS-CoV2 Mpro protease, particularly in the arrangement of key active site residues. We used virtual docking predictions to assess the hypothesis that existing drugs already approved for human use or clinical testing that are directed at the HCV NS3/4A protease might fit well into the active-site cleft of the SARS-CoV2 protease (Mpro). AutoDock docking scores for 12 HCV protease inhibitors and 9 HIV-1 protease inhibitors were determined and compared to the docking scores for an α-ketoamide inhibitor of Mpro, which has recently been shown to inhibit SARS-CoV2 virus replication in cell culture. We identified eight HCV protease inhibitors that bound to the Mpro active site with higher docking scores than the α-ketoamide inhibitor, suggesting that these protease inhibitors may effectively bind to the Mpro active site. These results provide the rationale for us to test the identified HCV protease inhibitors as inhibitors of the SARS-CoV2 protease, and as inhibitors of SARS-CoV2 virus replication. Subsequently these repurposed drugs could be evaluated as COVID-19 therapeutics.
| S-EPMC7263768 | biostudies-literature