Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

Determination of binding potential of HCV protease inhibitors against SARS-CoV-2 Papain-like protease with computational docking

Background: SARS-CoV-2, a novel coronavirus that causes a pandemic respiratory disease, has recently emerged from China. Since it’s a life-threatening virus, investigation of curative medications along with protective vaccines still maintains its importance. Drug repurposing is a reasonable and immediate approach to combat SARS-CoV-2 infection by identifying inhibitory molecules from marketed drugs. PL protease (PLpro.) is one of the essential enzymes for the progression of SARS-CoV-2 replication and life cycle. Objective: We aimed to investigate the potential of 4 HCV protease inhibitors as probable repurposing drugs in Covid-19 treatment. Methods: In order to understand the possible binding affinity of HCV protease inhibitors, Boceprevir, Grazoprevir, Simeprevir, and Telaprevir, against to PLpro, we performed docking analysis in silico. Docking study was accomplished using AutoDock 4.2 software. Potential druggable pockets on PLpro were predicted by DoGSiteScorer tool in order to explore any overlapping with binding regions and these pockets. Results: This analysis demonstrated Boceprevir, Grazoprevir, Simeprevir and Telaprevir interacted by PLpro with binding energies (kcal/mol) of -4.97, -4.24, -6.98, -1.08, respectively. Asn109, one of the interacted residues with both Boceprevir and Simeprevir, is a neighbouring residue to catalytic Cys111. Additionally, Telaprevir notably interacted with catalytic His272 in the active site. Conclusion: Present study explains the binding efficiency and repurposing potential of certain HCV protease inhibitors against to SARS-CoV-2 PLpro enzyme. Docking sites and potential druggability of ligands were also crosschecked by the estimation of druggable pockets. Thereby our results can promote promising preliminary data for research on drug development in the fight of Covid-19.

The Q41R mutation in HCV-protease enhances the reactivity towards MAVS by suppressing non-reactive pathways

Recent experimental findings pointed out a new mutation in HCV protease, Q41R, responsible for a significant enhancement of the enzyme’s reactivity towards the mitochondrial antiviral-signaling protein (MAVS). The Q41R mutation...

Repurposing the HCV NS3–4A protease drug boceprevir as COVID-19 therapeutics

α-Ketoamide HCV protease inhibitors covalently bind to SARS-CoV-2 3CLpro. Boceprevir is a particular promising repurposed drug as it potently inhibits cellular viral proliferation.

Hepatitis C Virus Drugs Simeprevir and Grazoprevir Synergize with Remdesivir to Suppress SARS-CoV-2 Replication in Cell Culture

SummaryEffective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS- CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold.HighlightsSeveral HCV protease inhibitors are predicted to inhibit SARS-CoV-2 Mpro and PLpro.Seven HCV drugs inhibit Mpro enzyme activity, four HCV drugs inhibit PLpro.Seven HCV drugs inhibit SARS-CoV-2 replication in Vero and/or human cells.HCV drugs simeprevir and grazoprevir synergize with remdesivir to inhibit SARS- CoV-2.eTOC blurbBafna, White and colleagues report that several available hepatitis C virus drugs inhibit the SARS-CoV-2 Mpro and/or PLpro proteases and SARS-CoV-2 replication in cell culture. Two drugs, simeprevir and grazoprevir, synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir antiviral activity as much as 10-fold.Abstract Figure

The conquest of hepatitis C: telaprevir and beyond

Taking one pill a day for 8–12 weeks cures 98%–99% of the cases of chronic hepatitis C infections. This is one of the compelling success stories of biomedical research and drug discovery. The discovery of the hepatitis C virus (HCV) as the infective agent opened the doors to unraveling its life cycle and the identification of multiple molecular targets for drug discovery. One such target is HCV protease, an enzyme, among others, essential for virus replication. Several pharmaceutical companies, including Vertex, launched projects to find inhibitors of this enzyme. The Vertex effort overcame multiple problems and created an inhibitor known as telaprevir and marketed as Incivek. Incivek was a remarkable market entry with sales of a billion dollars in its first year. However, the success story was short-lived as Gilead Sciences came up with remarkable combination drugs for Hepatitis C. including Harvoni. Being first with a much improved product is not a guarantee of long-term success.

Rational design of a new class of protease inhibitors for the potential treatment of coronavirus diseases

ABSTRACTThe coronavirus main protease, Mpro, is a key protein in the virus life cycle and a major drug target. Based on crystal structures of SARSCoV2 Mpro complexed with peptidomimetic inhibitors, we recognized a binding characteristic shared with proline-containing inhibitors of hepatitis C virus protease. Initial tests showed that this subclass of HCV protease inhibitors indeed exhibited activity against Mpro. Postulating a benefit for a preorganized backbone conformation, we designed new ketoamide-based Mpro inhibitors based on central proline rings. One of the designed compounds, ML1000, inhibits Mpro with low-nanomolar affinity and suppresses SARSCoV2 viral replication in human cells at sub-micromolar concentrations. Our findings identify ML1000 as a promising new pre-organized scaffold for the development of anti-coronavirus drugs.

Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A

ABSTRACT Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein. IMPORTANCE For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cis cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.

In Silico Evaluation of the Effectivity of Approved Protease Inhibitors against the Main Protease of the Novel SARS-CoV-2 Virus

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than −8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.