Unraveling the mechanisms of Sofosbuvir resistance in HCV NS3/4A protease: Structural and molecular simulation-based insights.

Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions are gradually decreasing the efficacy of DAAs. Among the most effective HCV NS3/4A protease drugs, Sofosbuvir also develops resistance due to mutations in the NS3 and NS5B regions. Four mutations at positions A156Y, L36P, Q41H, and Q80K are classified as high-level resistance mutations. The resistance mechanism of HCV NS3/4A protease toward Sofosbuvir caused by these mutations is still unclear, as there is less information available regarding the structural and functional effects of the mutations against Sofosbuvir. In this work, we combined molecular dynamics simulation, molecular mechanics/Generalized-Born surface area calculation, principal component analysis, and free energy landscape analysis to explore the resistance mechanism of HCV NS3/4A protease due to these mutations, as well as compare interaction changes in wild-type. Subsequently, we identified that the mutant form of HCV NS3/4A protease affects the activity of Sofosbuvir. In this study, the resistance mechanism of Sofosbuvir at the atomic level is proposed. The proposed drug-resistance mechanism will provide valuable guidance for the design of HCV drugs.

Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED.

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus (HCV) infection. Its structures have been elusive to the public until recently when one of the crystal forms is solved by microcrystal electron diffraction (MicroED). In this work, the MicroED structures of two distinct polymorphic crystal forms of paritaprevir are reported from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropyl sulfonamide and methyl pyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the HCV non-structural 3/4A (NS3/4A) serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acyl sulfonamide inhibitors to the HCV NS3/4A serine protease. In addition, this also demonstrates the opportunity to derive different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.

Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity.

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3.

Sequential Phosphorylation of Hepatitis C Virus NS5A Protein Requires the ATP-Binding Domain of NS3 Helicase.

The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.

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.

Hepatitis C Virus NS3/4A Inhibition and Host Immunomodulation by Tannins from Terminalia chebula: A Structural Perspective.

Terminalia chebula Retz. forms a key component of traditional folk medicine and is also reported to possess antihepatitis C virus (HCV) and immunomodulatory activities. However, information on the intermolecular interactions of phytochemicals from this plant with HCV and human proteins are yet to be established. Thus, by this current study, we investigated the HCV NS3/4A inhibitory and host immune-modulatory activity of phytocompounds from T. chebula through in silico strategies involving network pharmacology and structural bioinformatics techniques. To start with, the phytochemical dataset of T. chebula was curated from biological databases and the published literature. Further, the target ability of the phytocompounds was predicted using BindingDB for both HCV NS3/4A and other probable host targets involved in the immune system. Further, the identified targets were docked to the phytochemical dataset using AutoDock Vina executed through the POAP pipeline. The resultant docked complexes with significant binding energy were subjected to 50 ns molecular dynamics (MD) simulation in order to infer the stability of complex formation. During network pharmacology analysis, the gene set pathway enrichment of host targets was performed using the STRING and Reactome pathway databases. Further, the biological network among compounds, proteins, and pathways was constructed using Cytoscape 3.6.1. Furthermore, the druglikeness, side effects, and toxicity of the phytocompounds were also predicted using the MolSoft, ADVERpred, and PreADMET methods, respectively. Out of 41 selected compounds, 10 were predicted to target HCV NS3/4A and also to possess druglike and nontoxic properties. Among these 10 molecules, Chebulagic acid and 1,2,3,4,6-Pentagalloyl glucose exhibited potent HCV NS3/4A inhibitory activity, as these scored a lowest binding energy (BE) of -8.6 kcal/mol and -7.7 kcal/mol with 11 and 20 intermolecular interactions with active site residues, respectively. These findings are highly comparable with Asunaprevir (known inhibitor of HCV NS3/4A), which scored a BE of -7.4 kcal/mol with 20 key intermolecular interactions. MD studies also strongly suggest that chebulagic acid and 1,2,3,4,6-Pentagalloyl glucose as promising leads, as these molecules showed stable binding during 50 ns of production run. Further, the gene set enrichment and network analysis of 18 protein targets prioritized 10 compounds and were predicted to potentially modulate the host immune system, hemostasis, cytokine levels, interleukins signaling pathways, and platelet aggregation. On overall analysis, this present study predicts that tannins from T. chebula have a potential HCV NS3/4A inhibitory and host immune-modulatory activity. However, further experimental studies are required to confirm the efficacies.

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

Recent experimental findings pointed out a new mutation in the HCV protease, Q41R, responsible for a significant enhancement of the enzyme's reactivity towards the mitochondrial antiviral-signaling protein (MAVS). The Q41R mutation is located rather far from the active site, and its involvement in the overall reaction mechanism is thus unclear. We used classical molecular dynamics and QM/MM to study the acylation reaction of HCV NS3/4A protease variants bound to MAVS and the NS4A/4B substrate and uncovered the indirect mechanism by which the Q41R mutation plays a critical role in the efficient cleavage of the substrate. Our simulations reveal that there are two major conformations of the MAVS H1'(p) residue for the wild type protease and only one conformation for the Q41R mutant. The conformational space of H1'(p) is restricted by the Q41R mutation due to a π-π stacking between H1'(p) and R41 as well as a strong hydrogen bond between the backbone of H57 and the side chain of R41. Further QM/MM calculations indicate that the complex with the conformation ruled out by the Q41R substitution is a non-reactive species due to its higher free energy barrier for the acylation reaction. Based on our calculations, we propose a kinetic mechanism that explains experimental data showing an increase of apparent rate constants for MAVS cleavage in Q41R mutants. Our model predicts that the non-reactive conformation of the enzyme-substrate complex modulates reaction kinetics like an uncompetitive inhibitor.

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir.

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification.

HCV RdRp, sofosbuvir and beyond.

The therapeutic targeting of the nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) of the Hepatitis C Virus (HCV) with nucleotide analogs led to a deep understanding of this enzymes structure, function and substrate specificity. Unlike previously studied DNA polymerases including the reverse transcriptase of Human Immunodeficiency Virus, development of biochemical assays for HCV RdRp proved challenging due to low solubility of the full-length protein and inefficient acceptance of exogenous primer/templates. Despite the poor apparent specific activity, HCV RdRp was found to support rapid and processive transcription once elongation is initiated in vitro consistent with its high level of viral replication in the livers of patients. Understanding of the substrate specificity of HCV RdRp led to the discovery of the active triphosphate of sofosbuvir as a nonobligate chain-terminator of viral RNA transcripts. The ternary crystal structure of HCV RdRp, primer/template, and incoming nucleotide showed the interaction between the nucleotide analog and the 2'-hydroxyl binding pocket and how an unfit mutation of serine 282 to threonine results in resistance by interacting with the uracil base and modified 2'-position of the analog. Host polymerases mediate off-target toxicity of nucleotide analogs and the active metabolite of sofosbuvir was found to not be efficiently incorporated by host polymerases including the mitochondrial RNA polymerase (POLRMT). Knowledge from studying inhibitors of HCV RdRp serves to advance antiviral drug discovery for other emerging RNA viruses including the discovery of remdesivir as an inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the virus that causes COVID-19.

The spectrum between substrates and inhibitors: Pinpointing the binding mode of dengue protease ligands with modulated basicity and hydrophobicity.

Peptides can be inhibitors and substrates of proteases. The present study describes the inhibitor- vs. substrate-like properties of peptidic ligands of dengue protease which were designed to provide insight into their binding modes. Of particular interest was the localization of the cleavable peptide bond and the placement of hydrophobic elements in the binding site. The findings provide clues for the design of covalent inhibitors in which electrophilic functional groups bind to the catalytic serine, and in addition for the development of inhibitors that are less basic than the natural substrate and therefore have an improved pharmacokinetic profile. We observed a tendency of basic elements to favor a substrate-like binding mode, whereas hydrophobic elements decrease or eliminate enzymatic cleavage. This indicates a necessity to include basic elements which closely mimic the natural substrates into covalent inhibitors, posing a challenge from the chemical and pharmacokinetic perspective. However, hydrophobic elements may offer opportunities to develop non-covalent inhibitors with a favorable ADME profile and potentially improved target-binding kinetics.

Novel pyridinium-type fullerene derivatives as multitargeting inhibitors of HIV-1 reverse transcriptase, HIV-1 protease, and HCV NS5B polymerase.

In the present study, we newly synthesized four types of novel fullerene derivatives: pyridinium/ethyl ester-type derivatives 3b-3l, pyridinium/carboxylic acid-type derivatives 4a, 4e, 4f, pyridinium/amide-type derivative 5a, and pyridinium/2-morpholinone-type derivative 6a. Among the assessed compounds, cis-3c, cis-3d, trans-3e, trans-3h, cis-3l, cis-4e, cis-4f, trans-4f, and cis-5a were found to inhibit HIV-1 reverse transcriptase (HIV-RT), HIV-1 protease (HIV-PR), and HCV NS5B polymerase (HCV NS5B), with IC50 values observed in the micromolar range. Cellular uptake of pyridinium/ethyl ester-type derivatives was higher than that of corresponding pyridinium/carboxylic acid-type derivatives and pyridinium/amide-type derivatives. This result might indicate that pyridinium/ethyl ester-type derivatives are expected to be lead compounds for multitargeting drugs to treat HIV/HCV coinfection.

Understanding effect site pharmacology of uprifosbuvir, a hepatitis C virus nucleoside inhibitor: Case study of a multidisciplinary modeling approach in drug development.

Uprifosbuvir is a uridine nucleoside monophosphate prodrug inhibitor of the hepatitis C virus NS5B RNA polymerase. To quantitatively elucidate key metabolic pathways, assess the link between unmeasurable effect site concentrations and viral load reduction, and evaluate the influence of intrinsic and extrinsic factors on pharmacokinetics and pharmacodynamics, a model-informed drug development (MIDD) framework was initiated at an early stage. Originally scoped as a modeling effort focused on minimal physiologically based pharmacokinetic and covariate analyses, this project turned into a collaborative effort focused on gaining a deeper understanding of the data from drug metabolism, biopharmaceutics, pharmacometrics, and clinical pharmacology perspectives. This article presents an example of the practical execution of a MIDD-based, cooperative multidisciplinary modeling approach, creating a model that grows along with the team's integrated knowledge. Insights gained from this process could be used in forming optimal collaborations between disciplines in drug development for other investigative compounds.

Targeted design of drug binding sites in the main protease of SARS-CoV-2 reveals potential signatures of adaptation.

Several existing drugs are currently being tested worldwide to treat COVID-19 patients. Recent data indicate that SARS-CoV-2 is rapidly evolving into more transmissible variants. It is therefore highly possible that SARS-CoV-2 can accumulate adaptive mutations modulating drug susceptibility and hampering viral antigenicity. Thus, it is vital to predict potential non-synonymous mutation sites and predict the evolution of protein structural modifications leading to drug tolerance. As two FDA-approved anti-hepatitis C virus (HCV) drugs, boceprevir, and telaprevir, have been shown to effectively inhibit SARS-CoV-2 by targeting the main protease (Mpro), here we used a high-throughput interface-based protein design strategy to identify mutational hotspots and potential signatures of adaptation in these drug binding sites of Mpro. Several mutants exhibited reduced binding affinity to these drugs, out of which hotspot residues having a strong tendency to undergo positive selection were identified. The data further indicated that these anti-HCV drugs have larger footprints in the mutational landscape of Mpro and hence encompass the highest potential for positive selection and adaptation. These findings are crucial in understanding the potential structural modifications in the drug binding sites of Mpro and thus its signatures of adaptation. Furthermore, the data could provide systemic strategies for robust antiviral design and discovery against COVID-19 in the future.

Rational drug discovery: Ellagic acid as a potent dual-target inhibitor against hepatitis C virus genotype 3 (HCV G3) NS3 enzymes.

Structure-based virtual screening (SBVS) has served as a popular strategy for rational drug discovery. In this study, we aimed to discover novel benzopyran-based inhibitors that targeted the NS3 enzymes (NS3/4A protease and NS3 helicase) of HCV G3 using a combination of in silico and in vitro approaches. With the aid of SBVS, six novel compounds were discovered to inhibit HCV G3 NS3/4A protease and two phytochemicals (ellagic acid and myricetin) were identified as dual-target inhibitors that inhibited both NS3/4A protease and NS3 helicase in vitro (IC50  = 40.37 ± 5.47 nm and 6.58 ± 0.99 µm, respectively). Inhibitory activities against the replication of HCV G3 replicons were further assessed in a cell-based system with four compounds showed dose-dependent inhibition. Compound P8 was determined to be the most potent compound from the cell-based assay with an EC50 of 19.05 µm. The dual-target inhibitor, ellagic acid, was determined as the second most potent (EC50  = 32.37 µm) and the most selective in its inhibitory activity against the replication of HCV replicons, without severely affecting the viability of the host cells (selectivity index > 6.18).

A computational drug repurposing approach in identifying the cephalosporin antibiotic and anti-hepatitis C drug derivatives for COVID-19 treatment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 1.4 million deaths worldwide. Repurposing existing drugs offers the fastest opportunity to identify new indications for existing drugs as a stable solution against coronavirus disease 2019 (COVID-19). The SARS-CoV-2 main protease (Mpro) is a critical target for designing potent antiviral agents against COVID-19. In this study, we identify potential inhibitors against COVID-19, using an amalgam of virtual screening, molecular dynamics (MD) simulations, and binding-free energy approaches from the Korea Chemical Bank drug repurposing (KCB-DR) database. The database screening of KCB-DR resulted in 149 binders. The dynamics of protein-drug complex formation for the seven top scoring drugs were investigated through MD simulations. Six drugs showed stable binding with active site of SARS-CoV-2 Mpro indicated by steady RMSD of protein backbone atoms and potential energy profiles. Furthermore, binding free energy calculations suggested the community-acquired bacterial pneumonia drug ceftaroline fosamil and the hepatitis C virus (HCV) protease inhibitor telaprevir are potent inhibitors against Mpro. Molecular dynamics and interaction analysis revealed that ceftaroline fosamil and telaprevir form hydrogen bonds with important active site residues such as Thr24, Thr25, His41, Thr45, Gly143, Ser144, Cys145, and Glu166 that is supported by crystallographic information of known inhibitors. Telaprevir has potential side effects, but its derivatives have good pharmacokinetic properties and are suggested to bind Mpro. We suggest the telaprevir derivatives and ceftaroline fosamil bind tightly with SARS-CoV-2 Mpro and should be validated through preclinical testing.

Metabolism of Direct-acting Antiviral Agents (DAAs) in Hepatitis C Therapy: A Review of the Literature.

BACKGROUND: Hepatitis C virus (HCV) infection is still one of the leading causes of chronic liver disease, with chronically infected making up approximately 1% of the global population. Of those infected, 70% (55-85%) will develop chronic HCV infection. Chronic HCV infection causes substantial morbidity and mortality, with complications including cirrhosis, end-stage liver disease, hepatocellular carcinoma, and eventually death. OBJECTIVE: Therapeutic options for chronic HCV infection have evolved dramatically since 2014, with a translation from pegylated interferon and ribavirin (associated with suboptimal cure and high treatment-related toxicity) to oral direct-acting antiviral treatment. There are four classes of direct-acting antivirals which differ by their mechanism of action and therapeutic target. They are all pointed to proteins that form the cytoplasmic viral replication complex. Multiple studies have demonstrated that direct-acting antiviral therapy is extremely well tolerated, highly efficacious, with few side effects. METHODS: We performed an indexed MEDLINE search with keywords regarding specific direct-acting antiviral regimes and their pharmacokinetics, drug-drug interactions, and metabolism in specific settings of pregnancy, lactation, liver cirrhosis, liver transplantation and HCC risk, kidney failure and kidney transplantation. RESULTS: We present a comprehensive overview of specific direct-acting antiviral metabolism and drug-drug interaction issues in different settings. CONCLUSION: Despite its complex pharmacokinetics and the possibility of drug-drug interactions, direct-acting antivirals are highly efficacious in providing viral clearance, which is an obvious advantage compared to possible interactions or side effects. They should be administered cautiously in patients with other comorbidities, and with tight control of immunosuppressive therapy.

Targeting the RdRp of Emerging RNA Viruses: The Structure-Based Drug Design Challenge.

The RNA-dependent RNA polymerase (RdRp) is an essential enzyme for the viral replication process, catalyzing the viral RNA synthesis using a metal ion-dependent mechanism. In recent years, RdRp has emerged as an optimal target for the development of antiviral drugs, as demonstrated by recent approvals of sofosbuvir and remdesivir against Hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively. In this work, we overview the main sequence and structural features of the RdRp of emerging RNA viruses such as Coronaviruses, Flaviviruses, and HCV, as well as inhibition strategies implemented so far. While analyzing the structural information available on the RdRp of emerging RNA viruses, we provide examples of success stories such as for HCV and SARS-CoV-2. In contrast, Flaviviruses' story has raised attention about how the lack of structural details on catalytically-competent or ligand-bound RdRp strongly hampers the application of structure-based drug design, either in repurposing and conventional approaches.

Potential for Protein Kinase Pharmacological Regulation in Flaviviridae Infections.

Protein kinases (PKs) are enzymes that catalyze the transfer of the terminal phosphate group from ATP to a protein acceptor, mainly to serine, threonine, and tyrosine residues. PK catalyzed phosphorylation is critical to the regulation of cellular signaling pathways that affect crucial cell processes, such as growth, differentiation, and metabolism. PKs represent attractive targets for drugs against a wide spectrum of diseases, including viral infections. Two different approaches are being applied in the search for antivirals: compounds directed against viral targets (direct-acting antivirals, DAAs), or against cellular components essential for the viral life cycle (host-directed antivirals, HDAs). One of the main drawbacks of DAAs is the rapid emergence of drug-resistant viruses. In contrast, HDAs present a higher barrier to resistance development. This work reviews the use of chemicals that target cellular PKs as HDAs against virus of the Flaviviridae family (Flavivirus and Hepacivirus), thus being potentially valuable therapeutic targets in the control of these pathogens.

Discovery of BMS-986144, a Third-Generation, Pan-Genotype NS3/4A Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.

The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.