RNA-dependent RNA Polymerase Enzyme Research Articles

Reversible and irreversible inhibitors of coronavirus Nsp15 endoribonuclease

The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR DETERMINATION OF RELATED SUBSTANCES AND PURITY OF FAVIPIRAVIR DRUG SUBSTANCE

Favipiravir (6-fluoro-3-hydroxy-2-pyrazine carboxamide) is an antiviral drug that specifically and potently inhibits the RNA-dependent RNA polymerase enzyme of RNA viruses. A straightforward stabilityindicating high-performance liquid chromatography (RP-HPLC) approach can be used to identify favipiravir, its known contaminants, and degradation products. This approach is exact, accurate, and repeatable. The process was validated according to the current International Council for Harmonization requirements. Within the acceptance criteria, the suggested technique exhibits outstanding accuracy, linearity, precision, robustness, specificity, LOD, LOQ, and system applicability findings. For all known analytes over the concentration range from LOQ to 200% of the specification level, the calibration plot revealed a linear relationship. LOD and LOQ were found in the field to be 0.02-0.16 μg mL-1 and 0.06-0.40 μg mL-1 for all known analytes, respectively. A recovery study was used to determine the suggested method’s accuracy. Studies on solution stability and robustness produced results that fell within acceptable bounds . The devised technique is appropriate for routine analysis.

DEVELOPMENT AND VALIDATION OF A NOVEL STABILITY INDICATING REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR RELATED SUBSTANCES AND ASSAY ANALYSIS OF MOLNUPIRAVIR DRUG SUBSTANCE AND DRUG PRODUCT

Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of β-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 µg mL-1 and 0.12-0.20 µg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.

Inspection on the Mechanism of SARS-CoV-2 Inhibition by Penciclovir: A Molecular Dynamic Study.

In recent years, humanity has had to face a critical pandemic due to SARS-CoV-2. In the rapid search for effective drugs against this RNA-positive virus, the repurposing of already existing nucleotide/nucleoside analogs able to stop RNA replication by inhibiting the RNA-dependent RNA polymerase enzyme has been evaluated. In this process, a valid contribution has been the use of in silico experiments, which allow for a rapid evaluation of the possible effectiveness of the proposed drugs. Here we propose a molecular dynamic study to provide insight into the inhibition mechanism of Penciclovir, a nucleotide analog on the RNA-dependent RNA polymerase enzyme. Besides the presented results, in this article, for the first time, molecular dynamic simulations have been performed considering not only the RNA-dependent RNA polymerase protein, but also its cofactors (fundamental for RNA replication) and double-strand RNA.

Favipiravir: An Antiviral Drug

Favipiravir is an antiviral drug that has been shown to treat a variety of life-threatening infections, including Ebola, Lassa, and the COVID-19 virus. It's a pyrazine carboxamide derivative with antiviral action that targets RNA-dependent RNA polymerase enzymes, which are required for viral genome transcription and replication. Favipiravir is an antiviral previously indicated for influenza and Ebola, which has shown some promise in early trials for treatment of COVID-19. The nucleoside analogue favipiravir is rapidly metabolized in host cells which disrupts viral synthesis and leads to mutagenesis The mechanism of action of the Favipiravir and Side effects like QTc prolongation or teratogenicity pose risk to extensive community application discusses in this review. In this article, we have tried to provide a comprehensive, evidence-based review of this drug about synthesis, Pharmacology, Mechanism of Action, Antiviral activity, Consequences.

Effects of Some Interferon-Related Proteins on Influenza A Viruse RNA Polymerase Activity

Interferons (IFNs) are one of the most important components of innate immunity against viruses, especially those carrying the RNA genomes such as influenza viruses. Upon viral infection, the IFNs are rapidly secreted, inducing the expression of several genes in the target cells and establishing an antiviral state. In this study, the effects of proteins encoded by some IFN-related genes on influenza A virus RNA-dependent RNA polymerase enzyme were investigated. We evaluated the importance of these proteins in the pathogenesis of different influenza A virus types. The IFN-related genes were amplified by polymerase chain reaction from the HEK293 cDNA library and cloned into pCHA expression vector. The expression of genes and subcellular localizations of the proteins were determined by Western blotting and immunofluorescence staining, respectively. The effects of IFNs-related proteins on virus RdRP enzyme were determined by influenza A virus mini-replicons. The study revealed that the influenza A virus infections significantly altered the transcript level of the IFN-related CCL5, IFIT1, IFIT3, IFITM3, and OAS1 genes in HEK293 cells. It was determined that the alteration of the gene expression was also related to the virus type. The mini-replicon assays showed that the transient expression of CCL5, IFI27, OAS1, IFITM3, IFIT1, and IFIT3 have inhibitory effects on WSN and/or DkPen type virus RdRP enzymes. We observed that the proteins except OAS1 inhibited WSN type RdRP enzyme at a higher level than that of DkPen enzyme. It was concluded that influenza A virus infection significantly alters the IFN-related gene expression in the cells. Most of the proteins encoded from these genes showed an inhibitory effect on the virus RdRP enzymes in the HEK293 cells. The inhibition of the influenza virus RdRP with IFN-related proteins may be the result of direct or indirect interactions between the host proteins and the viral enzyme subunits.

Polyamidoamine-Remdesivir Conjugate: Physical Stability and Cellular Uptake Enhancement

Nucleoside analogue antiviral remdesivir works by inhibiting the RNA-dependent RNA polymerase enzyme and terminating the viral replication. Currently, remdesivir is under a clinical trial for its activity against SARS-CoV-2. In the blood, remdesivir will undergo an enzymatic reaction to become monophosphate analogue form which is difficult to penetrate into the cell membrane. PAMAM (polyamidoamine) dendrimer is a good carrier to encapsulate remdesivir as a water-insoluble drug (0,339 mg/mL). Entrapment of remdesivir in the PAMAM cavity avoided remdesivir molecules to not undergo the enzymatic reactions. This study aimed to synthesize, characterize and evaluate cellular uptake of PAMAM-Remdesivir conjugate. PAMAM-Remdesivir was prepared with various stirring times (3, 6, 12, 24, and 48 hours). The conjugates were characterized to observe the size and particle distribution using Particle Size Analyzer, encapsulating efficiency using UV-Vis Spectroscopy, interaction between PAMAM and remdesivir particle using Fourier Transform Infrared Spectroscopy and cellular uptake of PAMAM-RDV using Fluorescence Microscope. The optimized stirring time of PAMAM-Remdesivir conjugate was 24 hours wich resulted the particles charge of + 23,07 mV of zeta potential, 1008 nm of particle size, 0,730 of PDI, and 69% entrapment efficiency. In addition, the FTIR analysis showed that remdesivir molecules successfully conjugated to PAMAM. Thus, through strring optimization time, the remdesivir molecules were successfully entrapped to PAMAM cavity. The cellular uptake in Vero Cell of PAMAM-RDV conjugated fluorescein isothiocyanate was observed with fluorescence microscope and had a stronger intensity than remdesivir only solution.

Remdesivir - Current evidence & perspective in management of COVID-19 infection.

Coronavirus infection or COVID 19 was first reported in December 2019 in Wuhan, China, and has rapidly spread to more than 200 countries. The pandemic has also taken a toll of over 2.2 million. But the elusive search for an effective antidote is still on. Pending multiple and robust randomized controlled studies, some drugs are being used globally based on in-vitro studies, in -vivo evidence, observational studies, and small nonrandomized studies. Remdesivir is a nucleotide analog. It inhibits viral RNA-dependent RNA polymerase enzyme. Several studies have hitherto demonstrated the promising in-vitro and in-vivo antiviral activities of the molecule against severe acute respiratory syndrome coronavirus (SARS-CoV-1) and the Middle East respiratory syndrome coronavirus (MERS-CoV) strains. It has now exhibited potential in vitro activity against SARS-CoV-2 strains too. Based on pivotal studies, remdesivir is now being used to treat moderate to severe patients through emergency use authorizations and other access programs around the world. This review aims to summarize the evidence and clinical trials of remdesivir as a potential therapeutic option for COVID-19.

A Validated high Performance Liquid Chromatographic Method for the Quantification of Favipiravir by PDA Detector

Favipiravir is an antiviral agent showing activity for the treatment of various life threatening viruses such as Ebola virus, Lassa virus and also recent virus for COVID-19. It is a pyrazine carboxamide derivative with activity against RNA viruses which targets RNA-dependent RNA polymerase enzymes which are necessary for the transcription and replication of viral genomes. The lack of research work and no compendial methods available for the estimation of this drug influenced the current research investigation to give a simple, sensitive, rapid, precise, accurate and robust isocratic high performance liquid chromatographic and UV Spectroscopic method for the determination and quantification of Favipiravir. The elution was done by using SHIMADZU Prominence-i, LC-2030 C system equipped with Shim-Pack GIST C18 (250X 4.6 mm, 5μm) column with a mobile phase mixture of 10 mM potassium dihydrogen ortho phosphate buffer (pH 4.0) and acetonitrile in the ratio of 90:10 v/v at a flow rate of 1.0 ml/min. The ultraviolet detection was done at the wavelength of 315 nm by maintaining column temperature at 30°. The total run time was 8.0 min. Calibration plot showed best regression over the concentration range of 10-60 μg/ml of Favipiravir standard solutions. The LOD and LOQ was found to be 0.18 μg/ml and 0.53 μg/ml, respectively. The accuracy of the proposed method was determined by performing recovery studies and was found to be between 99.47-100.80%. The repeatability testing for both sample and standard solutions was found as %RSD<2.0% which is within the acceptable limits showing that the method is precise as well. The proposed method was successfully applied for the marketed formulations of Favipiravir tablets. In addition the main features of the proposed method are economic and eco-friendly with less retention time around 4.622 min.

The preclinical inhibitor GS441524 in combination with GC376 efficaciously inhibited the proliferation of SARS-CoV-2 in the mouse respiratory tract

ABSTRACT The unprecedented coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a serious threat to global public health. Development of effective therapies against SARS-CoV-2 is urgently needed. Here, we evaluated the antiviral activity of a remdesivir parent nucleotide analog, GS441524, which targets the coronavirus RNA-dependent RNA polymerase enzyme, and a feline coronavirus prodrug, GC376, which targets its main protease, using a mouse-adapted SARS-CoV-2 infected mouse model. Our results showed that GS441524 effectively blocked the proliferation of SARS-CoV-2 in the mouse upper and lower respiratory tracts via combined intranasal (i.n.) and intramuscular (i.m.) treatment. However, the ability of high-dose GC376 (i.m. or i.n. and i.m.) was weaker than GS441524. Notably, low-dose combined application of GS441524 with GC376 could effectively protect mice against SARS-CoV-2 infection via i.n. or i.n. and i.m. treatment. Moreover, we found that the pharmacokinetic properties of GS441524 is better than GC376, and combined application of GC376 and GS441524 had a synergistic effect. Our findings support the further evaluation of the combined application of GC376 and GS441524 in future clinical studies.

Mixed infection by a partitivirus and a negative-sense RNA virus related to mymonaviruses in the polypore fungus Bondarzewia berkeleyi

Virus communities of forest fungi remain poorly characterized. In this study, we detected two new viruses co-infecting an isolate of the polypore fungus Bondarzewia berkeleyi using high-throughput sequencing. One of them was a putative new partitivirus designated as Bondarzewia berkeleyi partitivirus 1 (BbPV1), with two linear dsRNA genome segments of 1928 and 1863 bp encoding a putative RNA-dependent RNA polymerase (RdRP) of 591 aa and a putative capsid protein of 538 aa. The other virus, designated as Bondarzewia berkeleyi negative-strand RNA virus 1 (BbNSRV1), had a non-segmented negative-sense RNA genome of 10,983 nt and was related to members of family Mymonaviridae. The BbNSRV1 genome includes six predicted open reading frames (ORFs) of 279, 425, 230, 174, 200 and 1970 aa. The longest ORF contained conserved regions corresponding to Mononegavirales RdRP and mRNA-capping enzyme region V constituting the mononegavirus Large protein. In addition, a low level of sequence identity was detected between the putative nucleocapsid protein-coding ORF2 of Lentinula edodes negative-strand RNA virus 1 and BbNSRV1. The viruses characterized in this study are the first ones described in Bondarzewia spp., and BbNSRV1 is the second mymona-like virus described in a basidiomycete host.

Molecular Docking and Pharmacophore-Based Virtual Screening of Novel Inhibitors for HCV NS5B RNA-Dependent RNA Polymerase Enzyme from Crude Sesame Essential Oil.

Concern has been expressed worldwide about the rising prevalence of HCV induced acute hepatitis and chronic liver diseases with associated cirrhosis and liver cancers. However, the available synthetic drugs are ineffective for all the HCV genotypes especially in genotype-1 patients with about 40% viral response rates and numerous side effects. Besides, the availability of veritable bioinformatics tools which includes molecular docking and virtual screening studies have shown that computational generated models nowadays assists in modern drug design and development of novel and more potent inhibitors through the understanding of protein (receptors) -ligand (drugs) interaction mechanisms. Non-structural proteins especially the 5B (NS5B) is an RNA-dependent RNA polymerase implicated in the synthesis and replication of the HCV RNA; and has been a potential target for its inhibitory activities. Due to the paucity of knowledge, we aimed to determine the differential inhibitory activity of essential oils present in the crude Sesame leaves extracts on HCV-NS5B RNA dependent RNA polymerase. Using in-silico studies- a Microsoft pharmacophore-based virtual screening and molecular docking tools on the iGEMDOCK vs 2.0 software was used to dock the essential oil ligands on the generated HCV NS5B (PDB ID: 4EO6) RNA-dependent RNA polymerase protein. GC-MS of the leaves confirmed carboxylic acids and phenolic groups in the essential oils especially some potent antioxidants like alpha-linolenic acid, linoleic acid, oleic acid, etc. Moreover, Alpha-Linolenic acid/ALA (-102.2/-103.4 kcal/mol) and Linoleic acid/LA (-94.8/-109.8 kcal/mol) showed higher inhibitory impacts among the six top different docked ligands, selected based on their high differential binding affinity and pharmacological interaction energy profiles against HCV NS5B RNA polymerase activities, by forming more H-bond interactions than the NS5B co-crystallized ligand. ADMET showed that ALA is well tolerated without any apparent toxicity in the body. Hence, ALA having the highest inhibitory impacts against the HCV NS5B goes to confirm the beneficial impacts of carboxylic acids from Sesame plant in maintaining liver cellular integrity and as a natural inhibitor of HCV NS5B RNA dependent RNA Polymerase enzyme activities.

Single-cell analysis reveals the relevance of foot-and-mouth disease virus persistence to emopamil-binding protein gene expression in host cells.

Foot-and-mouth disease virus (FMDV) infects host cells in either an acute or persistent manner. In this study, we examined the relevance of the establishment of FMDV persistence to the expression of the emopamil-binding protein (EBP) gene in 231 individual persistently infected baby hamster kidney (BHK-21) cells after passages 28, 38, and 68 (PI28, PI38, and PI68). At PI28, the stage at which persistent infection of FDMV becomes unstable, the percentage of cells carrying FMDV was 66.7%, while 80.2% of cells were EBP positive. Additionally, in 55.6% of the EBP-positive cells at PI28, EBP expression was upregulated approximately 149.9% compared to uninfected BHK-21 cells. This was the highest expression level among all cell passages measured. Interestingly, in a parallel experiment, the average EBP expression level in the whole cell population at PI28 was only slightly higher (108.2%) than that in uninfected BHK-21 cells. At PI38, 98.7% of the cells were positive for FMDV 3D (an RNA-dependent RNA polymerase enzyme gene), and its maximum expression level observed at this passage. The expression level of EBP in 78.2% of the total cells, however, was reduced significantly. At PI68, 95.8% of the cells were 3D positive, and the expression of both the EBP and 3D genes were at the lowest levels of all the passages. Our studies using single cells yielded data that are otherwise inaccessible a using whole cell population. These results suggest that the establishment of persistent infection by FMDV is a dynamic process that results from the continuous adaptation and coevolution of viruses and cells to reach an equilibrium.

Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) infection or HCV-related liver diseases are now shown to cause more than 350,000 deaths every year. Adaptability of HCV genome to vary its composition and the existence of multiple strains makes it more difficult to combat the emergence of drug-resistant HCV infections. Among the HCV polyprotein which has both the structural and non-structural regions, the non-structural protein NS5B RNA-dependent RNA polymerase (RdRP) mainly mediates the catalytic role of RNA replication in conjunction with its viral protein machinery as well as host chaperone proteins. Lack of such RNA-dependent RNA polymerase enzyme in host had made it an attractive and hotly pursued target for drug discovery efforts. Recent drug discovery efforts targeting HCV RdRP have seen success with FDA approval for sofosbuvir as a direct-acting antiviral against HCV infection. However, variations in drug-binding sites induce drug resistance, and therefore targeting allosteric sites could delay the emergence of drug resistance. In this study, we focussed on allosteric thumb site II of the non-structural protein NS5B RNA-dependent RNA polymerase and developed a five-feature pharmacophore hypothesis/model which estimated the experimental activity with a strong correlation of 0.971 & 0.944 for training and test sets, respectively. Further, the Güner-Henry score of 0.6 suggests that the model was able to discern the active and inactive compounds and enrich the true positives during a database search. In this study, database search and molecular docking results supported by experimental HCV viral replication inhibition assays suggested ligands with best fitness to the pharmacophore model dock to the key residues involved in thumbs site II, which inhibited the HCV 1b viral replication in sub-micro-molar range.

Structure of a backtracked state reveals conformational changes similar to the state following nucleotide incorporation in human norovirus polymerase.

The RNA-dependent RNA polymerase (RdRP) from norovirus (NV) genogroup II has previously been crystallized as an apoenzyme (APO1) in multiple crystal forms, as well as as a pre-incorporation ternary complex (PRE1) bound to Mn(2+), various nucleoside triphosphates and an RNA primer-template duplex in an orthorhombic crystal form. When crystallized under near-identical conditions with a slightly different RNA primer/template duplex, however, the enzyme-RNA complex forms tetragonal crystals (anisotropic data, dmin ≃ 1.9 Å) containing a complex with the primer/template bound in a backtracked state (BACK1) similar to a post-incorporation complex (POST1) in a step of the enzymatic cycle immediately following nucleotidyl transfer. The BACK1 conformation shows that the terminal nucleotide of the primer binds in a manner similar to the nucleoside triphosphate seen in the PRE1 complex, even though the terminal two phosphoryl groups in the triphosphate moiety are absent and a covalent bond is present between the α-phosphoryl group of the terminal nucleotide and the 3'-oxygen of the penultimate nucleotide residue. The two manganese ions bound at the active site coordinate to conserved Asp residues and the bridging phosphoryl group of the terminal nucleotide. Surprisingly, the conformation of the thumb domain in BACK1 resembles the open APO1 state more than the closed conformation seen in PRE1. The BACK1 complex thus reveals a hybrid state in which the active site is closed while the thumb domain is open. Comparison of the APO1, PRE1 and BACK1 structures of NV polymerase helps to reveal a more complete and complex pathway of conformational changes within a single RdRP enzyme system. These conformational changes lend insight into the mechanism of RNA translocation following nucleotidyl transfer and suggest novel approaches for the development of antiviral inhibitors.

Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.

Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

Sofosbuvir in the treatment of chronic hepatitis C: new dog, new tricks.

The existing standard of care for chronic hepatitis C virus (HCV) infection includes the use of pegylated interferon and ribavirin as primary components of treatment, with the addition of a direct-acting antiviral for genotype 1 infection. Sofosbuvir, an oral nucleotide inhibitor of the HCV nonstructural protein 5B RNA-dependent RNA polymerase enzyme, was recently approved for use in combination with ribavirin and/or pegylated interferon for chronic HCV infection, depending on the genotype. Sofosbuvir is orally administered, and peak plasma concentrations are not affected by food. The drug is renally eliminated and does not require adjustment in mild to moderate renal insufficiency or in any degree of hepatic impairment. Sofosbuvir is not metabolized by cytochrome P450 isoenzymes, nor does it induce or inhibit the metabolism of agents that are substrates of these enzymes. Sofosbuvir demonstrates a high barrier to resistance and was well tolerated by patients in clinical trials. Overall efficacy rates vary between 70% and 90%.

Discovery of a Novel Series of Potent Non-Nucleoside Inhibitors of Hepatitis C Virus NS5B

Hepatitis C virus (HCV) is a major global public health problem. While the current standard of care, a direct-acting antiviral (DAA) protease inhibitor taken in combination with pegylated interferon and ribavirin, represents a major advancement in recent years, an unmet medical need still exists for treatment modalities that improve upon both efficacy and tolerability. Toward those ends, much effort has continued to focus on the discovery of new DAAs, with the ultimate goal to provide interferon-free combinations. The RNA-dependent RNA polymerase enzyme NS5B represents one such DAA therapeutic target for inhibition that has attracted much interest over the past decade. Herein, we report the discovery and optimization of a novel series of inhibitors of HCV NS5B, through the use of structure-based design applied to a fragment-derived starting point. Issues of potency, pharmacokinetics, and early safety were addressed in order to provide a clinical candidate in fluoropyridone 19.

The RNA-dependent RNA polymerase essential for post-transcriptional gene silencing in Neurospora crassa interacts with replication protein A

Post-transcriptional gene silencing (PTGS) pathways play a role in genome defence and have been extensively studied, yet how repetitive elements in the genome are identified is still unclear. It has been suggested that they may produce aberrant transcripts (aRNA) that are converted by an RNA-dependent RNA polymerase (RdRP) into double-stranded RNA (dsRNA), the essential intermediate of PTGS. However, how RdRP enzymes recognize aberrant transcripts remains a key question. Here we show that in Neurospora crassa the RdRP QDE-1 interacts with Replication Protein A (RPA), part of the DNA replication machinery. We show that both QDE-1 and RPA are nuclear proteins and that QDE-1 is specifically recruited onto the repetitive transgenic loci. We speculate that this localization of QDE-1 could allow the in situ production of dsRNA using transgenic nascent transcripts as templates, as in other systems. Supporting a link between the two proteins, we found that the accumulation of short interfering RNAs (siRNAs), the hallmark of silencing, is dependent on an ongoing DNA synthesis. The interaction between QDE-1 and RPA is important since it should guide further studies aimed at understanding the specificity of the RdRP and it provides for the first time a potential link between a PTGS component and the DNA replication machinery.

Development of carboxylic acid replacements in indole- N-acetamide inhibitors of hepatitis C virus NS5B polymerase

Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here 2 series of indole- N-acetamides, bearing physicochemically diverse carboxylic acid replacements, which show potent affinity for the NS5B enzyme with reduced potential for formation of glucuronide conjugates. Preliminary optimization of these series furnished compounds that are potent in the blockade of subgenomic HCV RNA replication in HUH-7 cells.