RNA-dependent RNA Polymerase Inhibitors Research Articles

In Silico Analysis of 14-Deoxy 11, 12-Didehydro Andrographolide (AGP 2) from Sambiloto (Andrographis paniculata) as Drug Candidate Against SARS-CoV-2

The outbreak of the COVID-19 pandemic in the world has urged researchers to develop a vaccine or therapeutic drugs to fight this virus. This study aimed to assay 14 deoxy-11,12-didehydroandrographolide (AGP 2) ability as an inhibitor of 3-chymotrypsin like-protease (3CLPro), Papain-like protease (PLPro), and RNA-dependent RNA-polymerase (RdRp), the viral proteins of SARS-CoV-2 and to evaluate it safeness as a drug candidate. In-silico technique was performed in this study to analyze the binding interaction, complex stability between protein and ligand, and drug-likeness properties. The proteins and ligands were obtained from Protein Data Bank (PDB) and PubChem web tools, then using PyRx to identify the binding affinity score, PyMoL to visualize the 3D binding interaction, and WebGro web tools to analyze the stability of each complex. A drug-likeness evaluation was done using SwissADME, pkCSM, and Way2drug web tools. The result of this study showed that the binding affinity score for each complex is; AGP 2-3CLPro (-6.7 kcal/mol), AGP 2- PLPro (-6.4 kcal/mol), and AGP 2-RdRp (-7.0 kcal/mol) where the AGP 2-RdRp and AGP 2-3CLPro showed a stable form indicating the inhibitor ability of AGP 2. This study also demonstrates that the drug-likeness properties of AGP 2 are safe to use. Additionally, it has been proved that AGP 2 can be developed into a therapeutic drug with further studies.

Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their ‘open’ counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.

A hypothesis on designing strategy of effective RdRp inhibitors for the treatment of SARS-CoV-2.

Vaccines are used as one of the major weapons for the eradication of pandemic. However, the rise of different variants of the SARS-CoV-2 virus is creating doubts regarding the end of the pandemic. Hence, there is an urgent need to develop more drug candidates which can be useful for the treatment of COVID-19. In the present research for the scientific hypothesis, emphasis was given on the direct antiviral therapy available for the treatment of COVID-19. In lieu of this, the available molecular targets which include Severe Acute Respiratory Syndrome Chymotrypsin-like Protease (SARS-3CLpro), Papain-Like Cysteine Protease (PLpro), and RNA-Dependent RNA Polymerase (RdRp) were explored. As per the current scientific reports and literature, among all the available molecular targets, RNA-Dependent RNA Polymerase (RdRp) was found to be a crucial molecular target for the treatment of COVID-19. Most of the inhibitors which are reported against this target consisted of the free amine group and carbonyl group which might be playing an important role in the binding interaction with the RdRp protein. Among all the reported RdRp inhibitors, remdesivir, favipiravir, and molnupiravir were found to be the most promising drugs against COVID-19. Overall, the structural features of this RNA-Dependent RNA Polymerase (RdRp) inhibitors proved the importance of pyrrolo-triazine and pyrimidine scaffolds. Previous computational models of these drug molecules indicated that substitution with the polar functional group, hydrogen bond donor, and electronegative atoms on these scaffolds may increase the activity against the RdRp protein. Hence, in line with the proposed hypothesis, in the present research work for the evaluation of the hypothesis, new molecules were designed from the pyrrolo-triazine and pyrimidine scaffolds. Further, molecular docking and MD simulation studies were performed with these designed molecules. All these designed molecules (DM-1, DM-2, and DM-3) showed the results as per the proposed hypothesis. Among all the designed molecules, DM-1 showed promising results against the RdRp protein of SARS-CoV-2. In the future, these structural features can be used for the development of new RdRp inhibitors with improved activity. Also, in the future lead compound DM-1 can be explored against the RdRp protein for the treatment of COVID-19.

Small-molecule metabolites in SARS-CoV-2 treatment: a comprehensive review.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has quickly spread all over the world. In this respect, traditional medicinal chemistry, repurposing, and computational approaches have been exploited to develop novel medicines for treating this condition. The effectiveness of chemicals and testing methods in the identification of new promising therapies, and the extent of preparedness for future pandemics, have been further highly advantaged by recent breakthroughs in introducing noble small compounds for clinical testing purposes. Currently, numerous studies are developing small-molecule (SM) therapeutic products forinhibiting SARS-CoV-2 infection and replication, as well as managing the disease-related outcomes. Transmembrane serine protease (TMPRSS2)-inhibiting medicinal products can thus prevent the entry of the SARS-CoV-2 into the cells, and constrain its spreading along with the morbidity and mortality due to the coronavirus disease 2019 (COVID-19), particularly when co-administered with inhibitors such as chloroquine (CQ) and dihydroorotate dehydrogenase (DHODH). The present review demonstrates that the clinical-stage therapeutic agents, targeting additional viral proteins, might improve the effectiveness of COVID-19 treatment if applied as an adjuvant therapy side-by-side with RNA-dependent RNA polymerase (RdRp) inhibitors.

Discovery of non-nucleoside oxindole derivatives as potent inhibitors against dengue RNA-dependent RNA polymerase

A series of thiazole linked Oxindole-5-Sulfonamide (OSA) derivatives were designed as inhibitors of RNA-dependent RNA polymerase (RdRp) activity of Dengue virus. These were synthesized and then evaluated for their efficacy in ex-vivo virus replication assay using human cell lines. Among 20 primary compounds in the series, OSA-15 was identified as a hit. A series of analogues were synthesized by replacing the difluoro benzyl group of OSA-15 with different substituted benzyl groups. The efficacy of OSA-15derivatives was less than that of the parent compound, except OSA-15-17, which has shown improved efficacy than OSA-15. The further optimization was carried out by adding dimethyl (DM) groups to both the sulfonamide and oxindole NH’s to produce OSA-15-DM and OSA-15-17-DM. These two compounds were showing no detectable cytotoxicity and the latter was more efficacious. Further, both these compounds were tested for inhibition in all the serotypes of the Dengue virus using an ex-vivo assay. The EC50 of OSA-15-17-DM was observed in a low micromolar range between 2.5 and 5.0 µg/ml. Computation docking and molecular dynamics simulation studies confirmed the binding of identified hits to DENV RdRp. OSA15-17-DM blocks the RNA entrance and elongation site for their biological activity with high binding affinity. Overall, the identified oxindole derivatives are novel compounds that can inhibit Dengue replication, working as non-nucleoside inhibitors (NNI) to explore as anti-viral RdRp activity.

Insight into the Hantaan virus RNA-dependent RNA polymerase inhibition using in-silico approaches.

The Hantaan virus (HTN) is a member of the hantaviridae family. It is a segmented type, negative-strand virus (sNSVs). It causes hemorrhagic fever with renal syndrome, which includes fever, vascular hemorrhage, and renal failure. This illness is one of the most serious hemorrhagic diseases in the world, and it is a major public health concern due to its high mortality rate. The Hantaan virus RNA-dependent RNA polymerase complex (RdRp) is involved in viral RNA transcription and replication for the survival and transmission of this virus. Therefore, it is a primary target for antiviral drug development. Interference with the endonucleolytic "cap-snatching" reaction by the HTN virus RdRp endonuclease domain is a particularly appealing approach for drug discovery against this virus. This RdRp endonuclease domain of the HTN virus has a metal-dependent catalytic activity. We targeted this metal-dependent enzymatic activity to identify inhibitors that can bind and disrupt this endonuclease enzyme activity using in-silico approaches i.e., molecular docking, molecular dynamics simulation, predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) and drug-likeness studies. The docking studies showed that peramivir, and ingavirin compounds can effectively bind with the manganese ions and engage with other active site residues of this protein. Molecular simulations also showed stable binding of these ligands with the active site of HTN RdRp. Simulation analysis showed that they were in constant contact with the active site manganese ions and amino acid residues of the HTN virus endonuclease domain. This study will help in better understanding the HTN and related viruses.

Preliminary Computational Analysis of Pyrazinamide-Based Derivatives Reveals Possible Inhibition of SARS-CoV-2 RNA-Dependent RNA Polymerase, and Their Possible Use as Antiviral Agents

Pyrazinamide is a pyrazine analog currently used to treat tuberculosis. It shares a core structure similar to that of favipiravir, which is a promising drug candidate that may inhibit the SARS-CoV-2 RNA-dependent RNA polymerase. This feature could be an opportunity for further drug development of anti-COVID-19 medication starting from a pyrazinamide core structure. This study aimed to determine and predict the most effective pyrazinamide-based analogs against the SARS-CoV-2 RNA-dependent RNA polymerase by using combined ligand- and structure-based computational analysis. This study performed a rational in silico study to screen pyrazinamide-like molecules from a commercially available ZINC database, with a similarity score higher than 0.40, and then these screened for acceptable pharmacokinetic properties, and then to further conduct molecular docking analysis with SARS-CoV-2 RNA-dependent RNA polymerase. The results showed that compound 12, having a dichloropyrimidine core structure had a similarity score of 0.446. It exerted the most binding affinity with RNA-dependent RNA polymerase, with estimated docking scores of −5.72, −5.25, −7.06, −7.00 and −4.63 kcal/mol in intact, ribosylated, mono-phosphoribosylated, di-phosphoribosylated and tri-phosphoribosylated forms, respectively. Watson-Crick base-pairing of compound 12 indicated that it favored binding with the uracil nucleoside of the RNA template. Compound 12 was confirmed as the lead compound, being a pyrazinamide-like molecule, and so might be a most promising candidate molecule, as an adenine analog RNA-dependent RNA polymerase inhibitor. It is suggested that the antiviral effect of this lead compound should be studied further as part of a drug discovery and development process. HIGHLIGHTS In silico analysis was performed to screen pyrazinamide-like compounds against the RNA-dependent RNA polymerase Pharmacokinetics and drug-likeliness properties of fourteen preselected compounds were assessed by using SwissADME with favorable results Among these preselected compounds, compound 12 showed the best docking score in almost all of its phosphoribosylated forms GRAPHICAL ABSTRACT

Potent and biostable inhibitors of the main protease of SARS-CoV-2

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection invitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.

Discovery of New Zika Protease and Polymerase Inhibitors through the Open Science Collaboration Project OpenZika.

The Zika virus (ZIKV) is a neurotropic arbovirus considered a global threat to public health. Although there have been several efforts in drug discovery projects for ZIKV in recent years, there are still no antiviral drugs approved to date. Here, we describe the results of a global collaborative crowdsourced open science project, the OpenZika project, from IBM's World Community Grid (WCG), which integrates different computational and experimental strategies for advancing a drug candidate for ZIKV. Initially, molecular docking protocols were developed to identify potential inhibitors of ZIKV NS5 RNA-dependent RNA polymerase (NS5 RdRp), NS3 protease (NS2B-NS3pro), and NS3 helicase (NS3hel). Then, a machine learning (ML) model was built to distinguish active vs inactive compounds for the cytoprotective effect against ZIKV infection. We performed three independent target-based virtual screening campaigns (NS5 RdRp, NS2B-NS3pro, and NS3hel), followed by predictions by the ML model and other filters, and prioritized a total of 61 compounds for further testing in enzymatic and phenotypic assays. This yielded five non-nucleoside compounds which showed inhibitory activity against ZIKV NS5 RdRp in enzymatic assays (IC50 range from 0.61 to 17 μM). Two compounds thermally destabilized NS3hel and showed binding affinity in the micromolar range (Kd range from 9 to 35 μM). Moreover, the compounds LabMol-301 inhibited both NS5 RdRp and NS2B-NS3pro (IC50 of 0.8 and 7.4 μM, respectively) and LabMol-212 thermally destabilized the ZIKV NS3hel (Kd of 35 μM). Both also protected cells from death induced by ZIKV infection in in vitro cell-based assays. However, while eight compounds (including LabMol-301 and LabMol-212) showed a cytoprotective effect and prevented ZIKV-induced cell death, agreeing with our ML model for prediction of this cytoprotective effect, no compound showed a direct antiviral effect against ZIKV. Thus, the new scaffolds discovered here are promising hits for future structural optimization and for advancing the discovery of further drug candidates for ZIKV. Furthermore, this work has demonstrated the importance of the integration of computational and experimental approaches, as well as the potential of large-scale collaborative networks to advance drug discovery projects for neglected diseases and emerging viruses, despite the lack of available direct antiviral activity and cytoprotective effect data, that reflects on the assertiveness of the computational predictions. The importance of these efforts rests with the need to be prepared for future viral epidemic and pandemic outbreaks.

The first Chinese oral anti-COVID-19 drug Azvudine launched

The first Chinese oral anti-COVID-19 drug Azvudine launched

Chemoinformatic Design and Profiling of Derivatives of Dasabuvir, Efavirenz, and Tipranavir as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase and Methyltransferase

Zika virus (ZIKV) infection is one of the mosquito-borne flaviviruses of human importance with more than 2 million suspected cases and more than 1 million people infected in about 30 countries. There are reported inhibitors of the zika virus replication machinery, but no approved effective antiviral therapy including vaccines directed against the virus for treatment or prevention is currently available. The study investigated the chemoinformatic design and profiling of derivatives of dasabuvir, efavirenz, and tipranavir as potential inhibitors of the zika virus RNA-dependent RNA polymerase (RdRP) and/or methyltransferase (MTase). The three-dimensional (3D) coordinates of dasabuvir, efavirenz, and tipranavir were obtained from the PubChem database, and their respective derivatives were designed with DataWarrior-5.2.1 using an evolutionary algorithm. Derivatives that were not mutagenic, tumorigenic, or irritant were selected; docked into RdRP and MTase; and further subjected to absorption, distribution, metabolism, excretion, and toxicity (ADMET) evaluation with Swiss-ADME and pkCSM web tools. Some of the designed compounds are Lipinski’s rule-of-five compliant, with good synthetic accessibilities. Compounds 20d, 21d, 22d, and 1e are nontoxic with the only limitation of CYP1A2, CYP2C19, and/or CYP2C9 inhibition. Replacements of −CH3 and −NH– in the methanesulfonamide moiety of dasabuvir with −OH and −CH2– or −CH2CH2–, respectively, improved the safety/toxicity profile. Hepatotoxicity in 5d, 4d, and 18d is likely due to −NH– in their methanesulfonamide/sulfamic acid moieties. These compounds are potent inhibitors of N-7 and 2′-methylation activities of ZIKV methyltransferase and/or RNA synthesis through interactions with amino acid residues in the priming loop/“N-pocket” in the virus RdRP. Synthesis of these compounds and wet laboratory validation against ZIKV are recommended.

Pulmonary delivery of favipiravir inhalation solution for COVID-19 treatment: in vitro characterization, stability, in vitro cytotoxicity, and antiviral activity using real time cell analysis

Favipiravir, an RNA-dependent RNA polymerase (RdRp) inhibitor, is used to treat patients infected with influenza virus and most recently with SARS-CoV-2. However, poor accumulation of favipiravir in lung tissue following oral administration has required an alternative method of administration that directly targets the lungs. In this study, an inhalation solution of favipiravir at a concentration of 2 mg mL−1 was developed and characterized for the first time. The chemical stability of inhaled favipiravir solution in two different media, phosphate buffer saline (PBS) and normal saline (NS), was investigated under different conditions: 5 ± 3 °C, 25 ± 2 °C/60% RH ± 5% RH, and 40 ± 2 °C/75% RH ± 5% RH; in addition to constant light exposure. As a result, favipiravir solution in PBS revealed superior stability over 12 months at 5 ± 3 °C. Antiviral activity of favipiravir was assessed at the concentrations between 0.25 and 3 mg mL−1 with real time cell analyzer on Vero-E6 that were infected with SARS-CoV-2/B.1.36. The optimum concentration was found to be 2 mg mL−1, where minimum toxicity and sufficient antiviral activity was observed. Furthermore, cell viability assay against Calu-3 lung epithelial cells confirmed the biocompatibility of favipiravir at concentrations up to 50 μM (7.855 mg mL−1). The in vitro aerodynamic profiles of the developed inhaled favipiravir formulation, when delivered with soft-mist inhaler indicated good lung targeting properties. These results suggest that favipiravir solution prepared with PBS could be considered as a suitable and promising inhalation formulation for pulmonary delivery in the treatment of patients with COVID-19.

A motley of possible therapies of the COVID-19: reminiscing the origin of the pandemic.

The 2019 outbreak of corona virus disease began from Wuhan (China), transforming into a leading pandemic, posing an immense threat to the global population. The WHO coined the term nCOVID-19 for the disease on 11th February, 2020 and the International Committee of Taxonomy of Viruses named it SARS-CoV-2, on account of its similarity with SARS-CoV-1 of 2003. The infection is associated with fever, cough, pneumonia, lung damage, and ARDS along with clinical implications of lung opacities. Brief understanding of the entry target of virus, i.e., ACE2 receptors has enabled numerous treatment options as discussed in this review. The manuscript provides a holistic picture of treatment options in COVID-19, such as non-specific anti-viral drugs, immunosuppressive agents, anti-inflammatory candidates, anti-HCV, nucleotide inhibitors, antibodies and anti-parasitic, RNA-dependent RNA polymerase inhibitors, anti-retroviral, vitamins and hormones, JAK inhibitors, and blood plasma therapy. The text targets to enlist the investigations conducted on all the above categories of drugs, with respect to the COVID-19 pandemic, to accelerate their significance in hindering the disease progression. The data collected primarily targets recently published articles and most recent records of clinical trials, focusing on the last 10-year database. The current review provides a comprehensive view on the critical need of finding a suitable treatment for the currently prevalent COVID-19 disease, and an opportunity for the researchers to investigate the varying possibilities to find and optimized treatment approach to mitigate and ameliorate the chaos created by the pandemic worldwide.

Repurposing of the antihistamine mebhydrolin napadisylate for treatment of Zika virus infection

Zika virus (ZIKV) infection can lead to severe neurological disorders and fetal defects, which has become a public health problem worldwide. However, effective clinical treatment for ZIKV infection was still arduous. Antihistamines are attractive candidates for drug repurposing due to their low price and widespread availability. Here we screened FDA-approved antihistamine drugs to obtain anti-ZIKV candidate compounds and identified mebhydrolin napadisylate (MHL) that exhibits the potent inhibition of ZIKV infection in various cell lines in a histamine H1 receptor-independent manner. Mechanistic studies suggest that MHL acts as a ZIKV NS5 RNA-dependent RNA polymerase (RdRp) inhibitor, supported by a structure–activity relationship (SAR) analysis showing the correlation between the inhibitory effect upon viral RNA synthesis and ZIKV infectivity. Furthermore, MHL was shown to bind ZIKV NS5 RdRp in vitro and predicted to interact with key residues at the active site of ZIKV NS5 RdRp by molecular docking analysis. Our data together suggest that MHL suppresses ZIKV infection through the inhibition of ZIKV NS5 RdRp activity. This study highlights that MHL might be a promising clinical anti-ZIKV therapeutic.

Drug-Membrane Interactions: Effects of Virus-Specific RNA-Dependent RNA Polymerase Inhibitors Remdesivir and Favipiravir on the Structure of Lipid Bilayers.

The two RNA-dependent RNA polymerase inhibitors remdesivir and favipiravir were originally developed and approved as broad-spectrum antiviral drugs for the treatment of harmful viral infections such as Ebola and influenza. With the outbreak of the global SARS-CoV-2 pandemic, the two drugs were repurposed for the treatment of COVID-19 patients. Clinical studies suggested that the efficacy of the drugs is enhanced in the case of an early or even prophylactic application. Because the contact between drug molecules and the plasma membrane is essential for a successful permeation process of the substances and therefore for their intracellular efficiency, drug-induced effects on the membrane structure are likely and have already been shown for other substances. We investigated the impact of remdesivir and favipiravir on lipid bilayers in model and cell membranes via several biophysical approaches. The measurements revealed that the embedding of remdesivir molecules in the lipid bilayer results in a disturbance of the membrane structure of the tested phospholipid vesicles. Nevertheless, in a cell-based assay, the presence of remdesivir induced only weak hemolysis of the treated erythrocytes. In contrast, no experimental indication for an effect on the structure and integrity of the membrane was detected in the case of favipiravir. Regarding potential prophylactic or accompanying use of the drugs in the therapy of COVID-19, the physiologically relevant impacts associated with the drug-induced structural modifications of the membrane might be important to understand side effects and/or low effectivities.

Therapeutic efficacy of monoclonal antibodies and antivirals against SARS-CoV-2 Omicron BA.1 in Syrian hamsters.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the major antigen stimulating the host's protective immune response. Here we assessed the efficacy of therapeutic monoclonal antibodies (mAbs) against Omicron variant (B.1.1.529) sublineage BA.1 variants in Syrian hamsters. Of the FDA-approved therapeutic mAbs tested (that is, REGN10987/REGN10933, COV2-2196/COV2-2130 and S309), only COV2-2196/COV2-2130 efficiently inhibited BA.1 replication in the lungs of hamsters, and this effect was diminished against a BA.1.1 variant possessing the S-R346K substitution. In addition, treatment of BA.1-infected hamsters with molnupiravir (a SARS-CoV-2 RNA-dependent RNA polymerase inhibitor) or S-217622 (a SARS-CoV-2 protease inhibitor) strongly reduced virus replication in the lungs. These findings suggest that the use of therapeutic mAbs in Omicron-infected patients should be carefully considered due to mutations that affect efficacy, and demonstrate that the antiviral compounds molnupiravir and S-217622 are effective against Omicron BA.1 variants.

New Analogues of Uridine as Possible Anti-Viral Agents Specific to SARS-CoV-2.

The development of specific drugs against SARS-CoV-2 infection is a major challenge facing global science and healthcare. Despite numerous attempts, there are still no truly effective drugs. Currently, the main approach in the creation of drugs against COVID-19 is repurposing, i.e., re-profiling existing drugs approved for medical use, for example, the use of a drug for the treatment of Ebola—Remdesivir, and the use of a drug for the treatment of influenza—Favipiravir. However, it is already obvious that these drugs are not specific enough nor effective enough. Another promising approach is the creation of new molecules, but it should be noted immediately that implementation requires much more time and costs. However, the search for new SARS-CoV-2 specific antiviral agents continues. The aim of our work was the creation of new 5-substituted uridine derivatives as potential inhibitors of coronavirus RNA-dependent RNA polymerase. The substances were obtained in high yields by the Suzuki‒Miyaura reaction and characterized using modern physicochemical methods. However, testing of their antiviral activity against SARS-CoV-2 did not reveal a significant inhibitory effect.

Evaluation of Potential Anti-Hepatitis A Virus 3C Protease Inhibitors Using Molecular Docking.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis worldwide and occasionally causes acute liver failure and can lead to death in the absence of liver transplantation. Although HAV vaccination is available, the prevalence of HAV vaccination is not adequate in some countries. Additionally, the improvements in public health reduced our immunity to HAV infection. These situations motivated us to develop potentially new anti-HAV therapeutic options. We carried out the in silico screening of anti-HAV compounds targeting the 3C protease enzyme using the Schrodinger Modeling software from the antiviral library of 25,000 compounds to evaluate anti-HAV 3C protease inhibitors. Additionally, in vitro studies were introduced to examine the inhibitory effects of HAV subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in hepatoma cell lines using luciferase assays and real-time RT-PCR. In silico studies enabled us to identify five lead candidates with optimal binding interactions in the active site of the target HAV 3C protease using the Schrodinger Glide program. In vitro studies substantiated our hypothesis from in silico findings. One of our lead compounds, Z10325150, showed 47% inhibitory effects on HAV genotype IB subgenomic replicon replication and 36% inhibitory effects on HAV genotype IIIA HA11-1299 replication in human hepatoma cell lines, with no cytotoxic effects at concentrations of 100 μg/mL. The effects of the combination therapy of Z10325150 and RNA-dependent RNA polymerase inhibitor, favipiravir on HAV genotype IB HM175 subgenomic replicon replication and HAV genotype IIIA HA11-1299 replication showed 64% and 48% inhibitory effects of HAV subgenomic replicon and HAV replication, respectively. We identified the HAV 3C protease inhibitor Z10325150 through in silico screening and confirmed the HAV replication inhibitory activity in human hepatocytes. Z10325150 may offer the potential for a useful HAV inhibitor in severe hepatitis A.

Drug repurposing against the RNA-dependent RNA polymerase domain of dengue serotype 3 by virtual screening and molecular dynamics simulations

Dengue is an arboviral disease caused by the dengue flavivirus. The NS5 protein of flaviviruses is a potential therapeutic target, and comprises an RNA-dependent RNA polymerase (RDRP) domain that catalyses viral replication. The aim of this study was to repurpose FDA-approved drugs against the RDRP domain of dengue virus serotype 3 (DENV3) using structure-based virtual screening and molecular dynamics (MD) simulations. The FDA-approved drugs were screened against the RDRP domain of DENV3 using a two-step docking-based screening approach with Glide SP and Glide XP. For comparison, four reported DENV3 RDRP inhibitors were docked as standards. The hitlist was screened based on the docking score of the inhibitor with the lowest docking score (PubChem ID: 118797902; reported IC50 value: 0.34 µM). Five hits with docking scores and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) energy lower than those of 118797902 were selected. The stability of the hit-receptor complexes was investigated using 100 ns MD simulations in an explicit solvent. The results of MD simulations demonstrated that polydatin and betiatide remained stably bound to the receptor, and formed stable interactions with the RDRP domain of DENV3. The hit-receptor interactions were comparable to those of 118797902. The average Prime MM-GBSA energy of polydatin and betiatide was lower than that of 118797902 during simulation, indicating that their binding affinity to DENV3 RDRP was higher than that of the standard. The results of this study may aid in the development of serotype-selective drugs against dengue in the future. Communicated by Ramaswamy H. Sarma

2'- and 3'-Ribose Modifications of Nucleotide Analogues Establish the Structural Basis to Inhibit the Viral Replication of SARS-CoV-2.

Inhibition of RNA-dependent RNA polymerase (RdRp) by nucleotide analogues with ribose modification provides a promising antiviral strategy for the treatment of SARS-CoV-2. Previous works have shown that remdesivir carrying 1′-substitution can act as a “delayed chain terminator”, while nucleotide analogues with 2′-methyl group substitution could immediately terminate the chain extension. However, how the inhibition can be established by the 3′-ribose modification as well as other 2′-ribose modifications is not fully understood. Herein, we have evaluated the potential of several adenosine analogues with 2′- and/or 3′-modifications as obligate chain terminators by comprehensive structural analysis based on extensive molecular dynamics simulations. Our results suggest that 2′-modification couples with the protein environment to affect the structural stability, while 3′-hydrogen substitution inherently exerts “immediate termination” without compromising the structural stability in the active site. Our study provides an alternative promising modification scheme to orientate the further optimization of obligate terminators for SARS-CoV-2 RdRp.