Design, Synthesis, Evaluation and Molecular Dynamics Simulation of Dengue Virus NS5-RdRp Inhibitors.

Dengue virus (DENV) is a major mosquito-borne human pathogen in tropical countries; however, there are currently no targeted antiviral treatments for DENV infection. Compounds 27 and 29 have been reported to be allosteric inhibitors of DENV RdRp with potent inhibitory effects. In this study, the structures of compounds 27 and 29 were optimized using computer-aided drug design (CADD) approaches. Nine novel compounds were synthesized based on rational considerations, including molecular docking scores, free energy of binding to receptor proteins, predicted Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) parameters, structural diversity, and feasibility of synthesis. Subsequently, the anti-DENV activity was assessed. In the cytopathic effect (CPE) assay conducted on BHK-21 cells using the DENV2 NGC strain, both SW-b and SW-d demonstrated comparable or superior activity against DENV2, with IC50 values of 3.58 ± 0.29 µM and 23.94 ± 1.00 µM, respectively, compared to that of compound 27 (IC50 = 19.67 ± 1.12 µM). Importantly, both SW-b and SW-d exhibited low cytotoxicity, with CC50 values of 24.65 µmol and 133.70 µmol, respectively, resulting in selectivity indices of 6.89 and 5.58, respectively. Furthermore, when compared to the positive control compound 3'-dATP (IC50 = 30.09 ± 8.26 µM), SW-b and SW-d displayed superior inhibitory activity in an enzyme inhibitory assay, with IC50 values of 11.54 ± 1.30 µM and 13.54 ± 0.32 µM, respectively. Molecular dynamics (MD) simulations elucidated the mode of action of SW-b and SW-d, highlighting their ability to enhance π-π packing interactions between benzene rings and residue W795 in the S1 fragment, compared to compounds 27 and 29. Although the transacylsulphonamide fragment reduced the interaction between T794 and NH, it augmented the interaction between R729 and T794. In summary, our study underscores the potential of SW-b and SW-d as allosteric inhibitors targeting the DENV NS5 RdRp domain. However, further in vivo studies are warranted to assess their pharmacology and toxicity profiles.

Structure-Based Drug Design of RdRp Inhibitors against SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic since 2019, spreading rapidly and posing a significant threat to human health and life. With over 6 billion confirmed cases of the virus, the need for effective therapeutic drugs has become more urgent than ever before. RNA-dependent RNA polymerase (RdRp) is crucial in viral replication and transcription, catalysing viral RNA synthesis and serving as a promising therapeutic target for developing antiviral drugs. In this article, we explore the inhibition of RdRp as a potential treatment for viral diseases, analysing the structural information of RdRp in virus proliferation and summarizing the reported inhibitors' pharmacophore features and structure-activity relationship profiles. We hope that the information provided by this review will aid in structure-based drug design and aid in the global fight against SARS-CoV-2 infection.

Repurposing of potential antiviral drugs against RNA-dependent RNA polymerase of SARS-CoV-2 by computational approach.

The high incidences of COVID-19 cases are believed to be associated with high transmissibility rates, which emphasizes the need for the discovery of evidence-based antiviral therapies for curing the disease. The rationale of repurposing existing classes of antiviral small molecule therapeutics against SARS-CoV-2 infection has been expected to accelerate the tedious and expensive drug development process. While Remdesivir has been recently approved to be the first treatment option for specific groups of COVID-19 patients, combinatory therapy with potential antiviral drugs may be necessary to enhance the efficacy in different populations. Hence, a comprehensive list of investigational antimicrobial drug compounds such as Favipiravir, Fidaxomicin, Galidesivir, GC376, Ribavirin, Rifabutin, and Umifenovir were computationally evaluated in this study. We performed in silico docking and molecular dynamics simulation on the selected small molecules against RNA-dependent RNA polymerase, which is one of the key target proteins of SARS-CoV-2, using AutoDock and GROMACS. Interestingly, our results revealed that the macrocyclic antibiotic, Fidaxomicin, possesses the highest binding affinity with the lowest energy value of -8.97 kcal/mol binding to the same active sites of RdRp. GC376, Rifabutin, Umifenovir and Remdesivir were identified as the next best compounds. Therefore, the above-mentioned compounds could be considered good leads for further preclinical and clinical experimentations as potentially efficient antiviral inhibitors for combination therapies against SARS-CoV-2.

Beyond the vaccines: a glance at the small molecule and peptide-based anti-COVID19 arsenal.

Unprecedented efforts of the researchers have been witnessed in the recent past towards the development of vaccine platforms for the control of the COVID-19 pandemic. Albeit, vaccination stands as a practical strategy to prevent SARS-CoV-2 infection, supplementing the anti-COVID19 arsenal with therapeutic options such as small molecules/peptides and antibodies is being conceived as a prudent strategy to tackle the emerging SARS-CoV-2 variants. Noteworthy to mention that collective efforts from numerous teams have led to the generation of a voluminous library composed of chemically and mechanistically diverse small molecules as anti-COVID19 scaffolds. This review article presents an overview of medicinal chemistry campaigns and drug repurposing programs that culminated in the identification of a plethora of small molecule-based anti-COVID19 drugs mediating their antiviral effects through inhibition of proteases, S protein, RdRp, ACE2, TMPRSS2, cathepsin and other targets. In light of the evidence ascertaining the potential of small molecule drugs to approach conserved proteins required for the viral replication of all coronaviruses, accelerated FDA approvals are anticipated for small molecules for the treatment of COVID19 shortly. Though the recent attempts invested in this direction in pursuit of enrichment of the anti-COVID-19 armoury (chemical tools) are praiseworthy, some strategies need to be implemented to extract conclusive benefits of the recently reported small molecule viz. (i) detailed preclinical investigation of the generated anti-COVID19 scaffolds (ii) in-vitro profiling of the inhibitors against the emerging SARS-CoV-2 variants (iii) development of assays enabling rapid screening of the libraries of anti-COVID19 scaffold (iv) leveraging the applications of machine learning based predictive models to expedite the anti-COVID19 drug discovery campaign (v) design of antibody-drug conjugates.

Molnupiravir for the treatment of COVID-19.

Molnupiravir (MK-4482, EIDD-2801) is a promising broad-spectrum experimental antiviral developed by Merck & Co. It is a nucleoside analogue prodrug that undergoes rapid conversion into nucleoside triphosphate (NTP) by intracellular metabolic processes. NTP inhibits viral polymerase by acting as an alternative substrate. Molnupiravir was initially developed to treat influenza and Venezuelan equine encephalitis virus (VEEV) infection as it exerts its antiviral activity by inhibiting RNA-dependent RNA polymerase (RdRp). Currently, it is being developed for the treatment of SARS-CoV-2 infection. Molnupiravir has demonstrated potent in vitro antiviral activity against positive-sense RNA viruses including influenza viruses, SARS-CoV, SARS-CoV-2 and MERS-CoV with low cytotoxicity and a high resistance barrier. Molnupiravir has been evaluated in phase I, II and III trials where it has demonstrated good efficacy, dose-dependent pharmacokinetics and a sound safety profile. In an interim analysis of a phase III study, treatment with molnupiravir reduced the risk of hospitalization or death by 50% in patients with COVID-19; in the final analysis, the reduction was 30%. On the basis of positive results in clinical trials, molnupiravir has been authorized for emergency use by the U.K. Medicines and Healthcare products Regulatory Agency (MHRA) and the U.S. Food and Drug Administration (FDA) in adults with mild to moderate COVID-19.

5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

Approaches to the Potential Therapy of COVID-19: A General Overview from the Medicinal Chemistry Perspective.

In spite of advances in vaccination, control of the COVID-19 pandemic will require the use of pharmacological treatments against SARS-CoV2. Their development needs to consider the existence of two phases in the disease, namely the viral infection and the inflammatory stages. The main targets for antiviral therapeutic intervention are: (a) viral proteins, including the spike (S) protein characteristic of the viral cover and the viral proteases in charge of processing the polyprotein arising from viral genome translation; (b) host proteins, such as those involved in the processes related to viral entry into the host cell and the release of the viral genome inside the cell, the elongation factor eEF1A and importins. The use of antivirals targeted at host proteins is less developed but it has the potential advantage of not being affected by mutations in the genome of the virus and therefore being active against all its variants. Regarding drugs that address the hyperinflammatory phase of the disease triggered by the so-called cytokine storm, the following strategies are particularly relevant: (a) drugs targeting JAK kinases; (b) sphingosine kinase 2 inhibitors; (c) antibodies against interleukin 6 or its receptor; (d) use of the traditional anti-inflammatory corticosteroids.

Alphavirus antivirals targeting RNA-dependent RNA polymerase domain of nsP4 divulged using surface plasmon resonance.

Alphaviruses are continuously re-emerging and pose a global threat to human health and currently no antiviral drug is commercially available for alphaviral infections. Alphavirus non-structural protein nsP4, which possesses RNA-dependent RNA polymerase (RdRp) activity, is a potential antiviral target. To date, no antiviral drug is commercially available against alphaviruses. Since RdRp is the key virus-specific enzyme involved in viral genome replication, this study identifies and validates the antiviral efficacy of small molecules targeting alphavirus RdRp. Purified nsP4 was characterized using the surface plasmon resonance (SPR) assay, and the binding affinities of divalent metal ions, ribonucleotides, and in vitro transcribed viral RNA oligonucleotides were obtained in the micromolar (µm) range. Further, four potential inhibitors, piperine (PIP), 2-thiouridine (2TU), pyrazinamide (PZA), and chlorogenic acid (CGA), were identified against nsP4 RdRp using a molecular docking approach. The SPR assay validated the binding of PIP, 2TU, PZA, and CGA to purified nsP4 RdRp with KD of 0.08, 0.13, 0.66, and 9.87 µm, respectively. Initial testing of these molecules as alphavirus replication inhibitors was done using SINV-IRES-Luc virus. Detailed assessment of antiviral efficacy of molecules against CHIKV was performed by plaque reduction assay, qRT-PCR, and immunofluorescence assay. PIP, 2TU, PZA, and CGA showed antiviral potency against CHIKV with EC50 values of 6.68, 27.88, 36.26, and 53.62 µm, respectively. This study paves the way towards the development of novel broad-spectrum alphavirus antivirals targeting nsP4 RdRp.

RdRp inhibitors and COVID-19: Is molnupiravir a good option?

Rapid changes in the viral genome allow viruses to evade threats posed by the host immune response or antiviral drugs, and can lead to viral persistence in the host cells. RNA-dependent RNA polymerase (RdRp) is an essential enzyme in RNA viruses, which is involved in RNA synthesis through the formation of phosphodiester bonds. Therefore, in RNA viral infections such as SARS-CoV-2, RdRp could be a crucial therapeutic target. The present review discusses the promising application of RdRp inhibitors, previously approved or currently being tested in human clinical trials, in the treatment of RNA virus infections. Nucleoside inhibitors (NIs) bind to the active site of RdRp, while nonnucleoside inhibitors (NNIs) bind to allosteric sites. Given the absence of highly effective drugs for the treatment of COVID-19, the discovery of an efficient treatment for this pandemic is an urgent concern for researchers around the world. We review the evidence for molnupiravir (MK-4482, EIDD-2801), an antiviral drug originally designed for Alphavirus infections, as a potential preventive and therapeutic agent for the management of COVID-19. At the beginning of this pandemic, molnupiravir was in preclinical development for seasonal influenza. When COVID-19 spread dramatically, the timeline for development was accelerated to focus on the treatment of this pandemic. Real time consultation with regulators took place to expedite this program. We summarize the therapeutic potential of RdRp inhibitors, and highlight molnupiravir as a new small molecule drug for COVID-19 treatment.

Repositioning of RdRp Inhibitors Against HCV NS5B Polymerase Utilizing Structure-Based Molecular Docking.

OBJECTIVE: Hepatitis C Virus (HCV) is very dreadful as it can attack an estimated 71 million people around the world. The World Health Organization (WHO) reported that every year about 399000 people die due to HCV caused by chronic cirrhosis and liver cancer globally. There are many drugs available for the treatment of HCV. But drug resistance and toxicity are major issues. The quest for potential drugs utilizing repositioning would be a very useful and economical method to combat HCV. METHODS: One of the most common HCV targets is RNA-dependent RNA polymerase (RdRp). The RdRp is common in HCV, Dengue virus (DENV), Zika virus (ZIKV), and Yellow fever virus (YFV) belonging to the same family of Flaviviridae. An attempt has been made in the present study to reposition different DENV, ZIKV, and YFV RdRp inhibitors against HCV NS5B polymerase utilizing structure-based molecular docking which explores the affinity and mode of binding of these RdRp inhibitors. RESULTS: Several 87 compounds having dengue, yellow fever and zika RdRp inhibitory activities have been taken into consideration for the screening of potential RdRp leads utilizing docking simulation, which focuses on the affinity and mode of binding of sofosbuvir diphosphate, a standard HCV, RdRp inhibitor. CONCLUSION: The compounds 6 (N-sulfonylanthranilic acid derivative), 17 (R1479), 20 (DMB220), 23 (FD-83-KI26), 40 (CCG-7648), 50 (T-1106), 65 (mycophenolic acid), and 69 (DMB213) exhibited docking score within the range of -7.602 to -8.971 Kcal/Mol having almost same mode of interaction as compared to the reference drug molecule. The drugs mentioned above possess satisfactory affinity to bind the hepatitis C viral RdRp and thus may be used to treat the disease. Therefore, these predicted compounds may be potential leads for further testing of anti HCV activity and can be repurposed to combat HCV. The high throughput shotgun of drug repurposing utilizing structure-based docking simulation freeware would be a cost-effective way to screen the potential anti-HCV leads.

Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase.

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure-activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization.

RdRp (RNA-dependent RNA polymerase): A key target providing anti-virals for the management of various viral diseases.

With the arrival of the Covid-19 pandemic, anti-viral agents have regained center stage in the arena of medicine. Out of the various drug targets involved in managing RNA-viral infections, the one that dominates almost all RNA viruses is RdRp (RNA-dependent RNA polymerase). RdRp are proteins that are involved in the replication of RNA-based viruses. Inhibition of RdRps has been an integral approach for managing various viral infections such as dengue, influenza, HCV (Hepatitis), BVDV, etc. Inhibition of the coronavirus RdRp is currently rigorously explored for the treatment of Covid-19 related complications. So, keeping in view the importance and current relevance of this drug target, we have discussed the importance of RdRp in developing anti-viral agents against various viral diseases. Different reported inhibitors have also been discussed, and emphasis has been laid on highlighting the inhibitor's pharmacophoric features and SAR profile.

The Situation of Small Molecules Targeting Key Proteins in combatting SARS-CoV-2: Synthesis, Metabolic Pathway, Mechanism of Action, and Potential Therapeutic Applications.

Due to the high mortality rate of the 2019 coronavirus disease (COVID-19) pandemic, there is an immediate need to discover drugs that can help before a vaccine becomes available. Given that the process of producing new drugs is so long, the strategy of repurposing existing drugs is one of the promising options for the urgent treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19 disease. Although FDA has approved Remdesivir for the use in hospitalized adults and pediatric patients suffering from COVID-19, no fully effective and reliable drug has been yet identified worldwide to treat COVID-19 specifically. Thus, scientists are still trying to find antivirals specific to COVID-19. This work reviews the chemical structure, metabolic pathway, and mechanism of action of the existing drugs with potential therapeutic applications for COVID-19. Furthermore, we summarized the molecular docking stimulation of the medications related to key protein targets. These already established drugs could be further developed, and after their testing through clinical trials, they could be used as suitable therapeutic options for patients suffering from COVID-19.

Quinoline and Quinazoline Derivatives Inhibit Viral RNA Synthesis by SARS-CoV-2 RdRp.

Coronavirus disease 2019 (COVID-19) is a fatal respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of potential drugs is urgently needed to control the pandemic. RNA dependent RNA polymerase (RdRp) is a conserved protein within RNA viruses and plays a crucial role in the viral life cycle, thus making it an attractive target for development of antiviral drugs. In this study, 101 quinoline and quinazoline derivatives were screened against SARS-CoV-2 RdRp using a cell-based assay. Three compounds I-13e, I-13h, and I-13i exhibit remarkable potency in inhibiting RNA synthesis driven by SARS-CoV-2 RdRp and relatively low cytotoxicity. Among these three compounds, I-13e showed the strongest inhibition upon RNA synthesis driven by SARS-CoV-2 RdRp, the resistance to viral exoribonuclease activity and the inhibitory effect on the replication of CoV, thus holding potential of being drug candidate for treatment of SARS-CoV-2.

Evaluation of Small Molecule Combinations against Respiratory Syncytial Virus In Vitro.

Respiratory syncytial virus (RSV) is a major pathogen that causes severe lower respiratory tract infection in infants, the elderly and the immunocompromised worldwide. At present no approved specific drugs or vaccines are available to treat this pathogen. Recently, several promising candidates targeting RSV entry and multiplication steps are under investigation. However, it is possible to lead to drug resistance under the long-term treatment. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we tested in vitro two-drug combinations of fusion inhibitors (GS5806, Ziresovir and BMS433771) and RNA-dependent RNA polymerase complex (RdRp) inhibitors (ALS8176, RSV604, and Cyclopamine). The statistical program MacSynergy II was employed to determine synergism, additivity or antagonism between drugs. From the result, we found that combinations of ALS8176 and Ziresovir or GS5806 exhibit additive effects against RSV in vitro, with interaction volume of 50 µM2% and 31 µM2% at 95% confidence interval, respectively. On the other hand, all combinations between fusion inhibitors showed antagonistic effects against RSV in vitro, with volume of antagonism ranging from -50 µM2 % to -176 µM2 % at 95% confidence interval. Over all, our results suggest the potentially therapeutic combinations in combating RSV in vitro could be considered for further animal and clinical evaluations.

nCOVID-19 Pandemic: From Molecular Pathogenesis to Potential Investigational Therapeutics.

In December 2019, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related epidemic was first observed in Wuhan, China. In 2020, owing to the highly infectious and deadly nature of the virus, this widespread novel coronavirus disease 2019 (nCOVID-19) became a worldwide pandemic. Studies have revealed that various environmental factors including temperature, humidity, and air pollution may also affect the transmission pattern of COVID-19. Unfortunately, still, there is no specific drug that has been validated in large-scale studies to treat patients with confirmed nCOVID-19. However, remdesivir, an inhibitor of RNA-dependent RNA polymerase (RdRp), has appeared as an auspicious antiviral drug. Currently, a large-scale study on remdesivir (i.e., 200 mg on first day, then 100 mg once/day) is ongoing to evaluate its clinical efficacy to treat nCOVID-19. Good antiviral activity against SARS-CoV-2 was not observed with the use of lopinavir/ritonavir (LPV/r). Nonetheless, the combination of umifenovir and LPV/r was found to have better antiviral activity. Furthermore, a combination of hydroxychloroquine (i.e., 200 mg 3 times/day) and azithromycin (i.e., 500 mg on first day, then 250 mg/day from day 2-5) also exhibited good activity. Currently, there are also ongoing studies to evaluate the efficacy of teicoplanin and monoclonal and polyclonal antibodies against SARS-CoV-2. Thus, in this article, we have analyzed the genetic diversity and molecular pathogenesis of nCOVID-19. We also present possible therapeutic options for nCOVID-19 patients.

RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery.

COVID-19 respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global health issue since it emerged in December 2019. While great global efforts are underway to develop vaccines and to discover or repurpose therapeutic agents for this disease, as of this writing only the nucleoside drug remdesivir has been approved under Emergency Use Authorization to treat COVID-19. The RNA-dependent RNA polymerase (RdRP), a viral enzyme for viral RNA replication in host cells, is one of the most intriguing and promising drug targets for SARS-CoV-2 drug development. Because RdRP is a viral enzyme with no host cell homologs, selective SARS-CoV-2 RdRP inhibitors can be developed that have improved potency and fewer off-target effects against human host proteins and thus are safer and more effective therapeutics for treating COVID-19. This review focuses on biochemical enzyme and cell-based assays for RdRPs that could be used in high-throughput screening to discover new and repurposed drugs against SARS-CoV-2.

Functionalized 2,1-benzothiazine 2,2-dioxides as new inhibitors of Dengue NS5 RNA-dependent RNA polymerase.

Over recent years, many RNA viruses have been "re-discovered", including life-threatening flaviviruses, such as Dengue, Zika, and several encephalitis viruses. Since no specific inhibitors are currently available to treat these infections, there is a pressing need for new therapeutics. Among the flaviviral proteins, NS5 RNA-dependent RNA polymerase (RdRp) represents a validated target being essential for viral replication and it has no human analog. To date, few NS5 RdRp inhibitor chemotypes have been reported and no inhibitors are currently in clinical development. In this context, after an in vitro screening against Dengue 3 NS5 RdRp of our in-house HCV NS5B inhibitors focused library, we found that 2,1-benzothiazine 2,2-dioxides are promising non-nucleoside inhibitors of flaviviral RdRp with compounds 8 and 10 showing IC50 of 0.6 and 0.9 µM, respectively. Preliminary structure-activity relationships indicated a key role for the C-4 benzoyl group and the importance of a properly functionalized C-6 phenoxy moiety to modulate potency. Compound 8 acts as non-competitive inhibitor and its proposed pose in the so-called N pocket of the RdRp thumb domain allowed to explain the key contribution of the benzoyl and the phenoxy moieties for the ligand binding.

A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems.

In this work, we present and discuss a comprehensive set of both newly and previously synthesized compounds belonging to 5 distinct molecular classes of linear aromatic N-polycyclic systems that efficiently inhibits bovine viral diarrhea virus (BVDV) infection. A coupled in silico/in vitro investigation was employed to formulate a molecular rationale explaining the notable affinity of all molecules to BVDV RNA dependent RNA polymerase (RdRp) NS5B. We initially developed a three-dimensional common-feature pharmacophore model according to which two hydrogen bond acceptors and one hydrophobic aromatic feature are shared by all molecular series in binding the viral polymerase. The pharmacophoric information was used to retrieve a putative binding site on the surface of the BVDV RdRp and to guide compound docking within the protein binding site. The affinity of all compounds towards the enzyme was scored via molecular dynamics-based simulations, showing high correlation with in vitro EC50 data. The determination of the interaction spectra of the protein residues involved in inhibitor binding highlighted amino acids R295 and Y674 as the two fundamental H-bond donors, while two hydrophobic cavities HC1 (residues A221, I261, I287, and Y289) and HC2 (residues V216, Y303, V306, K307, P408, and A412) fulfill the third pharmacophoric requirement. Three RdRp (K263, R295 and Y674) residues critical for drug binding were selected and mutagenized, both in silico and in vitro, into alanine, and the affinity of a set of selected compounds towards the mutant RdRp isoforms was determined accordingly. The agreement between predicted and experimental data confirmed the proposed common molecular rationale shared by molecules characterized by different chemical scaffolds in binding to the BVDV RdRp, ultimately yielding compound 6b (EC50 = 0.3 µM; IC50 = 0.48 µM) as a new, potent inhibitor of this Pestivirus.

Anti-norovirus therapeutics: a patent review (2010-2015).

INTRODUCTION: Human noroviruses are the primary causative agents of acute gastroenteritis and are a pressing public health burden worldwide. There are currently no vaccines or small molecule therapeutics available for the treatment or prophylaxis of norovirus infections. An improved understanding of norovirus biology, as well as the pathogenic mechanisms underlying the disease, has provided the impetus for a range of intense exploratory drug discovery efforts targeting viral and host factors. AREAS COVERED: An overview of norovirus inhibitors disclosed in the patent literature (2010-present) and Clinicaltrials.gov is presented. The review is further enriched and supplemented by recent literature reports. EXPERT OPINION: Seminal discoveries made in recent years, including a better understanding of the pathobiology and life cycle of norovirus, the identification and targeting of multiple viral and host factors, the advent of a replicon system and a small animal model for the preclinical evaluation of lead compounds, and the availability of high resolution X-ray crystal structures that can be utilized in structure-based drug design and lead optimization campaigns, collectively suggest that a small molecule therapeutic and prophylactic for norovirus infection is likely to emerge in the not too distant future.