C60 fullerene against SARS-CoV-2 coronavirus: an in silico insight.

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.

Antiviral drug discovery: preparing for the next pandemic.

Clinically approved antiviral drugs are currently available for only 10 of the more than 220 viruses known to infect humans. The SARS-CoV-2 outbreak has exposed the critical need for compounds that can be rapidly mobilised for the treatment of re-emerging or emerging viral diseases, while vaccine development is underway. We review the current status of antiviral therapies focusing on RNA viruses, highlighting strategies for antiviral drug discovery and discuss the challenges, solutions and options to accelerate drug discovery efforts.

RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global pandemic. Although great efforts have been made to develop effective therapeutic interventions, only the nucleotide analog remdesivir was approved for emergency use against COVID-19. Remdesivir targets the RNA-dependent RNA polymerase (RdRp), an essential enzyme for viral RNA replication and a promising drug target for COVID-19. Recently, several structures of RdRp in complex with substrate RNA and remdesivir were reported, providing insights into the mechanisms of RNA recognition by RdRp. These structures also reveal the mechanism of RdRp inhibition by nucleotide inhibitors and offer a molecular template for the development of RdRp-targeting drugs. This review discusses the recognition mechanism of RNA and nucleotide inhibitor by RdRp, and its implication in drug discovery.

Novel Dihydroorotate Dehydrogenase Inhibitors with Potent Interferon-Independent Antiviral Activity against Mammarenaviruses In Vitro.

Mammarenaviruses cause chronic infections in rodents, which are their predominant natural hosts. Human infection with some of these viruses causes high-consequence disease, posing significant issues in public health. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenavirus drugs are limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe side effects. Dihydroorotate dehydrogenase (DHODH) inhibitors, which block de novo pyrimidine biosynthesis, have antiviral activity against viruses from different families, including Arenaviridae, the taxonomic home of mammarenaviruses. Here, we evaluate five novel DHODH inhibitors for their antiviral activity against mammarenaviruses. All tested DHODH inhibitors were potently active against lymphocytic choriomeningitis virus (LCMV) (half-maximal effective concentrations [EC50] in the low nanomolar range, selectivity index [SI] > 1000). The tested DHODH inhibitors did not affect virion cell entry or budding, but rather interfered with viral RNA synthesis. This interference resulted in a potent interferon-independent inhibition of mammarenavirus multiplication in vitro, including the highly virulent Lassa and Junín viruses.

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase.

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.

Peptides containing the PCNA interacting motif APIM bind to the ß-clamp and inhibit bacterial growth and mutagenesis.

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, ß-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The ß-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the ß-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the ß-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial ß-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study.

AIMS: A new human coronavirus (HCoV), which has been designated SARS-CoV-2, began spreading in December 2019 in Wuhan City, China causing pneumonia called COVID-19. The spread of SARS-CoV-2 has been faster than any other coronaviruses that have succeeded in crossing the animal-human barrier. There is concern that this new virus will spread around the world as did the previous two HCoVs-Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS)-each of which caused approximately 800 deaths in the years 2002 and 2012, respectively. Thus far, 11,268 deaths have been reported from the 258,842 confirmed infections in 168 countries. MAIN METHODS: In this study, the RNA-dependent RNA polymerase (RdRp) of the newly emerged coronavirus is modeled, validated, and then targeted using different anti-polymerase drugs currently on the market that have been approved for use against various viruses. KEY FINDINGS: The results suggest the effectiveness of Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir as potent drugs against SARS-CoV-2 since they tightly bind to its RdRp. In addition, the results suggest guanosine derivative (IDX-184), Setrobuvir, and YAK as top seeds for antiviral treatments with high potential to fight the SARS-CoV-2 strain specifically. SIGNIFICANCE: The availability of FDA-approved anti-RdRp drugs can help treat patients and reduce the danger of the mysterious new viral infection COVID-19. The drugs mentioned above can tightly bind to the RdRp of the SARS-CoV-2 strain and thus may be used to treat the disease. No toxicity measurements are required for these drugs since they were previously tested prior to their approval by the FDA.

The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.

Discovery of Influenza Polymerase PA-PB1 Interaction Inhibitors Using an In Vitro Split-Luciferase Complementation-Based Assay.

The limited therapeutic options and increasing drug-resistance call for next-generation influenza antivirals. Due to the essential function in viral replication and high sequence conservation among influenza viruses, influenza polymerase PA-PB1 protein-protein interaction becomes an attractive drug target. Here, we developed an in vitro split luciferase complementation-based assay to speed up screening of PA-PB1 interaction inhibitors. By screening 10,000 compounds, we identified two PA-PB1 interaction inhibitors, R160792 and R151785, with potent and broad-spectrum antiviral activity against a panel of influenza A and B viruses, including amantadine-, oseltamivir-, or dual resistant strains. Further mechanistic study reveals that R151785 inhibits PA nuclear localization, reduces the levels of viral RNAs and proteins, and inhibits viral replication at the intermediate stage, all of which are in line with its antiviral mechanism of action. Overall, we developed a robust high throughput-screening assay for screening broad-spectrum influenza antivirals targeting PA-PB1 interaction and identified R151785 as a promising antiviral drug candidate.

Psoromic Acid, a Lichen-Derived Molecule, Inhibits the Replication of HSV-1 and HSV-2, and Inactivates HSV-1 DNA Polymerase: Shedding Light on Antiherpetic Properties.

Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC50): 1.9 µM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC50: 2.6 µM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC50: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC50): 2.7 µM; SI: 114.8) compared with that of ACV (EC50: 2.8 µM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC50: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC50): 0.7 µM; inhibition constant (Ki): 0.3 µM) compared with reference drugs aphidicolin (IC50: 0.8 µM; Ki: 0.4 µM) and ACV triphosphate (ACV-TP) (IC50: 0.9 µM; Ki: 0.5 µM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.

Discovery and development of DNA polymerase IIIC inhibitors to treat Gram-positive infections.

Despite the growing global crisis caused by antimicrobial drug resistance among pathogenic bacteria, the number of new antibiotics, especially new chemical class of antibiotics under development is insufficient to tackle the problem. Our review focuses on an emerging class of antibacterial therapeutic agents that holds a completely novel mechanism of action, namely, inhibition of bacterial DNA polymerase IIIC. The recent entry of this new class into human trials may herald the introduction of novel drugs whose novel molecular target precludes cross-resistance with existing antibiotic classes. This review therefore examines the evolution of DNA pol IIIC inhibitors from the discovery of 6-(p-hydroxyphenylazo)uracil (HPUra) in the 1960s to the development of current first-in-class N7-substituted guanine drug candidate ACX-362E, now under clinical development for the treatment of Clostridioides difficile infection.

Design of N-Benzoxaborole Benzofuran GSK8175-Optimization of Human Pharmacokinetics Inspired by Metabolites of a Failed Clinical HCV Inhibitor.

We previously described the discovery of GSK5852 (1), a non-nucleoside polymerase (NS5B) inhibitor of hepatitis C virus (HCV), in which an N-benzyl boronic acid was essential for potent antiviral activity. Unfortunately, facile benzylic oxidation resulted in a short plasma half-life (5 h) in human volunteers, and a backup program was initiated to remove metabolic liabilities associated with 1. Herein, we describe second-generation NS5B inhibitors including GSK8175 (49), a sulfonamide- N-benzoxaborole analog with low in vivo clearance across preclinical species and broad-spectrum activity against HCV replicons. An X-ray structure of NS5B protein cocrystallized with 49 revealed unique protein-inhibitor interactions mediated by an extensive network of ordered water molecules and the first evidence of boronate complex formation within the binding pocket. In clinical studies, 49 displayed a 60-63 h half-life and a robust decrease in viral RNA levels in HCV-infected patients, thereby validating our hypothesis that reducing benzylic oxidation would improve human pharmacokinetics and lower efficacious doses relative to 1.

Alpha-carboxynucleoside phosphonates: direct-acting inhibitors of viral DNA polymerases.

Acyclic nucleoside phosphonates represent a well-defined class of clinically used nucleoside analogs. All acyclic nucleoside phosphonates need intracellular phosphorylation before they can bind viral DNA polymerases. Recently, a novel class of alpha-carboxynucleoside phosphonates have been designed to mimic the natural 2'-deoxynucleotide 5'-triphosphate substrates of DNA polymerases. They contain a carboxyl group in the phosphonate moiety linked to the nucleobase through a cyclic or acyclic bridge. Alpha-carboxynucleoside phosphonates act as viral DNA polymerase inhibitors without any prior requirement of metabolic conversion. Selective inhibitory activity against retroviral reverse transcriptase and herpesvirus DNA polymerases have been demonstrated. These compounds have a unique mechanism of inhibition of viral DNA polymerases, and provide possibilities for further modifications to optimize and fine tune their antiviral DNA polymerase spectrum.

New Strategy on Antimicrobial-resistance: Inhibitors of DNA Replication Enzymes.

BACKGROUND: Antimicrobial resistance is found in all microorganisms and has become one of the biggest threats to global health. New antimicrobials with different action mechanisms are effective weapons to fight against antibiotic-resistance. OBJECTIVE: This review aims to find potential drugs which can be further developed into clinic practice and provide clues for developing more effective antimicrobials. METHODS: DNA replication universally exists in all living organisms and is a complicated process in which multiple enzymes are involved in. Enzymes in bacterial DNA replication of initiation and elongation phases bring abundant targets for antimicrobial development as they are conserved and indispensable. In this review, enzyme inhibitors of DNA helicase, DNA primase, topoisomerases, DNA polymerase and DNA ligase were discussed. Special attentions were paid to structures, activities and action modes of these enzyme inhibitors. RESULTS: Among these enzymes, type II topoisomerase is the most validated target with abundant inhibitors. For type II topoisomerase inhibitors (excluding quinolones), NBTIs and benzimidazole urea derivatives are the most promising inhibitors because of their good antimicrobial activity and physicochemical properties. Simultaneously, DNA gyrase targeted drugs are particularly attractive in the treatment of tuberculosis as DNA gyrase is the sole type II topoisomerase in Mycobacterium tuberculosis. Relatively, exploitation of antimicrobial inhibitors of the other DNA replication enzymes are primeval, in which inhibitors of topo III are even blank so far. CONCLUSION: This review demonstrates that inhibitors of DNA replication enzymes are abundant, diverse and promising, many of which can be developed into antimicrobials to deal with antibioticresistance.

DNA Related Enzymes as Molecular Targets for Antiviral and Antitumoral Chemotherapy. A Natural Overview of the Current Perspectives.

BACKGROUND: The discovery of new chemotherapeutic agents still remains a continuous goal to achieve. DNA polymerases and topoisomerases act in nucleic acids metabolism modulating different processes like replication, mitosis, damage repair, DNA topology and transcription. It has been widely documented that Polymerases serve as molecular targets for antiviral and antitumoral chemotherapy. Furthermore, telomerase is a ribonucleoprotein with exacerbated activity in most of the tumor cell lines, becoming as an emergent target in Cancer treatment. METHODS: We undertook an exhaustive search of bibliographic databases for peer-reviewed research literature related to the last decade. The characteristics of screened bibliography describe structure activity relationships and show the principal moieties involved. This work tries to summarize the investigation about natural and semi-synthetic products with natural origin with the faculty to inhibit key enzymes that play a crucial role in DNA metabolism. RESULTS: Eighty-five data references were included in this review, showing natural products widely distributed throughout the plant kingdom and their bioactive properties such as tumor growing inhibitory effects, and anti-AIDS activity. CONCLUSION: The findings of this review confirm the importance to find new drugs and biologically active natural products, and their potential medicinally useful benefits.

Identification of new promising plant-based lead compounds for inhibition of prokaryotic replicative DNA polymerases: combination of in silico and in vitro studies.

Emerging widespread bacterial resistance to current antibiotics with traditional targets is one of the major global concerns. Therefore, so many investigations are exploring the potential of other druggable macromolecules of bacteria such as replication machinery components that are not addressed by previous antibiotics. DNA polymerase is the major part of this machine. However, a few studies have been done on it so far. In this respect, we report the discovery of four new plant-based leads against DNA polymerase (pol) IIIC (three leads) and pol IIIE (one lead) of Gram-positive and negative bacteria by combining a sequentially constrained high-throughput virtual screenings on Traditional Chinese Medicine Database with in vitro assays. The compounds displayed relatively good levels of inhibitory effect. They were active against their designated targets at micromolar concentrations. The IC50 values for them are ranged from 25 to 111 µM. In addition, they showed minimum inhibitory concentrations in the range of 8-128 µg/mL against five representatives of pathogenic bacteria species. However, they were inactive against Pseudomonas aeruginosa. Given these results, these leads hold promise for future modification and optimization to be more effective in lower concentrations and also against most of the important bacterial species. Communicated by Ramaswamy H. Sarma.

Synthesis of Guanine α-Carboxy Nucleoside Phosphonate (G-α-CNP), a Direct Inhibitor of Multiple Viral DNA Polymerases.

The synthesis of guanine α-carboxy nucleoside phosphonate (G-α-CNP) is described. Two routes provide access to racemic G-α-CNP 9, one via base construction and the other utilizing Tsuji-Trost allylic substitution. The latter methodology was also applied to the enantiopure synthesis of both antipodes of G-α-CNP, each of which showing interesting antiviral DNA polymerase activity. Additionally, we report an improved multigram scale preparation of the cyclopentene building block 10, starting material for the preferred Tsuji-Trost route to 9.

Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities.

DNA polymerase Î¸ (Pol Î¸) is a multifunctional enzyme. It is nonessential in normal cells, but its upregulation in cancer cells correlates with cellular resistance to oxidative damage and poor prognosis. Pol Î¸ possesses polymerase activity and poorly characterized lyase activity. We examined the Pol Î¸ lyase activity on various abasic sites and determined that the enzyme is inactivated upon attempted removal of the oxidized abasic site commonly associated with C4'-oxidation (pC4-AP). Covalent modification of Pol Î¸ by the DNA lesion enabled determination of the primary nucleophile (Lys2383) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol Î¸ uses a single active site for polymerase and lyase activity. Mutation of Lys2383 significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys2383 is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol Î¸ and suggests a path forward for designing inhibitors of this attractive anticancer target.

New Thiazoline-Tetralin Derivatives and Biological Activity Evaluation.

In this study, novel N'-(3-cyclohexyl/phenyl-4-(substituted phenyl)thiazole-2(3H)-ylidene)-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (4a-4k) derivatives were synthesized and their anticancer potency were evaluated on human breast adenocarcinoma cell line (MCF-7), human lung carcinoma cell line (A549) and mouse embryoblast cell line (NIH/3T3) using the MTT method, DNA synthesis inhibition and flow cytometric analysis. Compound 4e bearing 4-methoxyphenyl moiety exhibited the highest antitumor efficiency against MCF-7 cell line with higher DNA synthesis inhibition and apoptotic cell percentages (ealy+late apoptotic cell). On the other hand, compounds 4f, 4g, and 4h bearing 4-bromo, 4-chloro and 4-florophenyl moieties, respectively caused excellent apoptosis levels against A549 cell line when treated with lower concentration even than cisplatin. Anticholinesterase activity of the compounds were also tested, compound 4h showed 49.92% inhibition of acetylcholinesterase (AChE).

Development of a Prodrug of Levofloxacin to Avoid Chelation with Al3+ and of Pemetrexed Dimedoxomil Esters for Oral Administration.

An ethoxycarbonyl 1-ethyl hemiacetal ester of levofloxacin (LVFX-EHE) avoids insoluble chelate formation with metal-containing drugs in the intestinal tract and is rapidly hydrolyzed to the parent drug. Furthermore, the minimum inhibitory concentration confirms that LVFX-EHE is less likely to cause pseudomembranous colitis because of less susceptibility to normal intestinal bacteria flora. Pemetrexed dimedoxomil, the prodrug of pemetrexed, was synthesized via reaction with medoxomil bromide after modification of L-glutamate with the tert-butyloxycarbonyl protecting group (BOC), followed by hydrolysis of the BOC moiety with trifluoroacetic acid (TFA) in CH2Cl2 at a temperature of 0°C for 2 h. A serum pemetrexed concentration of >2 µg/mL was observed after oral administration of pemetrexed dimedoxomil at a dose of 60 mg/kg to rats.