Translocation pause of remdesivir-containing primer/template RNA duplex within SARS-CoV-2's RNA polymerase complexes.

The mechanism of remdesivir incorporation into the RNA primer by the RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains to be fully established at the molecular level. Here, we compare molecular dynamics (MD) simulations after incorporation of either remdesivir monophosphate (RMP) or adenosine monophosphate (AMP). We find that the Mg2+-pyrophosphate (PPi) binds more tightly to the polymerase when the added RMP is at the third primer position than in the AMP added complex. The increased affinity of Mg2+-PPi to the RMP-added primer/template (P/T) RNA duplex complex introduces a new hydrogen bond of a substituted cyano group in RMP with the K593 sidechain. The new interactions disrupt a switching mechanism of a hydrogen bond network that is essential for translocation of the P/T duplex product and for opening of a vacant NTP-binding site necessary for next primer extension. Furthermore, steric interactions between the sidechain of S861 and the 1'-cyano group of RMP at position i+3 hinders translocation of RMP to the i + 4 position, where i labels the insertion site. These findings are particularly valuable to guide the design of more effective inhibitors of SARS-CoV-2 RNA polymerase.

Synthesis and antiviral activity of fatty acyl conjugates of remdesivir against severe acute respiratory syndrome coronavirus 2 and Ebola virus.

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 µM; IC50(Calu3) = 0.24 µM), 3'-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 µM; IC50(Calu3) = 0.18 µM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 µM; IC50(Calu3) = 0.25 µM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 µM; IC50(Calu3) = 0.06 µM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.

Protective Effect of Remdesivir Against Pulmonary Fibrosis in Mice.

Pulmonary fibrosis is a known sequela of severe or persistent lung damage. Existing clinical, imaging and autopsy studies have shown that the lungs exhibit a pathological pulmonary fibrosis phenotype after infection with coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pulmonary fibrosis may be one of the most serious sequelae associated with coronavirus disease 2019 (COVID-19). In this study, we aimed to examine the preventative effects of the antiviral drug remdesivir on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of remdesivir on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of remdesivir in lung fibroblasts and alveolar epithelial cells in vitro. The preventive remdesivir treatment was started on the day of bleomycin installation, and the results showed that remdesivir significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro experiments showed that remdesivir dose-dependently suppressed TGF-ß1-induced lung fibroblast activation and improved TGF-ß1-induced alveolar epithelial to mesenchymal transition. Our results indicate that remdesivir can preventatively alleviate the severity of pulmonary fibrosis and provide some reference for the prevention of pulmonary fibrosis in patients with COVID-19.

Molecular Dynamics Simulations Reveal the Interaction Fingerprint of Remdesivir Triphosphate Pivotal in Allosteric Regulation of SARS-CoV-2 RdRp.

The COVID-19 pandemic has now strengthened its hold on human health and coronavirus' lethal existence does not seem to be going away soon. In this regard, the optimization of reported information for understanding the mechanistic insights that facilitate the discovery towards new therapeutics is an unmet need. Remdesivir (RDV) is established to inhibit RNA-dependent RNA polymerase (RdRp) in distinct viral families including Ebola and SARS-CoV-2. Therefore, its derivatives have the potential to become a broad-spectrum antiviral agent effective against many other RNA viruses. In this study, we performed comparative analysis of RDV, RMP (RDV monophosphate), and RTP (RDV triphosphate) to undermine the inhibition mechanism caused by RTP as it is a metabolically active form of RDV. The MD results indicated that RTP rearranges itself from its initial RMP-pose at the catalytic site towards NTP entry site, however, RMP stays at the catalytic site. The thermodynamic profiling and free-energy analysis revealed that a stable pose of RTP at NTP entrance site seems critical to modulate the inhibition as its binding strength improved more than its initial RMP-pose obtained from docking at the catalytic site. We found that RTP not only occupies the residues K545, R553, and R555, essential to escorting NTP towards the catalytic site, but also interacts with other residues D618, P620, K621, R624, K798, and R836 that contribute significantly to its stability. From the interaction fingerprinting it is revealed that the RTP interact with basic and conserved residues that are detrimental for the RdRp activity, therefore it possibly perturbed the catalytic site and blocked the NTP entrance site considerably. Overall, we are highlighting the RTP binding pose and key residues that render the SARS-CoV-2 RdRp inactive, paving crucial insights towards the discovery of potent inhibitors.

Prevention and therapy of SARS-CoV-2 and the B.1.351 variant in mice.

Improving clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small-molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAbs) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). It is not known whether combination RDV/mAb will improve outcomes over single-agent therapies or whether antibody therapies will remain efficacious against variants. Here, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 h after infection and have therapeutic efficacy in vivo against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared with single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.

Can remdesivir and its parent nucleoside GS-441524 be potential oral drugs? An in vitro and in vivo DMPK assessment.

Remdesivir (RDV) is the only US Food and Drug Administration (FDA)-approved drug for treating COVID-19. However, RDV can only be given by intravenous route, and there is a pressing medical need for oral antivirals. Significant evidence suggests that the role of the parent nucleoside GS-441524 in the clinical outcomes of RDV could be largely underestimated. We performed an in vitro and in vivo drug metabolism and pharmacokinetics (DMPK) assessment to examine the potential of RDV, and particularly GS-441524, as oral drugs. In our in vitro assessments, RDV exhibited prohibitively low stability in human liver microsomes (HLMs, t 1/2 = âˆ¼1 min), with the primary CYP-mediated metabolism being the mono-oxidation likely on the phosphoramidate moiety. This observation is poorly aligned with any potential oral use of RDV, though in the presence of cobicistat, the microsomal stability was drastically boosted to the level observed without enzyme cofactor NADPH. Conversely, GS-441524 showed excellent metabolic stability in human plasma and HLMs. In further in vivo studies in CD-1 mice, GS-441524 displayed a favorable oral bioavailability of 57%. Importantly, GS-441524 produced adequate drug exposure in the mice plasma and lung, and was effectively converted to the active triphosphate, suggesting that it could be a promising oral antiviral drug for treating COVID-19.

Genetic conservation of SARS-CoV-2 RNA replication complex in globally circulating isolates and recently emerged variants from humans and minks suggests minimal pre-existing resistance to remdesivir.

Remdesivir (RDV) exhibits potent antiviral activity against SARS-CoV-2 and is currently the only drug approved for the treatment of COVID-19. However, little is currently known about the potential for pre-existing resistance to RDV and the possibility of SARS-CoV-2 genetic diversification that might impact RDV efficacy as the virus continue to spread globally. In this study, >90,000 SARS-CoV-2 sequences from globally circulating clinical isolates, including sequences from recently emerged United Kingdom and South Africa variants, and >300 from mink isolates were analyzed for genetic diversity in the RNA replication complex (nsp7, nsp8, nsp10, nsp12, nsp13, and nsp14) with a focus on the RNA-dependent RNA polymerase (nsp12), the molecular target of RDV. Overall, low genetic variation was observed with only 12 amino acid substitutions present in the entire RNA replication complex in ≥0.5% of analyzed sequences with the highest overall frequency (82.2%) observed for nsp12 P323L that consistently increased over time. Low sequence variation in the RNA replication complex was also observed among the mink isolates. Importantly, the coronavirus Nsp12 mutations previously selected in vitro in the presence of RDV were identified in only 2 isolates (0.002%) within all the analyzed sequences. In addition, among the sequence variants observed in ≥0.5% clinical isolates, including P323L, none were located near the established polymerase active site or sites critical for the RDV mechanism of inhibition. In summary, the low diversity and high genetic stability of the RNA replication complex observed over time and in the recently emerged SARS-CoV-2 variants suggests a minimal global risk of pre-existing SARS-CoV-2 resistance to RDV.

Validation of LC-MS/MS methods for determination of remdesivir and its metabolites GS-441524 and GS-704277 in acidified human plasma and their application in COVID-19 related clinical studies.

Remdesivir (RDV) is a phosphoramidate prodrug designed to have activity against a broad spectrum of viruses. Following IV administration, RDV is rapidly distributed into cells and tissues and simultaneously metabolized into GS-441524 and GS-704277 in plasma. LC-MS/MS methods were validated for determination of the 3 analytes in human plasma that involved two key aspects to guarantee their precision, accuracy and robustness. First, instability issues of the analytes were overcome by diluted formic acid (FA) treatment of the plasma samples. Secondly, a separate injection for each analyte was performed with different ESI modes and organic gradients to achieve sensitivity and minimize carryover. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a run time of 3.4 min. The calibration ranges were 4-4000, 2-2000, and 2-2000 ng/mL, respectively for RDV, GS-441524 and GS-704277. The intraday and interday precision (%CV) across validation runs at 3 QC levels for all 3 analytes was less than 6.6%, and the accuracy was within ±11.5%. The long-term storage stability in FA-treated plasma was established to be 392, 392 and 257 days at -70 °C, respectively for RDV, GS-441524 and GS-704277. The validated method was successfully applied in COVID-19 related clinical studies.

COVID-19, Companion Animals, Comparative Medicine, and One Health.

The COVID-19 pandemic in 2020 has stimulated open collaboration between different scientific and clinical disciplines like never before. Public and private partnerships continue to form in order to tackle this unprecedented global challenge. This paper highlights the importance of open collaboration and cooperation between the disciplines of medicine, veterinary medicine, and animal health sciences in the fight against COVID-19. Since the pandemic took the whole world by surprise, many existing drugs were rapidly repurposed and tested in COVID-19 clinical trials and some of the trials are revealing promising results, it is clear that the long-term solution will come in the form of vaccines. While vaccines are being developed, the antiviral agent Remdesivir (RDV, GS-5734) is being repurposed for use in human clinical trials but this is being done without acknowledging the significant efforts that went into development for treating cats with feline infectious peritonitis (FIP), a highly fatal immune-mediated vasculitis in cats which is caused by a feline coronavirus. There are many other antiviral drugs and immune modulating treatments that are currently being trialed that have animal health origins in terms of discovery and clinical development. Closer collaboration between the animal health and human health sectors is likely to accelerate progress in the fight against COVID-19. There is much that we do not yet know about COVID-19 and its causative agent SARS-CoV-2 but we will learn and progress much faster if we increase interdisciplinary collaboration and communication between human and animal health researchers and taking a genuine "One Health" approach to this and other emerging viral pathogens. Enhanced knowledge of zoonotic coronaviruses can significantly enhance our ability to fight current and future emerging coronaviruses. This article highlights the acute need for One Health and comparative medicine and the crucial importance of building on and recognizing veterinary research for addressing future human pandemics.

COVID-19: The Potential Role of Copper and N-acetylcysteine (NAC) in a Combination of Candidate Antiviral Treatments Against SARS-CoV-2.

BACKGROUND: On March 11, 2020, the World Health Organization (WHO) declared the outbreak of coronavirus disease (COVID-19) a pandemic. Since then, thousands of people have suffered and died, making the need for a treatment of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) more crucial than ever. MATERIALS AND METHODS: The authors carried out a search in PubMed, ClinicalTrials.gov and New England Journal of Medicine (NEJM) for COVID-19 to provide information on the most promising treatments against SARS-CoV-2. RESULTS: Possible COVID-19 agents with promising efficacy and favorable safety profile were identified. The results support the combination of copper, N-acetylcysteine (NAC), colchicine and nitric oxide (NO) with candidate antiviral agents, remdesivir or EIDD-2801, as a treatment for patients positive for SARS-CoV-2. CONCLUSION: The authors propose to study the effects of the combination of copper, NAC, colchicine, NO and currently used experimental antiviral agents, remdesivir or EIDD-2801, as a potential treatment scheme for SARS-COV-2.