A Systematic Review of the Role of Purinergic Signalling Pathway in the Treatment of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global health concern. Three years since its origin, despite the approval of vaccines and specific treatments against this new coronavirus, there are still high rates of infection, hospitalization, and mortality in some countries. COVID-19 is characterised by a high inflammatory state and coagulation disturbances that may be linked to purinergic signalling molecules such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine (ADO), and purinergic receptors (P1 and P2). These nucleotides/nucleosides play important roles in cellular processes, such as immunomodulation, blood clot formation, and vasodilation, which are affected during SARS-CoV-2 infection. Therefore, drugs targeting this purinergic pathway, currently used for other pathologies, are being evaluated in preclinical and clinical trials for COVID-19. In this review, we focus on the potential of these drugs to control the release, degradation, and reuptake of these extracellular nucleotides and nucleosides to treat COVID-19. Drugs targeting the P1 receptors could have therapeutic efficacy due to their capacity to modulate the cytokine storm and the immune response. Those acting in P2X7, which is linked to NLRP3 inflammasome activation, are also valuable candidates as they can reduce the release of pro-inflammatory cytokines. However, according to the available preclinical and clinical data, the most promising medications to be used for COVID-19 treatment are those that modulate platelets behaviour and blood coagulation factors, mainly through the P2Y12 receptor.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases.

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0-500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 12 modified) substrates. The screening results correlated well with literature data, and additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

Clarifying the Use of Benzylidene Protecting Group for D-(+)-Ribono-1,4-Lactone, an Essential Building Block in the Synthesis of C-Nucleosides.

In the last two years, nucleosides analogues, a class of well-established bioactive compounds, have been the subject of renewed interest from the scientific community thanks to their antiviral activity. The COVID-19 global pandemic, indeed, spread light on the antiviral drug Remdesivir, an adenine C-nucleoside analogue. This new attention of the medical community on Remdesivir prompts the medicinal chemists to investigate once again C-nucleosides. One of the essential building blocks to synthetize these compounds is the D-(+)-ribono-1,4-lactone, but some mechanistic aspects linked to the use of different carbohydrate protecting groups remain unclear. Here, we present our investigations on the use of benzylidene as a ribonolactone protecting group useful in the synthesis of C-purine nucleosides analogues. A detailed 1D and 2D NMR structural study of the obtained compounds under different reaction conditions is presented. In addition, a molecular modeling study at the B3LYP/6-31G* level of theory with the SM8 solvation model for CHCl3 and DMSO to support the obtained results is used. This study allows for clarifying mechanistic aspects as the side reactions and structural rearrangements liked to the use of the benzylidene protecting group.

Remdesivir induces persistent mitochondrial and structural damage in human induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalized coronavirus disease of 2019 (COVID-19) patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model. METHODS AND RESULTS: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19, respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential, and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype. CONCLUSIONS: Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients.

Pharmacokinetics and tissue distribution of remdesivir and its metabolites nucleotide monophosphate, nucleotide triphosphate, and nucleoside in mice.

Remdesivir (RDV) exerts anti-severe acute respiratory coronavirus 2 activity following metabolic activation in the target tissues. However, the pharmacokinetics and tissue distributions of the parent drug and its active metabolites have been poorly characterized to date. Blood and tissue levels were evaluated in the current study. After intravenous administration of 20 mg/kg RDV in mice, the concentrations of the parent drug, nucleotide monophosphate (RMP) and triphosphate (RTP), as well as nucleoside (RN), in the blood, heart, liver, lung, kidney, testis, and small intestine were quantified. In blood, RDV was rapidly and completely metabolized and was barely detected at 0.5 h, similar to RTP, while its metabolites RMP and RN exhibited higher blood levels with increased residence times. The area under the concentration versus time curve up to the last measured point in time (AUC0-t) values of RMP and RN were 4558 and 136,572 h∙nM, respectively. The maximum plasma concentration (Cmax) values of RMP and RN were 2896 nM and 35,819 nM, respectively. Moreover, RDV presented an extensive distribution, and the lung, liver and kidney showed high levels of the parent drug and metabolites. The metabolic stabilities of RDV and RMP were also evaluated using lung, liver, and kidney microsomes. RDV showed higher clearances in the liver and kidney than in the lung, with intrinsic clearance (CLint) values of 1740, 1253, and 127 mL/(min∙g microsomal protein), respectively.

Discovery, Design, Synthesis, and Application of Nucleoside/Nucleotides.

For decades, nucleosides and nucleotides have formed the cornerstone of antiviral, antiparasitic and anticancer therapeutics and have been used as tools in exploring nucleic acid structure and function [...].

Possible Targets and Therapies of SARS-CoV-2 Infection.

The global spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes COVID-19 has become a source of grave medical and socioeconomic concern to human society. Since its first appearance in the Wuhan region of China in December 2019, the most effective measures of managing the spread of SARS-CoV-2 infection have been social distancing and lockdown of human activity; the level of which has not been seen in our generations. Effective control of the viral infection and COVID-19 will ultimately depend on the development of either a vaccine or therapeutic agents. This article highlights the progresses made so far in these strategies by assessing key targets associated with the viral replication cycle. The key viral proteins and enzymes that could be targeted by new and repurposed drugs are discussed.