Potent halt of SARS-CoV-2 replication using clitocine-induced emergent-viral-RNA editing and disruption

Potently inhibiting the functions of the replication proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or inducing erroneous editing of newly replicated SARS-CoV-2 RNA strands during replication, is among the most effective anti-SARS-CoV-2 approaches. Nucleosides and their phosphorylated derivatives (i.e., nucleotides) are considered ideal options for stopping various infections of coronavirus disease 2019 (COVID-19). Clitocine (CLT) is a phosphorylatable natural nucleoside analog that is isolated mainly from two edible mushrooms, Clitocybe inversa and Leucopaxillus giganteus, and it is uniquely characterized by a nitro group on its pyrimidine ring. CLT and many of its derivatives are well known mainly for their potent anticancer activities. Leveraging the use of its favorable and rare chemical structure in medicinal chemistry and drug development, herein, we biochemically evaluated and computationally explained the potential capacity of CLT to mutate and disable the emergent viral RNA strands during the coronaviral replication processes against the SARS-CoV-2 particle and two of its essential replication proteins, the RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) enzymes, which are crucial for a complete and functioning replication machinery. The in vitro anti-RdRp, anti-ExoN, and anti-SARS-CoV-2 assays demonstrated the potent inhibitory activities of the CLT molecule and its expected major active metabolite, CLT triphosphate (CLT-TP), on coronaviral replication, with very interesting 50% inhibitory concentration (IC₅₀) values against the nirmatrelvir-resistant SARS-CoV-2 variant "NRSV" (0.19, 0.20, and 0.38 µM for CLT and 0.15, 0.18, and 0.34 µM for CLT-TP, respectively), using the structurally similar and approved anti-SARS-CoV-2 medication molnupiravir (MNP) and its known active triphosphate metabolite, β-D-N ⁴-hydroxycytidine 5'-triphosphate (NHC-TP), respectively, as reference drugs. The subsequent in silico simulations and computations, by implementing mainly the relevant molecular docking and molecular dynamics (MD) simulations, adequately expounded these biochemical findings. Through this new study, we also succeeded in uncovering and explaining the molecular-level mechanistic hallmarks of CLT/CLT-TP with respect to disrupting and impairing the coronaviral replication, as well as militating COVID-19 in a comprehensive way. In conclusion, the presented results need to be further confirmed by robust preclinical and clinical studies, along with the use of CLT as a promising pioneering parent anti-SARS-CoV-2 nucleoside analog by medicinal chemists for the synthesis of a wide spectrum of anti-RdRp nitronucleoside derivatives with ExoN-resistant chain-terminating activities (i.e., with ExoN-proofreading-escaping capacities) for optimum activity against coronaviruses.