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

ABSTRACT: 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.

SUBMITTER: Srivastava M

PROVIDER: S-EPMC8417884 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCEIn vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.

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

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