On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV-2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study

Abstract

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3′-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.

Trapping a non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase

“…91 Other computational studies also tested the design of inhibitors with ribose sugar modifications or removal of the OH function groups entirely. 40,41…”

“…91 Other computational studies also tested the design of inhibitors with ribose sugar modifications or removal of the OH function groups entirely. 40,41 In general, the nucleotide selection can be achieved by initial NTP-template base pairing and then the ribose recognition. A cluster of residues responsible for the ribose hydroxyl recognition have been suggested also for the PV RdRp, in which motif A and D move toward the active site upon closing to assist nucleotide insertion and selection.…”

“…18,35,36 Computational docking has often been employed, which predominantly focuses on nucleotide immediate binding to an apo-form RdRp structure, 37,38 non-differentiating the active open or closed form, and initially largely overlooking the functional RdRp (nsp12) elongation complex composed additionally of nsp8, nsp7, and RNA strands. 39 Moreover, while atomic molecular dynamics (MD) studies have provided insights into interactions of nucleotide analogues with the viral RdRp, they often directly utilize the insertion state, 40–42 ignoring the initial nucleotide analogue binding stage that can be essential for nucleotide screening or selectivity upon entry. Meanwhile, single-molecule studies have offered a glimpse into the dynamics of the elongation cycle, revealing that RdRp can adopt fast, slow, or very slow catalytic pathways with variable rates contingent upon the kinetic pathway.…”

On the Role of Temperature in the Depolymerization of PET by FAST‐PETase: An Atomistic Point of View on Possible Active Site Pre‐Organization and Substrate‐Destabilization Effects

“…The above-described protocol was successfully adopted in previous investigations. [36][37][38][39] The intrinsic conformational behavior of the 4PET in solution (34×30×38 Å 3 TIP3P water box) was further investigated, via three different MDs at 30 °C (303 K), 50 °C (323 K) and 150 °C (425 K). The final production, in each condition, was carried out for 150 ns.…”

Trapping non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase

“…Computational docking has often been employed, which predominantly focus on nucleotide immediate binding to an apo-form RdRp structure [34,35], non-differentiating the active open or closed form, and overlooking initially the functional RdRp (nsp12) elongation complex composed additionally of nsp8, nsp7, and RNA strands. Moreover, while atomic molecular dynamics (MD) studies have provided insights into interactions of nucleotide analogues with the viral RdRp, they often utilize directly the insertion state [36][37][38], ignoring the initial nucleotide analogue binding stage that can be essential for nucleotide screening or selectivity upon entry. Meanwhile, single-molecule studies have offered a glimpse into the dynamics of the elongation cycle, revealing that RdRp can adopt fast, slow, or very slow catalytic pathways with variable rates contingent upon the kinetic pathway [39].…”