SARS-CoV-2 Nsp16 activation mechanism and a cryptic pocket with pan-coronavirus antiviral potential

Abstract

Coronaviruses have caused multiple epidemics in the past two decades, in addition to the current COVID-19 pandemic that is severely damaging global health and the economy. Coronaviruses employ between twenty and thirty proteins to carry out their viral replication cycle including infection, immune evasion, and replication. Among these, nonstructural protein 16 (Nsp16), a 2’-O-methyltransferase, plays an essential role in immune evasion. Nsp16 achieves this by mimicking its human homolog, CMTr1, which methylates mRNA to enhance translation efficiency and distinguish self from other. Unlike human CMTr1, Nsp16 requires a binding partner, Nsp10, to activate its enzymatic activity. The requirement of this binding partner presents two questions that we investigate in this manuscript. First, how does Nsp10 activate Nsp16? While experimentally-derived structures of the active Nsp16/Nsp10 complex exist, structures of inactive, monomeric Nsp16 have yet to be solved. Therefore, it is unclear how Nsp10 activates Nsp16. Using over one millisecond of molecular dynamics simulations of both Nsp16 and its complex with Nsp10, we investigate how the presence of Nsp10 shifts Nsp16’s conformational ensemble in order to activate it. Second, guided by this activation mechanism and Markov state models (MSMs), we investigate if Nsp16 adopts inactive structures with cryptic pockets that, if targeted with a small molecule, could inhibit Nsp16 by stabilizing its inactive state. After identifying such a pocket in SARS-CoV-2 Nsp16, we show that this cryptic pocket also opens in SARS-CoV-1 and MERS, but not in human CMTr1. Therefore, it may be possible to develop pan-coronavirus antivirals that target this cryptic pocket.Statement of SignificanceCoronaviruses are a major threat to human health. These viruses employ molecular machines, called proteins, to infect host cells and replicate. Characterizing the structure and dynamics of these proteins could provide a basis for designing small molecule antivirals. In this work, we use computer simulations to understand the moving parts of an essential SARS-CoV-2 protein, understand how a binding partner turns it on and off, and identify a novel pocket that antivirals could target to shut this protein off. The pocket is also present in other coronaviruses but not in the related human protein, so it could be a valuable target for pan-coronavirus antivirals.

NSP16 2′-O-MTase in Coronavirus Pathogenesis: Possible Prevention and Treatments Strategies

“…As highlighted earlier, NSP16 needs to be combined with NSP10 to maintain stability and 2 -O-MTase activity [53]. Based on the structural similarity of coronavirus NSP16 with nucleoside analogs, the simulated inhibition mechanism and binding mode can be used for the development of potential therapeutic drugs, a cocktail of anti-coronavirus drugs, broad-spectrum antiviral drugs, or similar or candidate drugs, which have proven effective in clinical trials [78]. Thus, the development of molecules to inhibit cap 2 -O methylation and restore host antiviral response is one approach.…”

“…In addition to targeting the 2 -O-MTase active site, other approaches also utilize indirect factors necessary for coronavirus NSP16 activity. The specificity of the 2 -O-MTase active site is one of the potential methods, and can significantly impact viral infection with minimal toxicity [78]. Coronavirus NSP16 is a SAM-dependent 2 -O-MTase, which is thought to methylate the ribose 2 -OH of the first transcribed nucleotide of viral RNA cap structures [62].…”

The Role of Coronavirus RNA-Processing Enzymes in Innate Immune Evasion

“…Notably the GTP analog ribavirin triphosphate has been reported to suppress dengue virus 2′-O MTase activity [ 117 , 118 ]. The design of SAM analogues that also interacts with this adjacent cap-binding site is a challenge for the generation of more potent and specific inhibitors compared to sinefungin [ 115 , 119 ]. In this direction, several groups have performed virtual screening and molecular docking in order to identify alternative candidate drugs [ 120 , 121 , 122 ].…”

The pivotal roles of the host immune response in the fine-tuning the infection and the development of the vaccines for SARS-CoV-2

“…Recent studies showing that various SARS-CoV-2 proteins including some structural and the nonstructural proteins may contribute to the virus immune evasion strategies ( Table 1 ) which enhance the virus replication and promotes its pathogenesis and spread. 62 , 63 , 65 , 67 , 69 , 70 SARS-CoV-2-ORF8 plays important functions during virus replication and in viral immune evasion. 67 This may affect the downstream pathogenesis of the virus.…”

“…This interaction creates some unique binding pocket sites for the virus activation and contributes to the viral immune evasions. 65 SARS-CoV-NSP-1 binds to various host cell ribosomes subunits resulting in the shutdown of some host cell genes both in-vitro and in vivo . 62 SARS-CoV-2-ORF-9 C is a transmembrane protein that recently showed to play important immune evasion roles during the virus infection.…”