Crystal structure of SARS‐CoV‐2 nsp10–nsp16 in complex with small molecule inhibitors, SS148 and WZ16

Abstract

SARS‐CoV‐2 nsp10–nsp16 complex is a 2′‐O‐methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross‐screening of the inhibitors that we previously reported for SARS‐CoV‐2 nsp14 MTase activity against nsp10–nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10–nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S‐adenosyl‐L‐methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA‐dependent SAM‐competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.

In Vitro Reconstitution and Analysis of SARS-CoV-2/Host Protein–Protein Interactions

“…The interactions identified in this study were previously reported, which confirms that the LTE-expressed proteins were functional, and the assay could efficiently detect their interactions. − For instance, the crystal structure of the NSP10-NSP16 complex involved in viral RNA capping has been revealed earlier, and this complex formation is required to activate the 2′- O -methyltrasferase function of the NSP16 protein. , In our assay, the alpha signal intensity obtained for this complex was 5 × 10 4 CPS, which was 25× higher than the background signal.…”

“…29−33 For instance, the crystal structure of the NSP10-NSP16 complex involved in viral RNA capping has been revealed earlier, and this complex formation is required to activate the 2′-O-methyltrasferase function of the NSP16 protein. 34,35 In our assay, the alpha signal intensity obtained for this complex was 5 × 10 4 CPS, which was 25× higher than the background signal.…”

Discovery and structural characterization of monkeypox virus methyltransferase VP39 inhibitors reveal similarities to SARS-CoV-2 nsp14 methyltransferase

“…In fact, only a handful of inhibitors of viral MTases, mostly targeting flaviviruses 26,27 , were known prior to the COVID-19 pandemic. However, recently many inhibitors of SARS-CoV-2 MTases were reported by us and others 19,[28][29][30][31][32][33] .…”

Structure-Based Discovery of Inhibitors of the SARS-CoV-2 Nsp14 N7-Methyltransferase

“…These targets are well-precedented in antiviral research, with successful drugs treating analogous enzymes for HIV, HCV, RSV, HBV, HCMV, HSV, HPV, and human influenza virus, − among others, and these SARS-CoV-2 enzymes have been the focus of enormous efforts among many groups. ,,,− Other SARS-CoV-2 enzymes have attracted less attention, likely because there is less precedence for their targeting as antivirals. Nevertheless, enzymes like the macrodomain , and the papain-like protease of Nsp3, and the MTases Nsp10–Nsp16 complex and Nsp14 play key roles in the virulence of SARS-CoV-2. − While they have little precedence as antiviral drug targets, they seem attractive as novel enzymes for antiviral drug discovery.…”

“…1,4,5,10−13 Other SARS-CoV-2 enzymes have attracted less attention, likely because there is less precedence for their targeting as antivirals. Nevertheless, enzymes like the macrodomain 14,15 and the papain-like protease of Nsp3, 16 and the MTases Nsp10− Nsp16 complex 17 and Nsp14 play key roles in the virulence of SARS-CoV-2. 18−21 While they have little precedence as antiviral drug targets, they seem attractive as novel enzymes for antiviral drug discovery.…”