Fragment Binding to the Nsp3 Macrodomain of SARS-CoV-2 Identified Through Crystallographic Screening and Computational Docking

Abstract

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signalling

“…Given the urgent need for antiviral therapies for COVID-19 and the fact that the macrodomain is an attractive therapeutic target (Alhammad & Fehr, 2020;Brosey et al, 2021;Rack et al, 2020;Schuller et al, 2020), we attempted to repurpose compounds that already have regulatory approval as potential Nsp3 macrodomain inhibitors. For this, we performed a structure-based virtual screen of a library of 6364 compounds that have been approved for human use by any regulatory agency in the world, against the deposited crystal structures of the SARS-CoV-2 Nsp3 macrodomain bound to ADP-ribose (PDB 6W02) (Fig.…”

“…Successful viral evasion or suppression of the host interferon response is key for the establishment of a viral infection and is often mediated by several viral factors acting on multiple host targets (Garcia-Sastre, 2017). The coronavirus Nsp3 macrodomain is thought to represent an important mediator of coronavirus pathogenesis by reversing host antiviral ADP-ribosylation and is thus an attractive drug target (Alhammad & Fehr, 2020;Brosey et al, 2021;Rack et al, 2020;Schuller et al, 2020). However, little is known about the molecular targets of IFN-responsive PARPs and how reversal of these modifications by the Nsp3 macrodomain may exert its pro-viral effects.…”

“…To counteract antiviral ADP-ribosylation by host PARPs, SARS-CoV-2 and other coronaviruses encode a macrodomain within non-structural protein 3 (Nsp3) that hydrolyses ADP-ribose modifications (Alhammad & Fehr, 2020;Alhammad et al, 2021;Leung et al, 2018). Importantly, inactivating mutations within this domain lead to reduced viral replication and increased activation of the host IFN response (Fehr et al, 2015;Fehr et al, 2016;Grunewald et al, 2019), strongly indicating that pharmacological inhibition of the Nsp3 macrodomain may be of substantial therapeutic value (Alhammad & Fehr, 2020;Brosey et al, 2021;Rack et al, 2020;Schuller et al, 2020).…”

Crystal structures of the σ2receptor template large-library docking for selective chemotypes activein vivo

“…As this is also thought to be true of σ 1 ligands, and because most σ 2 ligands cross-react with the σ 1 receptor, probe ligands selective for σ 2 over σ 1 would help illuminate σ 2 biology and could be leads for novel therapeutics. However, given the relatively recent determination of the σ 2 receptor's molecular identity relatively little is known regarding its molecular architecture, ligand recognition, or amenability to methods like virtual screening for ligand identification [23][24][25][26][27][28][29][30] . Here, we employed a biochemical and structural approach combined with computational docking to address these issues.…”

Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain

“…This flexibility offers opportunities for rational targeting this protein module by small molecule inhibitors. Indeed, recent study has reported a number of potential small molecule binders discovered through crystallographic fragment screening 31 . In line with this, our unprecedented discovery of the binding of GS-441524, an active metabolite of remdesivir, supported this hypothesis that the ADP-ribose pocket of the macrodomain indeed represents a druggable site.…”