Targeting SARS-CoV-2 Nsp3 macrodomain structure with insights from human poly(ADP-ribose) glycohydrolase (PARG) structures with inhibitors

Abstract

Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly (ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal-ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.

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

“…Given the urgent need for antiviral therapies for COVID-19 and the fact that the macrodomain is an attractive therapeutic target ( 8 , 14 , 15 , 16 ), 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 6365 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 ( 3 ). 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 ( 8 , 14 , 15 , 16 ). 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 ( 8 , 9 , 10 ). Importantly, inactivating mutations within this domain lead to reduced viral replication and increased activation of the host IFN response ( 11 , 12 , 13 ), strongly indicating that pharmacological inhibition of the Nsp3 macrodomain may be of substantial therapeutic value ( 8 , 14 , 15 , 16 ).…”

A molecular toolbox for ADP-ribosyl binding proteins

“…In light of the current situation regarding the COVID-19 pandemic, we chose to demonstrate the applicability of this binding system for screening of small molecule inhibitors against the macrodomain of SARS-CoV-2 nsp3. Presently, efforts are being made by researchers worldwide to find inhibitors against this macrodomain (Bonfiglio et al, 2020;Brosey et al, 2021;Cantini et al, 2020;Michalska et al, 2020;Ni et al, 2021;Russo et al, 2021;Schuller et al, 2021;Virdi et al, 2020). The nsp3 macrodomain of coronaviruses was shown to be critical for the viral replication (Fehr et al, 2015(Fehr et al, , 2016, and therefore small molecule inhibitors might show promise as therapeutic agents to fight infections caused by SARS-CoV-2 (COVID-19) and other viruses.…”

X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

“…In general, DNA repair is the focal point for cellular regulation during DNA replication stress, development, differentiation, and responses to environmental damage. For example, the poly‐ADP ribosylation (PARylation) response to DNA breaks is linked to program cell death by an apoptosis‐inducing factor 178 and to regulating innate immune responses, so viral enzymes removing PARylation are an antiviral target 179 . Thus, structure‐based inhibitors can probe DNA repair and its interconnections for cell biology as well as provide foundations for potential drugs.…”