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

Ni, Xiaoling; Schroeder, Martin; Oliéric, V.; Sharpe, M.E.; Olmos, Victor Hipolito; Proschak, Ewgenij; Merk, Daniel; Knapp, S.; Chaikuad, A.

doi:10.1101/2021.03.04.433966

Cited by 1 publication

(1 citation statement)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Application Example: Screening For Inhibitors Against the Sars-cov-2 Nsp3 Macrodomainmentioning

confidence: 99%

A molecular toolbox for ADP-ribosyl binding proteins

Sowa

Galera‐Prat

Wazir

et al. 2021

Preprint

View full text Add to dashboard Cite

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or in viral infections, which makes them attractive targets for the development of inhibitors. Our goal was to develop a robust and accessible assay technology that is suitable for high-throughput screening and applicable to a wide range of proteins acting as either hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. As a foundation of our work, we developed a C-terminal protein fusion tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups as well as chemical ADP-ribosyl analogs. By fusion of the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we were able to generate robust FRET signals and the interaction with 22 previously described ADP-ribosyl-binders was confirmed. To demonstrate the applicability of this binding assay for high-throughput screening, we utilized it to screen for inhibitors of the SARS-CoV-2 nsp3 macrodomain and identified the drug suramin as a moderate yet unspecific inhibitor of this protein. To complement the binding technology, we prepared high-affinity ADP-ribosyl binders fused to a nanoluciferase, which enabled simple blot-based detection of mono- and poly-ADP-ribosylated proteins. These tools can be expressed recombinantly in E. coli using commonly available agents and will help to investigate ADP-ribosylation systems and aid in drug discovery.

show abstract