Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

Abstract

ADP-ribosylation is a post-translational modification catalyzed by writer enzymes -ADP-ribosyltransferases. The modification is part of many signaling events, can modulate the function and stability of target proteins, and often results in the recruitment of reader proteins that bind to the ADP-ribosyl groups. Erasers are integral actors in these signaling events and reverse the modification. ADP-ribosylation can be targeted with therapeutics and many inhibitors against writers exist, with some being in clinical use. Inhibitors against readers and erasers are sparser and development of these has gained momentum only in recent years. Drug discovery has been hampered by the lack of specific tools, however many significant advances in the methods have recently been reported. We discuss assays used in the field with a focus on methods allowing efficient identification of small molecule inhibitors and profiling against enzyme families. While human proteins are focused, the methods can be also applied to bacterial toxins and virus encoded erasers that can be targeted to treat infectious diseases in the future.

Medicinal Chemistry Perspective on Targeting Mono-ADP-Ribosylating PARPs with Small Molecules

“…The lack of access to a complete panel of sensitive, high-throughput PARP assays ,− to fully evaluate selectivity is a common weakness that leads to profiling of PARP inhibitors only against a handful of family members, rarely including representative mono-ARTs. This also emerged from the present perspective, where only a few research teams profiled their compounds against a larger panel of PARPs and TNKS, but the recent assay development efforts will likely make the profiling against a PARP panel more routine in the future. , The lack of selective inhibitors has until now hampered the unveiling of the pathophysiological roles of many mono-ARTs.…”

Design, synthesis and evaluation of inhibitors of the SARS-CoV-2 nsp3 macrodomain

“…A FRET method was utilized for the profiling of 15c a panel of human and viral macrodomains to determine their specificity [22] , [27] , [28] . The assay is based on the site-specific introduction of cysteine-linked mono-ADP ribose to the C-terminal Gαi peptide (GAP) by Pertussis toxin subunit1 (PtxS1) fused to YFP.…”

Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV2 nsp3 Macrodomain

“…A FRET method was utilized for the profiling of 15c a panel of human and viral macrodomains to determine their specificity [22, 25, 26]. The assay is based on the site-specific introduction of cysteine-linked mono-ADP ribose to the C-terminal Gαi peptide (GAP) by Pertussis toxin subunit1 (PtxS1) fused to YFP.…”