Small-Molecule Screening Assay for Mono-ADP-Ribosyltransferases
Mono-ADP-ribosyltransferases of the PARP/ARTD enzyme family are enzymes catalyzing the transfer of a single ADP-ribose unit to target proteins. The enzymes have various roles in vital cellular processes such as DNA repair and transcription, and many of the enzymes are linked to cancer-relevant functions. Thus inhibition of the enzymes is a potential way to discover and develop new drugs against cancer. Here we describe an activity-based screening assay for mono-ADP-ribosyltransferases. The assay utilizes the natural substrate of the enzymes, NAD+, and it is based on chemically converting the leftover substrate to a fluorophore and measuring its relative concentration after the enzymatic reaction. The assay is homogenous, robust, and cost-effective and, most importantly, applicable to mono-ADP-ribosyltransferases as well as poly-ADP-ribosyltransferases for screening of small-molecule inhibitors against the enzymes.
Key wordsPARP ARTD Mono-ADP-ribosyltransferase Screening assay Inhibitor
- 14.Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, Austin C, Baell J, Bejcek B, TDY C, Dahlin JL, Devanaryan V, Foley TL, Glicksman M, Hall MD, Hass JV, Inglese J, Iversen PW, Kahl SD, Kales SC, Lal-Nag M, Li Z, McGee J, McManus O, Riss T, Trask OJ Jr, Weidner JR, Xia M, Xu X (eds) (2004) Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda, MDGoogle Scholar