Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity.

Galera-Prat, Albert; Alaviuhkola, Juho; Alanen, Heli I; Lehtiö, Lari

Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different enzymes. We engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their activity using a homogeneous NAD conversion assay, facilitating its automation to lower the assay volume and costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity.

PROTEIN engineering; CATALYTIC domains; DRUG target; POLY(ADP-ribose) polymerase; DRUG discovery; ENZYMES

PEDS: Protein Engineering, Design & Selection, 2022, Vol 35, p1

1741-0126

Academic Journal

10.1093/protein/gzac006