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bioRxiv · 10.1101/2022.04.11.487883

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

Galera-Prat, A.·Alaviuhkola, J.·Alanen, H. I.·Lehtiö, L.
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Abstract

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 such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family. We here expanded the methods and 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 enhanced activity using a simple and easily accessible homogeneous NAD+ conversion assay, facilitating its automation to reduce the assay volume and lowering the assay costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity and it can be applied to compound screening campaigns as well as inhibitor profiling.

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Galera-Prat, A., Alaviuhkola, J., Alanen, H. I., Lehtiö, L.. 2022-04-11. Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity. https://doi.org/10.1101/2022.04.11.487883

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