Development of a FRET-based assay that facilitates the identification of chemical probes to study the function of DTX3L-PARP9 complex

Abstract

Protein-protein interactions (PPIs) are unique molecular events that contribute to play important role in various cellular events. PPIs dysregulation can arise various cancers such as prostate and breast cancer. PARP9 is a member of macrodomain containing ADP-ribosyl transferase family but has no ADP-ribosylation activity. When PARP9 forms complex with DTX3L, this complex can ADPribosylate ubiquitin. The interaction between PARP9 and DTX3L could be an interesting drug target as its inhibition would uncouple the activities of these proteins. Notably the PARP9-DTX3L complex is overexpressed in cancers such as, prostate cancer.

The aim of this project is to develop a FRET-based assay which will help to identify inhibitors against the interaction between PARP9 and DTX3L. Ratiometric FRET (rFRET) signal used as an indicator to confirm the interaction between fluorophore tagged proteins (CFP-PARP9 and YFP-DTX3L). When the proteins interact a high rFRET signal is observed, while when it is inhibited a decrease in rFRET signal is observed.

Statistical validation of the assay was performed on day -to-day and plate-to- plate to determine its reproducibility and repeatability. The validation of assay showed robust Z’ value with an average 0.71. The screening was performed from four sets of small compound libraries to evaluate the effect of small molecules on the interaction. From the primary screening, seventeen compounds were identified with 20% hit limit. After that, to confirm about the inhibition of selected compound, rescreening was performed, and one potential inhibitor was selected. For further characterization of the compound, differential scanning fluorimetry (DSF) was performed to evaluate the effect of the compound on protein stability. The result of this experiment was that upon the addition of the compound, the melting temperature of both PARP9 and DTX3L decreased which is an indication of disruption of the structure of proteins.

The developed assay can be utilized in the future to discover inhibitors of the interaction between PARP9- DTX3L. The truncated constructs generated in the study can also be used for structural studies to reveal the molecular details of the interactions. This and experimentally determined binding modes of the discovered inhibitors will provide further possibilities for the structure-based development of improved chemical probes.