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

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.

[1,2,4]Triazolo[3,4-b]benzothiazole scaffold as versatile nicotinamide mimic allowing nanomolar inhibition of different PARP enzymes

“…43 The conditions for the proximity enhanced mono-ART essays (Table 4) were also as reported recently. 46 PARP6 (400 nM) inhibition was measured using the standard buffer (50 mM sodium phosphate pH 7.0) for the enhanced activity assay using 500 nM NAD + and 18 hour incubation in RT.…”

“…We therefore had to improve the assay method and developed a homogeneous proximity enhanced assay for mono-ARTs. 46 This new assay was used here to test the selected compounds against PARP7-PARP16 as it allowed us to measure robust IC50 values for the discovered potent inhibitors while using less enzyme (Figure S3). 35,43 Compound 16 was confirmed as a weak inhibitor of mono-ARTs while showing potent inhibition of poly-ARTs PARP1-2 and TNKS2.…”

“…The proteins used in tables 1-3 were produced as described earlier. Dockerin constructs to allow enhanced activity assays for mono-ARTs (Table 4) were produced in E. coli with an N-terminal MBP and a C-terminal dockerin domain from Hungateiclostridium thermocellum Cel48s as described recently 46 .…”

“…Table 4. Profile of the selected compounds against the PARP enzymes, IC50 (pIC50±SEM, n=3), where mono-ARTs PARP7-PARP16 are measured using a proximity enhanced assay, 46 potency of the compounds in rescuing cells from PARP10 overexpression along with 95% confidence interval for the EC50, and ADME profiling…”

Structure-guided design and characterization of a clickable, covalent PARP16 inhibitor

“…1D), however, only at high concentrations of NAD + (400 mM; 4-40-fold the amount used for the other PARPs) and PARP16 (1 mM added to the plate), conditions that force self-modication similar to assays developed by others. 17,27 We are therefore unable to determine a cellular IC 50 value for DB008 against PARP16. However, the IC 50 is not the best measure of potency for covalent inhibitors because incubation time can dramatically shi IC 50 values due to the timedependent nature of their inhibition.…”