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.

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

“…1D), however, only at high concentrations of NAD + (400 μM; 4–40-fold the amount used for the other PARPs) and PARP16 (1 μM added to the plate), conditions that force self-modification 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 shift IC 50 values due to the time-dependent nature of their inhibition.…”

[1,2,4]Triazolo[3,4-b]benzothiazole Scaffold as Versatile Nicotinamide Mimic Allowing Nanomolar Inhibition of Different PARP Enzymes

“…It should be noted that compounds 21 and 27 had reached the sensitivity limit of the mono-ART assay and that the values reported in Table were artificially high due to the enzyme concentrations needed for a robust conversion of NAD + . We therefore had to improve the assay method and developed a homogeneous proximity enhanced assay for mono-ARTs . This new assay was used here to test the selected compounds against PARP7–PARP16 as it allowed us to measure robust IC 50 values for the discovered potent inhibitors while using less enzyme (Figure S3).…”

“…We therefore had to improve the assay method and developed a homogeneous proximity enhanced assay for mono-ARTs. 47 This new assay was used here to test the selected compounds against PARP7−PARP16 as it allowed us to measure robust IC 50 values for the discovered potent inhibitors while using less enzyme (Figure S3). As emerged from Table 4, both 3-amino derivatives 21 and 27 were potent inhibitors of multiple human mono-ARTs.…”

“…Dockerin constructs to allow enhanced activity assays for mono-ARTs (Table 4) were produced in Escherichia coli with an N-terminal maltose binding protein (MBP) and a Cterminal dockerin domain from Hungateiclostridium thermocellum Cel48s as described recently. 47 Human PARP6 (Uniprot #Q2NL67-1) was cloned into a modified pFASTBac1 vector (Addgene #30116) with N-terminal 6× His-MBP tag with a TEV protease site by SLIC cloning. The construct was sequence verified by dideoxy sequencing.…”

“…Profile of the Selected Compounds against the PARP Enzymes, IC 50 (pIC 50 ± SEM, n = 3), where Mono-ARTs PARP7-PARP16 are Measured Using a Proximity-Enhanced Assay,47 Potency of the Compounds in Rescuing Cells from PARP10 Overexpression along with 95% Confidence Interval for the EC 50 , and ADME Profiling >Denotes less than 50% inhibition in the reported highest concentration. c Value for olaparib tested in parallel: 0.016 (3.0).…”