Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes

Abstract

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.

Selective pharmaceutical inhibition of PARP14 mitigates allergen-induced IgE and mucus overproduction in a mouse model of pulmonary allergic response

“…Treatment of ovalbumin-sensitized Parp14 −/− mice with the NAD + analogue PJ34 further reduced a subset of cytokine readouts but did not affect some other cytokine levels (37). The published interpretation of this result is at best complex and fraught, as the IC50 of PJ34 for PARP14 (1-10 μM) is ~100-fold less favorable than for PARP1 and PARP2 (48). Moreover, inactivation of PARP1 either by genetic or a more specific pharmacological agent blunted ovalbumin-induced allergic lung inflammation and type 2 cytokines (49–53).…”

“…Among the intracellular PARP family proteins, the NAD-binding pockets are structurally similar. Attempts to use existing PARP inhibitors as surrogates suffer from the drawbacks that their IC50 for PARP14 requires the use of concentrations that both are more inhibitory for poly-PARP enzymes such as PARP1 and PARP2 and from cellular toxicities at the concentrations affecting PARP14 (36,48). Moreover, Parp1 gene inactivation studies as well as use of PARP1, 2-specific inhibition using olaparib suggest that ovalbumin-induced allergic lung inflammation depends on the polymerase PARP1 (49)(50)(51)(52)(53).…”

Targeted Degradation of PARP14 Using a Heterobifunctional Small Molecule

“…RBN012811 was designed based upon a potent and selective PARP14 catalytic inhibitor, RBN012042 (Figure 2 and Table S1; PDB ID: 7 L9Y). We exploited a solvent‐exposed vector to add a linker and thalidomide moiety, and the resulting compound maintained its selectivity and potency for PARP14 based upon biochemical and biophysical assays [20–21] . We measured metabolic stability of the compounds in mouse liver microsomes, and while RBN012042 had low turnover, RBN012811 had high turnover (Table S2).…”

Adp-Ribosylation as Post-Translational Modification of Proteins: Use of Inhibitors in Cancer Control

“…In the beginning years, the developed inhibitors were designed targeting a group of ART enzymes; in recent years, novel compounds have been developed specific for a single ART enzyme, with effects at nanomolar concentration. For instance, there are new inhibitors targeting just ARTD10 [120,[127][128][129][130], ARTD11 [122,[131][132][133][134][135][136][137][138][139][140][141][142][143][144]. or ARTD14/PARP7 [142][143][144].…”

“…ARTD14/PARP7 is amplified and highly expressed in squamous cell carcinomas, in ovarian and lung tumors, and is associated with poor survival [135][136][137]. Protein substrates MARylation, such as α-tubulin, by ARTD14/PARP7, has a role in microtubule control in ovarian cancer [138,143,144].…”