Abstract
Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/β-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/β-catenin pathway–related cancers, such as colorectal cancers.
Highlights
ADP-ribosyltransferases (ARTs) are a family of 17 enzymes with a structurally conserved catalytic domain that uses NAD+ as the cofactor to transfer ADP-ribosyl group to the substrate protein [1, 2]
Talazoparib and olaparib display similar biochemical potency on their main cellular targets (PARP1/2) and differential potency across the larger Poly(ADP-ribosyl) polymerases (PARPs) family [17]. To assess if these observations extend to endogenous cellular settings, we developed biotinylated chemical probes for talazoparib and olaparib and applied them in NCI-H1048 cellular lysate to competitively profile poly(ADP-ribosyl) polymerase 1 (PARP1) or TNKS1 engagement with their corresponding parent inhibitors (Fig. 1)
Olaparib and talazoparib exhibited similar ability to compete PARP1 binding to either chemical probe, whereas talazoparib uniquely exhibited an approximately 1000-fold greater ability to prevent binding to TNKS1 (Fig. 1, right)
Summary
ADP-ribosyltransferases (ARTs) are a family of 17 enzymes with a structurally conserved catalytic domain that uses NAD+ as the cofactor to transfer ADP-ribosyl group to the substrate protein [1, 2]. Using surface plasmon resonance (SPR), we measured kinetic dissociation constants of several PARP inhibitors (talazoparib, olaparib, niraparib, and veliparib) to the catalytic domains of PARP1 and TNKS1. We were able to characterize the TNKS1 ART domain interactions with talazoparib, olaparib, and niraparib using HDX–MS (Fig. S2).
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