Slow Dissociation from the PARP1-HPF1 Complex Drives Inhibitor Potency

PARP1, upon binding to damaged DNA, is activated to perform poly ADP-ribosylation (PARylation) on itself and Other proteins, which leads to relaxation of chromatin and recruitment of DNA repair factors. HPF1 was recently discovered as a protein cofactor of PARP1 that directs preferential PARylation of histones over Other targets by contributing to and altering the PARP1 active site. Inhibitors of PARP1 (PARPi) are used in the treatment of BRCA-/- cancers, but the basis for their potency in cells, especially in the context of HPF1, is not fully understood. Here, we demonstrate the simple one-step association for eight different PARPi to PARP1 with measured rates of association (k_on) of 0.8-6 μM^-1 s^-1. We find only minor differences in these on rates when comparing PARP1 with the PARP1-HPF1 complex. By characterizing the rates of dissociation (k_off) and the binding constants (K_D) for two more recently discovered PARPi, we find, for example, that saruparib has a half-life for dissociation of 22.5 h and fluzoparib has higher affinity for PARP1 in the presence of HPF1, just like the structurally related compound olaparib. By using the measured K_D and k_on to calculate k_off, we find that the potency of PARPi in cells correlates best with the k_off from the PARP1-HPF1 complex. Our data suggest that dissociation of a drug compound from the PARP1-HPF1 complex should be the parameter of choice for guiding the development of next-generation PARPi.