Probing intracellular determinants of PARP inhibitor selectivity and pharmacology with CeTEAM

PARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by exploiting synthetic lethality and selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. The cellular target engagement by accumulation of mutant (CeTEAM) method provides insights into drug binding in cellulo by means of conditionally stabilized biosensors, thus offering a dynamic view of pharmacological events in living cells. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry biosensors to systematically characterize potential PARPi binding and cell cycle alterations at the single cell level. Our results reveal that most PARPi are generally equipotent for both PARPs or have slight biases only towards PARP1, not PARP2. AZD5305, a reported PARP1-selective inhibitor, was the exception and appears ~1600-fold more potent towards PARP1. Surprisingly, niraparib was >10-fold more selective for PARP1, despite reported equipotent biochemical activity. Meanwhile, the next generation PARPi, senaparib, was a potent PARP1/2 binder and DNA trapper. We also assessed the effect of the PARP1/2 active site component, HPF1, on intracellular PARPi binding and see that HPF1 depletion elicits slight deviations in apparent binding potency, while contributing additively to PARP-DNA trapping. These results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity. Furthermore, the PARP1/2 CeTEAM platform should facilitate the discovery of selective PARPi for better targeted therapies.