Structure-Guided Design and In-Cell Target Profiling of a Cell-Active Target Engagement Probe for PARP Inhibitors

Inhibition of the
poly­(ADP-ribose) polymerase (PARP) family of
enzymes has become an attractive therapeutic strategy in oncology
and beyond; however, chemical tools to profile PARP engagement in
live cells are lacking. Herein, we report the design and application
of PARPYnD, the first photoaffinity probe (AfBP) for
PARP enzymes based on triple PARP1/2/6 inhibitor AZ9482, which induces multipolar spindle (MPS) formation in breast cancer
cells. PARPYnD is a robust tool for profiling PARP1/2
and is used to profile clinical PARP inhibitor olaparib, identifying
several novel off-target proteins. Surprisingly, while PARPYnD can enrich recombinant PARP6 spiked into cellular lysates and inhibits
PARP6 in cell-free assays, it does not label PARP6 in intact cells.
These data highlight an intriguing biomolecular disparity between
recombinant and endogenous PARP6. PARPYnD provides a
new approach to expand our knowledge of the targets of this class
of compounds and the mechanisms of action of PARP inhibitors in cancer.

聚(ADP-核糖)聚合酶（poly(ADP-ribose) polymerase, PARP）家族酶的抑制作用，已成为肿瘤学及其他领域极具吸引力的治疗策略；然而，目前仍缺乏可在活细胞中表征PARP结合状态的化学工具。本文报道了基于三重PARP1/2/6抑制剂AZ9482的光亲和探针（AfBP）PARPYnD的设计与应用，该抑制剂可在乳腺癌细胞中诱导多极纺锤体（MPS）形成。PARPYnD是一款用于表征PARP1/2的稳健工具，我们利用其对临床PARP抑制剂奥拉帕利进行了表征，鉴定出多种新型脱靶蛋白。令人意外的是，尽管PARPYnD可富集掺入细胞裂解液中的重组PARP6，并在无细胞实验中抑制PARP6活性，但它无法在完整细胞中标记PARP6。上述数据揭示了重组PARP6与内源性PARP6之间存在有趣的生物分子差异。PARPYnD为拓展我们对该类化合物靶点以及PARP抑制剂抗癌作用机制的认知提供了新途径。