Chemical Biology-Based Tools to Uncover the Function of PARP16 in cancer

项目摘要/摘要 PARP16，PARP家族中的一员，负责执行翻译后 被称为ADP-核糖化的修饰，正在成为两种癌症亚型的新治疗靶点。在……里面 在卵巢癌中，PARP16被证明是负向调节蛋白质翻译以维持蛋白稳定。 PARP16的基因缺失(即敲除或敲除)导致全球蛋白质翻译增加， 迫使卵巢癌细胞进入蛋白毒性应激状态，最终导致癌细胞死亡。在……里面 小细胞肺癌(SCLC)，PARP16被确定为最近FDA批准的PARP1的非靶点 他唑帕利布对小细胞肺癌的疗效可能与其靶向作用有关。 PARP1和PARP16。然而，单独敲除PARP16也降低了小细胞肺癌的存活率。这两个都是 癌症研究指出PARP16是可操作的肿瘤学靶点，然而，PARP16催化活性的作用 在癌症中的作用还没有得到充分的描述。我们团队最近开发出了第一个以半胱氨酸为靶点的 名为DB008的共价PARP抑制剂，在蛋白质组范围内对PARP16显示出良好的选择性 共价结合模式。虽然DB008以纳摩尔效力抑制PARP16的催化活性，但这两种 DB008处理卵巢后观察到上述PARP16基因敲除/敲除表型 癌症和小细胞肺癌，表明非催化活性(即蛋白质-蛋白质相互作用) PARP16可能调节蛋白质动态平衡和肿瘤生长，而不是PARP16酶 活动。为了验证这一假设，我的目标是开发两种基于DB008的新型化学探针，以评估 PARP16在肿瘤中的非催化作用。在目标1中，我将合成一种靶向PARP16蛋白水解物 嵌合体(PROTAC)，将在卵巢癌和小细胞肺癌中化学敲除PARP16。PARP16 PROTAC将评估PARP16及其蛋白质-蛋白质相互作用的缺失是否会减少癌细胞 用基因敲除方法观察存活率。完成这一目标将验证PARP16作为一种新的 癌症靶点，并提供领先的临床前候选药物。在目标2中，我介绍了一种新的邻近标记 确定癌症中内源性PARP16相互作用因素的策略。这是通过将DB008转换为 笼式可光交联探头，使用紫外光覆盖并释放一种活性交联物， 将以共价方式标记相互作用的蛋白质，然后使用点击化学进行浓缩并通过质量进行鉴定 光谱分析。完成这一目标将有助于理解PARP16如何调节翻译和细胞 癌症的生存能力。目标2中描述的技术为共价抑制剂提供了一个新的用例，即 可推广到PAPS以外的其他酶家族。总而言之，这项建议将产生无价 用于揭示PARP16在癌症中的作用机制的化学生物学工具，同时还提供了一种 潜在的主要候选药物，用于对抗PARP16介导的疾病。

PROJECT SUMMARY/ABSTRACT PARP16, a member of the PARP family of enzymes responsible for carrying out the post-translational modification known as ADP-ribosylating, is emerging as a novel therapeutic target in two cancer subtypes. In ovarian cancer, PARP16 was shown to negatively regulate protein translation in order to maintain proteostasis. Genetic deletion (i.e., knockout or knockdown) of PARP16 resulted in an increase in global protein translation, forcing ovarian cancer cells to enter a state of proteotoxic stress that ultimately leads to cancer cell death. In small cell lung cancer (SCLC), PARP16 was identified as an off-target to the recently FDA-approved PARP1 inhibitor, talazoparib, suggesting that the efficacy of talazoparib in SCLC may be due to dual targeting of PARP1 and PARP16. However, knockdown of PARP16 alone also decreased SCLC viability. Both of these cancer studies point to PARP16 as actionable oncology target, however, the role of PARP16 catalytic activity in cancer has not been fully characterized. Our group has recently developed the first cysteine-targeted covalent PARP inhibitor called DB008, that displays excellent proteome-wide selectivity for PARP16 in the covalent binding mode. While DB008 inhibits PARP16 catalytic activity with nanomolar potency, neither of the aforementioned PARP16 knockout/knockdown phenotypes were observed with DB008 treatment in ovarian cancer and SCLC, suggesting that the non-catalytic activity (i.e., protein-protein interactions) of PARP16 may regulate protein homeostasis and cancer growth as opposed to PARP16 enzymatic activity. To test this hypothesis, I aim to develop two novel chemical probes, based on DB008, to evaluate the non-catalytic functions of PARP16 in cancer. In Aim 1, I will synthesize a PARP16 proteolysis targeting chimera (PROTAC) that will chemically knockdown PARP16 in ovarian cancer and SCLC. The PARP16 PROTAC will evaluate whether depletion of PARP16 and its protein-protein interactions reduces cancer cell viability as observed with genetic knockdown methods. Completion of this aim will validate PARP16 as a new cancer target and provide a lead preclinical drug candidate. In Aim 2, I introduce a novel proximity labeling strategy for identifying interactors of endogenous PARP16 in cancer. This is done by converting DB008 into a caged photo-crosslinkable probe that uses UV light to uncage and release a reactive crosslinking species that will covalently tag interacting proteins, which can then be enriched using click chemistry and identified by mass spectrometry. Completion of this aim will provide understanding for how PARP16 regulates translation and cell viability in cancer. The technology described in Aim 2 presents a new use case for covalent inhibitors that is generalizable to other enzymes families beyond PARPs. In summary, this proposal will generate invaluable chemical biology tools for uncovering the mechanism of action of PARP16 in cancer while also providing a potential lead drug candidate for combating PARP16-mediated diseases.