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

基于化学生物学的工具揭示 PARP16 在癌症中的功能

• 批准号: 10750279
• 负责人: Daniel Bejan
• 金额: $ 4.77万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-17 至 2025-07-16
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750279
• 关键词: ADP ribosylation; Acrylamides; Active Sites; Adenosine Diphosphate Ribose; Alkynes; Automobile Driving; Binding; Binding Proteins; Biological Assay; Biology; Cell Death; Cell Survival; Cells; Chemicals; Chemistry; Chimeric Proteins; Comparative Study; Cysteine; Diazomethane; Disease; Drug Targeting; Endoplasmic Reticulum; Environment; Enzymes; Exhibits; FDA approved; Family; Genetic; Goals; Growth; Human; Hydrogen Peroxide; Knock-out; Label; Lead; Length; Light; Link; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Maps; Mass Spectrum Analysis; Mediating; Methods; Nicotinamide adenine dinucleotide; Oncology; Phenotype; Phosphotransferases; Physiology; Poly(ADP-ribose) Polymerase Inhibitor; Post-Translational Protein Processing; Process; Protac; Protein Biosynthesis; Proteins; Proteome; Proteomics; Research Personnel; Role; Signal Transduction; Stress; Technology; Testing; Translations; Ubiquitin; Ultraviolet Rays; Western Blotting; cancer cell; cancer subtypes; cross reactivity; crosslink; drug candidate; inhibitor; insight; interest; knock-down; lung cancer cell; member; nanomolar; new therapeutic target; novel; paralogous gene; pomalidomide; pre-clinical; preservation; protein degradation; protein protein interaction; proteostasis; proteotoxicity; small cell lung carcinoma; small molecule; tandem mass spectrometry; tool; tumor progression; ubiquitin-protein ligase; ultraviolet irradiation; ADP ribosylation; Acrylamides; Active Sites; Adenosine Diphosphate Ribose; Alkynes; Automobile Driving; Binding; Binding Proteins; Biological Assay; Biology; Cell Death; Cell Survival; Cells; Chemicals; Chemistry; Chimeric Proteins; Comparative Study; Cysteine; Diazomethane; Disease; Drug Targeting; Endoplasmic Reticulum; Environment; Enzymes; Exhibits; FDA approved; Family; Genetic; Goals; Growth; Human; Hydrogen Peroxide; Knock-out; Label; Lead; Length; Light; Link; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Maps; Mass Spectrum Analysis; Mediating; Methods; Nicotinamide adenine dinucleotide; Oncology; Phenotype; Phosphotransferases; Physiology; Poly(ADP-ribose) Polymerase Inhibitor; Post-Translational Protein Processing; Process; Protac; Protein Biosynthesis; Proteins; Proteome; Proteomics; Research Personnel; Role; Signal Transduction; Stress; Technology; Testing; Translations; Ubiquitin; Ultraviolet Rays; Western Blotting; cancer cell; cancer subtypes; cross reactivity; crosslink; drug candidate; inhibitor; insight; interest; knock-down; lung cancer cell; member; nanomolar; new therapeutic target; novel; paralogous gene; pomalidomide; pre-clinical; preservation; protein degradation; protein protein interaction; proteostasis; proteotoxicity; small cell lung carcinoma; small molecule; tandem mass spectrometry; tool; tumor progression; ubiquitin-protein ligase; ultraviolet irradiation

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.

项目摘要/摘要 PARP16，PARP酶家族的成员，负责执行翻译后 在两种癌症亚型中，被称为ADP-核糖基化的修饰正在成为一种新型的治疗靶标。在 卵巢癌，PARP16被证明对维持蛋白质的蛋白质翻译负面调节。 PARP16的遗传缺失（即敲除或敲除）导致全球蛋白质翻译的增加， 迫使卵巢癌细胞进入蛋白质毒性应激的状态，最终导致癌细胞死亡。在 小细胞肺癌（SCLC），PARP16被确定为最近FDA批准的PARP1的脱靶 抑制剂Talazoparib，表明Talazoparib在SCLC中的功效可能是由于双重靶向 PARP1和PARP16。但是，仅击倒PARP16也降低了SCLC的生存能力。这两个 癌症研究指出PARP16是可行的肿瘤学靶标，但是，PARP16催化活性的作用 在癌症中尚未充分表征。我们的小组最近开发了第一个以半胱氨酸为目标的 共价PARP抑制剂称为DB008，在PARP16中显示出极好的蛋白质组选择性 共价结合模式。 DB008抑制PARP16纳摩尔效力的催化活性，但两种 卵巢中的DB008处理观察到上述PARP16敲除/敲低表型 癌症和SCLC，表明非催化活性（即蛋白质 - 蛋白质相互作用） PARP16可能调节蛋白质稳态和癌症的生长，而不是PARP16酶促的 活动。为了检验这一假设，我的目标是基于DB008开发两个新型的化学探针，以评估 PARP16在癌症中的非催化功能。在AIM 1中，我将合成PARP16蛋白水解靶向 嵌合体（Protac）将在卵巢癌和SCLC中化学敲低PARP16。 PARP16 Protac将评估PARP16及其蛋白质 - 蛋白质相互作用的耗竭是否会减少癌细胞 使用遗传敲低方法观察到的生存能力。此目标的完成将验证PARP16作为新的 癌症目标并提供铅临床前药物。在AIM 2中，我介绍了一个新颖的接近标签 鉴定癌症内源性PARP16相互作用者的策略。这是通过将DB008转换为 使用紫外线揭开笼子的光合探针，并释放一个反应性的交联物种 将共价标记相互作用的蛋白 光谱法。此目标的完成将为PARP16如何调节翻译和细胞提供理解 癌症的生存能力。 AIM 2中描述的技术提出了一种新的用例，用于共价抑制剂 可以推广到其他酶家庭以外的酶家庭。总而言之，该提案将产生无价的 化学生物学工具，用于发现PARP16在癌症中的作用机理，同时也提供了 潜在的铅药物来对抗PARP16介导的疾病。