PO.CH02.01 · 化学
化学蛋白质组学识别出与新一代poly(ADP-ribose)聚合酶抑制剂saruparib相互作用的转录相关蛋白网络
Chemical proteomics identifies a transcription-related network of proteins that interact with the next-generation poly(ADP-ribose) polymerase inhibitor saruparib
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摘要 Abstract
中文摘要
引言：卵巢癌是妇科相关死亡率的主要原因之一，往往由于晚期诊断所致。Poly(ADP-ribose)聚合酶1抑制剂（PARPi）已获FDA批准用于治疗卵巢癌、乳腺癌、胰腺癌和前列腺癌。PARPi在同源重组修复缺陷的细胞中导致合成致死。尽管PARPi是一种有前景的癌症治疗药物，但耐药性以及血液学毒性等副作用仍是未满足的临床挑战。
方法：在本研究中，我们利用PARP1充足的以及经CRISPR构建的PARP1缺陷的同源HEYA8和HAP1细胞系，使用衍生化的Olaparib和新一代PARPi Saruparib进行化学蛋白质组学。在细胞裂解液中经点击化学后下拉与药物结合的蛋白和蛋白复合物，随后通过LC-MS/MS识别Saruparib的新靶标。
结果：随后的分析揭示了一组具有统计学显著性（p值 ≤ 0.03）的247个独特蛋白，与未修饰PARPi竞争相比富集≥2倍。在这247个蛋白中，我们比较了两个细胞系中的不同条件，以识别与Saruparib及其经典靶标PARP1相互作用的蛋白。182个仅在Saruparib下拉中发现，192个仅在PARP1充足条件下（无论是否有药物）发现，159个在Saruparib下拉和PARP1充足条件下均发现。若干与TFIID复合物（由前起始转录因子组成）相关的蛋白显示仅在Saruparib下拉中富集，无论PARP1是否充足，提示转录机器可能失调。然而，TFIID复合物中的一个蛋白BRD2显示其富集依赖于PARP1充足性，提示潜在的药物和PARP双重特异性。BRD2是BET蛋白家族的成员，在转录调控和基因表达中发挥作用。我们进一步通过与HEK293T全细胞裂解液的共免疫沉淀实验验证了内源性BRD2与PARP1之间的相互作用。
结论：我们的发现揭示了一个转录相关蛋白（包括BRD2）的新格局，可能为与PARPi Saruparib的联合治疗提供靶标。
查看英文原文 English abstract
Introduction: Ovarian cancer is one of the leading causes of gynecological-related mortality rates, often due to late-stage diagnosis. Poly(ADP-ribose) polymerase 1 inhibitors (PARPi) have been FDA approved for treatment in ovarian, breast, pancreatic, and prostate cancers. PARPi's lead to synthetic lethality in cells deficient in homologous recombination repair. Although PARPi's are a promising cancer therapeutic, drug resistance as well as side effects like hematological toxicity remain unmet clinical challenges.
Methods: In this study we utilized PARP1 -proficient and generated CRISPR PARP1 -deficient isogenic HEYA8 and HAP1 cell lines to perform chemical proteomics using derivatized Olaparib and a next-generation PARPi, Saruparib. Proteins and protein complexes engaged with the drug were pulled down after click chemistry in cell lysates, followed by LC-MS/MS to identify new targets of Saruparib.
Results: The subsequent analysis revealed a statistically significant set (p-value ≤ 0.03) of 247 unique proteins that were ≥2-fold enriched when compared to the unmodified PARPi competition. Within those 247 proteins, we compared the different conditions in both cell lines to identify proteins that interacted with Saruparib as well as its canonical target, PARP1. 182 were found exclusively in the Saruparib pulldown, 192 were found only in PARP1-proficient conditions regardless of drug, and 159 were found in both the Saruparib pulldown and PARP1-proficient conditions. Several proteins associated with the TFIID complex, composed of pre-initiation transcription factors, were shown to be enriched only in the Saruparib pulldown regardless of PARP1-proficiency, indicating a potential deregulation of the transcriptional machinery. However, one protein of the TFIID complex, BRD2, was shown to have enrichment dependent on PARP1-proficiency, indicating both potential drug and PARP specificity. BRD2 is a member of the BET family of proteins, which plays a role in transcriptional regulation and gene expression. We further validated the interaction between endogenous BRD2 and PARP1 through co-immunoprecipitation experiments with HEK293T whole cell lysates.
Conclusions: Our findings have uncovered a novel landscape of transcription-related proteins, including BRD2, that might provide targets for combination therapy with the PARPi Saruparib.
利益披露 Disclosure
R. H. Martin, None..
L. Nguy, None..
T. Da Costa Nepomuceno, None..
O. Deng, None..
U. Rix, None.