Development of PARP-Isoform Selective Inhibitory Chemical Probes

项目摘要/摘要 高度异构体选择性聚(ADP-核糖)聚合酶(PARP)生化探针的开发将填补 在我们对PARP酶的生物学功能的理解中存在着关键的知识差距。PAP是一种 核酶家族，催化底物蛋白质的多(ADP-核糖基)聚合(PAR化)，如 组蛋白。PAR化促进DNA修复蛋白的募集。因此，PARP抑制剂(PARPI)是 被开发为一类新型抗癌药物，可作为单一药物用于治疗BRCA缺陷肿瘤 以及作为DNA损伤剂的联合治疗。PARP超级家族由17名成员组成。 临床PARPI通常与PARP-1和PARP-2以及其中一些的混杂抑制有关 甚至可以抑制其他的帕普。这成为它们非靶标血液毒性的潜在原因。由于非- 目前已知的PARPI对多种PARP-异构体的特异性靶向，对其 药理/临床特征已变得高度复杂。进一步研究表明，PARP-1单独抑制 足以抑制MDA-MB-436肿瘤移植瘤的生长。PARP-1和PARP-2耗尽导致 胚胎致死性和侵袭性T细胞淋巴瘤。阻碍PARP评估的挑战之一- 在细胞环境中，异构体特有的分子相互作用图景是缺乏有效和高度的PARP-异构体 选择性化学探头。因此，开发高选择性的PARP抑制探针迫在眉睫 需要的。我们计划通过开发异构体选择性和潜在无毒的新型PARP抑制剂来解决这一问题 将允许询问单个PAP下行信号事件的变化的化学探测器 一个分子水平。这里产生的探针也将作为下一代临床前的模板 探员们。我们将应用一组高效和新颖的UTT-先导化合物来开发精致的PARP-异构体 选择性化学探针，将有助于我们了解单个PAP在两种情况下的生物学功能 正常细胞和病变细胞。作为概念验证，我们已经合成并表征了几个PARPI 针对PARP-1和PARP-2的纳摩尔效力，对PARP-2的偏好提高约30倍。基座 在这一科学前提和初步数据的基础上，我们提出了以下具体目标：(A)鉴定PARP-1 和PARP-2选择性生化探针；(B)进行体外PARP酶测定和异构体选择性 筛选，和(C)评估CAPAN-1(BRCA2-/-和)中的异构体选择性最佳PARPI的细胞毒性 BRCA2cor)和SUM149(BRCA1-/-和BRCA1cor)细胞。这些研究有望扩大我们的知识 关于开发的PARP化学探针抑制特定PARP-异构体的影响。到赠款结束时 在项目期间，我们将准备评估细胞活性和高度选择性的PARP化学探针的高价值 基于细胞的实验。随后，我们计划研究parp-异构体的特定下游信号。 事件，新的PARylated底物蛋白的鉴定，靶标接触和表型变化，以及 以验证其他PAP是否为可行的药理靶点。

Project Summary/Abstract The development of highly isoform-selective poly(ADP-ribose) polymerase (PARP) biochemical probes will fill crucial knowledge gaps that exist in our understanding of biological functions of PARP enzymes. PARPs are a family of nuclear enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) of substrate proteins such as histones. PARylation facilitates recruitment of DNA repair proteins. Consequently, PARP inhibitors (PARPi) are developed as a novel class of anticancer drugs that are used as single agents to treat BRCA-deficient tumors and as combination therapy with DNA damaging agents. The PARP superfamily is comprised of 17 members. Clinical PARPi are often associated with promiscuous inhibition of both PARP-1 and PARP-2 and some of them even inhibit other PARPs. This becomes a potential cause for their off-target hematologic toxicity. Due to non- specific targeting of multiple PARP-isoforms by currently known PARPi, a thorough interpretation of their pharmacological/clinical profiles has become highly complex. Further it was shown that PARP-1 inhibition alone is sufficient to repress the growth of MDA-MB-436 tumor xenograft. Depletion of both PARP-1 and PARP-2 led to embryonic lethality and aggressive T-cell lymphomas. One of the challenges impeding evaluation of PARP- isoform specific molecular interaction landscape in cellular context is the lack of potent and highly PARP-isoform selective chemical probes. Therefore, development of highly isoform selective PARP inhibitory probes is urgently needed. Weplan to address this by developing isoform-selective and potentially non-toxic novel PARP inhibitory chemical probes that will allow interrogation of changes in downstream signaling events of individual PARPs at a molecular level. The probes generated herein will also serve as template for the next generation preclinical agents. We will apply highly potent and novel set of UTT-lead compounds to develop exquisitely PARP-isoform selective chemical probes that will facilitate our understanding of biological functions of individual PARPs in both normal and diseased cells. As a proof-of-concept, we have synthesized and characterized several PARPi with nanomolar potency against both PARP-1 and PARP-2, with ~30-fold higher preference toward PARP-2. Based on this scientific premise and preliminary data, we propose the following specific aims: (A) to identify PARP-1 and PARP-2 selective biochemical probes; (B) to conduct in vitro PARP enzyme assays and isoform selectivity screening, and (C) to evaluate cytotoxicity of an isoform selective best PARPi in CAPAN-1 (BRCA2-/- and BRCA2cor) and SUM149 (BRCA1-/- and BRCA1cor) cells. These studies are expected to expand our knowledge on the impact of inhibiting a specific PARP-isoform by developed PARP chemical probes. By the end of the grant project period, we will be poised to evaluate cell-active and highly PARP-selective chemical probes in high value cell-based experiments. Subsequently, we plan to investigate PARP-isoform specific downstream signaling events, identification of new PARylated substrate proteins, on-target engagement and phenotypic change, and to validate if other PARPs are viable pharmacological targets.