RAD51 inhibitors for chemotherapy and radiation therapy

描述（由申请人提供）：本项目的一个中心假设是，同源重组（HR） DNA修复水平升高导致人类肿瘤对某些化疗和放疗产生耐药性，而对HR的特异性抑制可能有助于克服这种耐药性。我们正在进行的研究计划包括一个多步骤筛选，以确定人类RAD51的小分子抑制剂，这是参与HR的中心蛋白。该项目的初始开发部分（由1R21CA124557-01A1资助）已经确定了几种先导化合物，它们可以阻断RAD51细丝的形成，抑制纯化系统中RAD51介导的重组，并降低细胞中的HR。这项工作也成功地验证了RAD51作为癌症治疗的靶点，并促进了检测技术的优化。目前的建议以几种方式建立在这项工作的基础上，包括支持药物化学、hr特异性细胞分析和小鼠模型的体内测试。目标是产生能够使人类肿瘤对普通肿瘤疗法敏感的药物制剂。在目标1中，我们建议使用改进的实验底物和条件筛选6800个药理上非常有利的化合物。命中化合物的滴定将确定那些活性最高的化合物，这将根据它们抑制RAD51细丝形成的能力和它们与RAD51蛋白的结合亲和力来确定。在Aim 2中，先导化合物将在纯化的体外系统中测试其抑制HR各方面的能力。这些生化分析中最活跃的化合物将发展到基于细胞的分析，以确定哪些可以在低浓度下特异性抑制体内HR，同时不影响其他DNA修复途径。随后将测试活性化合物对交联化疗药物和/或电离辐射（IR）使癌细胞敏感的能力。这些基于细胞的检测将在癌细胞系和非永生化的正常细胞上进行，以确定哪些细胞发挥肿瘤特异性作用。在Aim 3（将在UIC进行）中，将对早期目标和现有先导化合物的化学亚结构进行优化。商业上可用的化学相关化合物将首先进行测试。最优先的候选化合物将通过有针对性的化学修饰来优化，旨在提高rad51抑制活性和药理学性质。ADMET（吸收、分布、代谢、排泄和毒性）特性将被测量为最有希望的候选者。在Aim 4中，我们将在小鼠模型中进一步测试优先级最高的候选化合物。首先，确定化合物在小鼠体内的最大耐受剂量（MTD）。其次，将测试化合物对人类肿瘤异种移植物对顺铂或放射治疗的敏感性。

DESCRIPTION (provided by applicant): A central hypothesis of this project is that elevated levels of homologous recombinational (HR) DNA repair cause human tumors to be resistant to certain chemotherapies and radiotherapy, and that specific inhibition of HR may help overcome this resistance. Our on-going research plan involves a multistep screen for identifying small molecule inhibitors of human RAD51, which is the central protein involved in HR. An initial developmental portion of this project (funded by 1R21CA124557-01A1) has identified several lead compounds that block RAD51 filament formation, inhibit RAD51-mediated recombination in a purified system, and reduce HR in cells. This work also successfully validated RAD51 as a target for cancer therapy, and it facilitated optimization of assay techniques. The current proposal builds on this work in several ways including the support of medicinal chemistry, HR-specific cell-based assays, and in-vivo testing in a mouse model. The goal is to generate pharmacologic agents capable of sensitizing human tumors to common oncologic therapies. In Aim 1, we propose to screen a library of 6800 very pharmacologically favorable compounds, using improved assay substrates and conditions. Titrations of hit compounds will identify those with highest activities, which will be determined based on their ability to inhibit RAD51 filament formation and based on their binding affinity to RAD51 protein. In Aim 2, lead compounds will be tested for the ability to inhibit various aspects of HR in a purified system in-vitro system. The most active compounds in these biochemical assays will advance to cell-based assays, to determine which can specifically inhibit in-vivo HR at low concentrations while not affecting other DNA repair pathways. Active compounds will subsequently be tested for the ability to sensitize cancer cells to cross-linking chemotherapeutic drugs and/or ionizing radiation (IR). These cell-based assays will be performed on both cancer cell lines and non-immortalized normal cells, to identify which exert tumor-specific effects. In Aim 3 (which will be performed at UIC), the chemical sub-structures of lead compounds from earlier aims and from existing lead compounds will be optimized. Chemically-related compounds that are commercially available will first be tested. The highest priority candidate compounds will be optimized via targeted chemical modifications aimed at improving both RAD51-inhibitory activity and pharmacologic properties. ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties will be measured for the most promising candidates. In Aim 4, we will test the highest priority candidate compounds candidate compounds further in a mouse model. First, the maximal tolerated dose (MTD) of compounds will be determined in mice. Second, compounds will be tested for the ability to sensitize human tumor xenografts to treatment with cisplatin or radiation.