RAD51 inhibitors for chemotherapy and radiation therapy

描述（由申请人提供）：该项目的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具有抗性，并且HR的特异性抑制可能有助于克服这种抗性。我们正在进行的研究计划包括一个多步骤的筛选，以确定小分子抑制剂的人RAD 51，这是中央蛋白质参与HR. An本项目的初步开发部分（由1 R21 CA 124557 - 01 A1资助）已经确定了几个先导化合物，阻止RAD 51细丝的形成，抑制RAD 51介导的重组在一个纯化的系统，并减少HR细胞。这项工作还成功地验证了RAD 51作为癌症治疗的靶点，并促进了分析技术的优化。目前的提案以几种方式建立在这项工作的基础上，包括药物化学的支持，HR特异性基于细胞的测定，以及小鼠模型中的体内测试。目标是产生能够使人类肿瘤对常见肿瘤治疗敏感的药理学试剂。在目标1中，我们建议使用改进的测定底物和条件筛选6800种非常有利的化合物的库。命中化合物的滴定将鉴定具有最高活性的那些，其将基于它们抑制RAD 51细丝形成的能力和基于它们对RAD 51蛋白的结合亲和力来确定。在目的2中，将测试先导化合物在纯化系统体外系统中抑制HR的各个方面的能力。这些生化测定中最具活性的化合物将进入基于细胞的测定，以确定哪些化合物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使癌细胞对交联化疗药物和/或电离辐射（IR）敏感的能力。这些基于细胞的测定将在癌细胞系和非永生化正常细胞上进行，以鉴定哪些发挥肿瘤特异性作用。在目标3（将在UIC进行）中，将优化早期目标和现有先导化合物的先导化合物的化学亚结构。将首先测试市售的化学相关化合物。最高优先级的候选化合物将通过有针对性的化学修饰进行优化，旨在改善RAD 51抑制活性和药理学特性。将测量最有希望的候选药物的ADMET（吸收、分布、代谢、排泄和毒性）特性。在目标4中，我们将在小鼠模型中进一步测试最高优先级的候选化合物。首先，将在小鼠中确定化合物的最大耐受剂量（MTD）。其次，将测试化合物使人肿瘤异种移植物对顺铂或放射治疗敏感的能力。

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.