RAD51 inhibitors for chemotherapy and radiation therapy

描述(申请人提供)：该项目的一个中心假设是，同源重组(HR)DNA修复水平的提高会导致人类肿瘤对某些化疗和放射治疗产生耐药性，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的筛选，以确定人类RAD51的小分子抑制剂，这是参与HR的中心蛋白质。该项目的初步开发部分(由1R21CA124557-01A1资助)已经确定了几种先导化合物，它们可以阻断RAD51丝的形成，抑制RAD51在纯化系统中介导的重组，并降低细胞中的HR。这项工作还成功地验证了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、基于HR特定细胞的分析以及在小鼠模型中的体内测试。其目标是开发出能够使人类肿瘤对常见肿瘤疗法敏感的药理学药物。在目标1中，我们建议使用改进的检测底物和条件来筛选6800个药理上非常有利的化合物的文库。HIT化合物的滴定将识别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。在目标2中，将在体外纯化系统中测试先导化合物抑制HR的各个方面的能力。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，以确定哪些化合物可以在低浓度下特异性地抑制体内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)增敏的能力。这些基于细胞的分析将在癌细胞系和未永生化的正常细胞上进行，以确定哪些细胞发挥了肿瘤特异性作用。在目标3(将在UIC执行)中，来自早期目标和现有铅化合物的铅化合物的化学亚结构将得到优化。商业上可获得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旨在改善RAD51抑制活性和药理性质的有针对性的化学修饰进行优化。将对最有希望的候选药物进行ADMET(吸收、分布、新陈代谢、排泄和毒性)属性的测量。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，将确定化合物在小鼠身上的最大耐受量(MTD)。其次，将测试化合物对顺铂或放射治疗人肿瘤异种移植瘤的敏化能力。

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.