Discovery and development of Ku-targeted small molecule inhibitors: A novel mechanism of DNA-PK inhibition

DNA依赖蛋白激酶(DNA-PK)是癌症治疗的有效靶点 在DNA损伤反应(DDR)和非同源末端连接(NHEJ)双链断裂(DSB)中 修复小路。各种抗癌治疗策略，包括电离辐射(IR) 通过诱导DNA双链断裂而产生的效果。遗传学和药理学研究都表明，调节 DDR和DSB修复途径对DNA损伤治疗剂的疗效有深远的影响 支持在肿瘤治疗中靶向DNA-PK的前提。到目前为止，DNA-PK抑制剂的发展已经 完全专注于针对DNA-PKcs活性部位，其中三个目前处于早期临床试验阶段。 我们已经开发了通过Ku 70/80与DNA末端结合的DNA-PK激活要求 异二聚体通过一种新的机制识别抑制DNA-PK的小分子Ku抑制剂。初步 数据显示，Ku-抑制剂在纳摩尔浓度下消除DNA-PK的催化活性，并增强 细胞对DSB诱导疗法的敏感性。我们还证明了观察到的细胞效应是一种 直接靶向抑制Ku的功能。基于严格的已公布和初步数据，我们 假设通过靶向Ku-DNA结合介导的DNA-PK抑制将抑制DDR和 NHEJ途径导致癌细胞对DNA破坏性抗癌剂敏感。至 针对这一假设，我们提出了三个具体目标。在目标1中，我们将开发出高度有效和有选择性的 通过靶向Ku-DNA相互作用来抑制DNA-PK。已经建立了纳米分子抑制剂，化学 努力将重点放在优化物理化学和药代动力学特性上，以增加细胞摄取 和生物利用度，同时保持良好的效力和选择性。在目标2中，我们将确定分子 Ku抑制剂的作用机制及化学抑制对细胞的影响 DDR和修复路径。在目标3中，我们将询问DSB的调制如何通过HRR和DDR进行修复 Ku抑制引起的信号转导影响临床相关的单独和联合治疗的抗癌疗效 肺癌和卵巢癌的模型。我们还将评估常见癌症基因突变对 基因组稳定性和维持途径(包括BRCA1和BRCA2)走向利用合成致死 提高药物和辐射疗效的相互作用。完成这些研究将提供必要的 继续发现和开发新的Ku靶向DNA-PK抑制剂的信息。其影响 因此，这项研究的范围超出了产生新知识、试剂和模型的范围，以提供新的 针对目前难以有效治疗的多种癌症的分子靶向治疗选择。

The DNA-dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics involved in the DNA-damage response (DDR) and non-homologous end joining (NHEJ) double strand break (DSB) repair pathways. Various anti-cancer therapeutic strategies, including ionizing radiation (IR) impart their efficacy by inducing DNA DSBs. Both genetic and pharmacologic studies have demonstrated that modulating the DDR and DSB repair pathways has a profound impact on the efficacy of DNA damaging therapeutic agents supporting the premise of targeting DNA-PK in cancer therapy. Development of DNA-PK inhibitors thus far has focused entirely on targeting the DNA-PKcs active site, three of which are currently in early phase clinical trials. We have exploited the requirement for DNA-PK activation of binding to DNA termini via the Ku 70/80 heterodimer to identify small molecule Ku inhibitors that inhibit DNA-PK via a novel mechanism. Preliminary data show that Ku-inhibitors abrogate DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing therapeutics. We have also proven that the observed cellular effects are a function of direct on-target Ku inhibition. Based on the rigorous published and preliminary data we hypothesize that DNA-PK inhibition mediated by targeting Ku-DNA binding, will inhibit the DDR and NHEJ pathways resulting in sensitization of cancer cells to DNA damaging anti-cancer agents. To address this hypothesis, we propose three specific aims. In Aim 1 we will develop highly potent and selective DNA-PK inhibitors by targeting the Ku-DNA interaction. Having established nanomolar inhibitors, chemistry efforts will focus on optimizing the physicochemical and pharmacokinetic properties to increase cellular uptake and bioavailability while retaining excellent potency and selectivity. In Aim 2 we will determine the molecular mechanism of action (MOA) of Ku inhibitors and elucidate how chemical inhibition of Ku impacts the cellular DDR and repair pathways. In Aim 3 we will interrogate how modulation of DSB repair via HRR and DDR signaling due to Ku inhibition impacts anticancer efficacy alone and in combination therapy in clinically relevant models of lung and ovarian cancer. We will also assess the impact of common cancer genetic mutations in genome stability and maintenance pathways (including BRCA1 and BRCA2) towards exploiting synthetic lethal interactions to enhance drug and radiation efficacy. Completion of these studies will provide essential information for the continued discovery and development of novel Ku-targeted DNA-PK inhibitors. The impact of this research thus extends beyond the generation of new knowledge, reagents and models to provide new molecularly targeted treatment options for a wide array of cancers that are currently difficult to treat effectively.