Identification of small molecule inhibitors to exonuclease 1 for breast cancer treatment

我们的总体目标是充分响应PAR-20-271， 多功能DNA修复酶外切核酸酶1（EXO 1）的抑制剂，可用作研究 工具（化学探针），并作为开发潜在癌症治疗药物的临床前起点 药在化学探针门户网站或其他文献中没有列出EXO 1特异性小分子抑制剂。 我们将通过发现研究实现我们的目标，从实施初级高通量筛选（HTS） 我们已经开发了，通过一个良好的开发“关键路径”的二级检测来验证命中， 通过购买经验证的化学品的市售类似物进行早期命中-先导物优化 支架和有限的药物化学。EXO 1代表一个可药物化的目标，因为它包含 双链断裂反应和修复（DSBRR）功能必需的外切核酸酶活性 停滞的复制叉，这是细胞抵消内源性DNA损伤的关键途径， 复制应力与正常细胞相比，癌细胞携带显著更高的双链 断裂和复制应激，这产生了治疗癌症的治疗窗口。为了利用这一点，目前 治疗方法主要针对在修复途径或检查点信号传导途径中起作用的蛋白质 控制修复。许多癌细胞已经在DSBRR中有缺陷;因此，EXO 1抑制将导致癌症 通过合成致死机制的细胞特异性细胞死亡。此外，EXO 1 is将显示更大 特异性，因为PARP参与了广泛的其他细胞 而EXO 1则没有。我们的研究小组是第一个克隆人类EXO 1基因并表征 它的生化特性。我们已经大规模表达和纯化了全长和活性EXO 1酶， 我开发了一个强大的基于荧光的酶抑制试验，并在我们自己的核心进行了试点HTS 设施。因此，与桑福德伯纳姆Prebys医学发现研究所的Prebys中心合作， 我们处于有利地位，1）通过对精心策划的 ~ 320，000化合物库; 2）验证效力和选择性的命中; 3）执行“结构-活性关系” （SAR）-按目录”和有限的重点药物化学和基准吸收，分布，代谢， 和最佳探针的排泄（ADME）/药代动力学（PK）表征;和4）确定最佳探针的模式。 作用（MOA）和生物学效应。我们所有的目标都是响应和 在PAR-20-271范围内。新型EXO 1 is的开发不仅可以让我们提供一个关键的工具， （一）e.化学探针）来测试对复制修复界面的机械见解，但也将支持 开发了一种新的化疗药物，该药物既能阻断上游DNA复制步骤， 下游DSBRR途径，有可能诱导乳腺癌和其他癌症的临床合成致死性 DSBRR缺陷型癌症。

ABSTRACT Our overall goal, which is fully responsive to PAR-20-271, is to develop a selective and effective inhibitor of the multi-functional DNA repair enzyme exonuclease 1 (EXO1) that can be used both as a research tool (chemical probe) and as a pre-clinical starting point toward the development of a potential cancer therapeutic drug. There is no EXO1-specific small molecule inhibitor listed in the Chemical Probe Portal or other literature. We will achieve our goal through discovery research, from implementing a primary high-throughput screen (HTS) that we have already developed, to validating hits via a well-developed “critical path” of secondary assays, to performing early hit-to-lead optimization via purchase of commercially available analogs of validated chemical scaffolds and limited focused medicinal chemistry. EXO1 represents a druggable target, as it contains functionally essential exonuclease activity for double-strand break response and repair (DSBRR) for processing of stalled replication forks, which are critical pathways by which cells counteract endogenous DNA damage and replication stress. Compared to normal cells, cancer cells carry a significantly higher burden of double-strand breaks and replication stress, which generates a therapeutic window for treating cancer. To exploit this, current therapeutic approaches primarily target proteins acting in repair pathways or in checkpoint signaling pathways controlling repair. Many cancer cells are already defective in DSBRR; thus, EXO1 inhibition will cause cancer cell-specific cell death through a synthetic lethality mechanism. Furthermore, EXO1is will display greater specificity than currently used PARP inhibitors, because PARPs participate in a wide array of other cellular processes, whereas EXO1 does not. Our group was the first to clone the human EXO1 gene and to characterize its biochemical properties. We have expressed and purified the full-length and active EXO1 enzyme at scale, developed a robust fluorescence-based enzyme inhibition assay, and performed a pilot HTS in our own core facility. Thus, in collaboration with the Prebys Center of Sanford Burnham Prebys Medical Discovery Institute, we are well positioned to 1) identify inhibitors of EXO1 exonuclease by performing HTS of a well-curated ~320,000 compound library; 2) validate hits for potency and selectivity; 3) perform “structure-activity relationship (SAR)-by-catalog” and limited focused medicinal chemistry and benchmark absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) characterization of best probes; and 4) determine the mode of action (MOA) and biological effects of validated EXO1i candidate probes. All of our Aims are responsive to and within the scope of PAR-20-271. The development of novel EXO1is will not only allow us to provide a critical tool (i. e. chemical probe) to test mechanistic insights into the replication-repair interface but will also support development of a novel chemotherapeutic drug that blocks both upstream DNA replication steps and the downstream DSBRR pathway, with the potential to induce clinical synthetic lethality in breast cancer and other DSBRR-deficient cancers.