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

鉴定用于乳腺癌治疗的核酸外切酶 1 小分子抑制剂

• 批准号: 10735307
• 负责人: DAVID A. HORNE
• 金额: $ 57.54万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-02 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735307
• 关键词: BRCA1 gene; Benchmarking; Binding; Biochemical; Biological; Biological Assay; Biological Process; Breast; Breast Cancer Cell; Breast Cancer Treatment; Cancer Etiology; Cancerous; Catalogs; Cell Death; Cell physiology; Cells; Chemicals; Clinical; Collaborations; Core Facility; Critical Pathways; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA redundancy; DNA replication fork; Dedications; Development; Dose; Drug Kinetics; EXO1 gene; Enzyme Inhibition; Enzymes; Excision; Exclusion; Excretory function; Exonuclease; Failure; Family member; Fluorescence; Fluorescence Resonance Energy Transfer; Genes; Genetic; Goals; Growth; Human; Human Cloning; In Vitro; Invaded; Kinetics; Knock-out; Label; Lead; Length; Libraries; Literature; Malignant Neoplasms; Measures; Medical; Medicine; Metabolism; Mutate; Mutation; Normal Cell; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Poly(ADP-ribose) Polymerase Inhibitor; Poly(ADP-ribose) Polymerases; Positioning Attribute; Powder dose form; Predisposition; Process; Property; Proteins; Qualifying; RNA primers; Reaction; Research; Rodent; Signal Pathway; Single-Stranded DNA; Specificity; Structure-Activity Relationship; Testing; Therapeutic; Toxic effect; Validation; Work; absorption; analog; anti-cancer therapeutic; brca gene; cancer cell; cheminformatics; counterscreen; cytotoxic; cytotoxicity; druggable target; exodeoxyribonuclease; gene repair; high throughput screening; hormone related cancer; improved; inhibitor; insight; lead optimization; malignant breast neoplasm; meter; novel; novel therapeutics; nuclease; pre-clinical; prototype; repaired; replication stress; response; scaffold; scale up; small molecule; small molecule inhibitor; synergism; tool

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.

摘要 我们的总体目标完全响应 PAR-20-271，是开发一种选择性且有效的 多功能 DNA 修复酶核酸外切酶 1 (EXO1) 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方法的临床前起点 药品。化学探针门户或其他文献中没有列出 EXO1 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）开始 我们已经开发出通过二次分析的完善“关键路径”来验证命中， 通过购买经过验证的化学物质的市售类似物来执行早期的先导化合物优化 支架和有限的重点药物化学。 EXO1 代表可药物靶标，因为它包含 用于加工的双链断裂响应和修复 (DSBRR) 功能必需的核酸外切酶活性 停滞的复制叉，这是细胞抵抗内源性 DNA 损伤的关键途径 复制压力。与正常细胞相比，癌细胞携带明显更高的双链负担 断裂和复制压力，这产生了治疗癌症的治疗窗口。为了利用这一点，当前 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。许多癌细胞的 DSBRR 已经存在缺陷；因此，抑制EXO1会导致癌症 通过合成致死机制实现细胞特异性细胞死亡。此外，EXO1is 将显示更大的 特异性高于目前使用的 PARP 抑制剂，因为 PARP 参与多种其他细胞 处理，而 EXO1 不处理。我们的团队是第一个克隆人类 EXO1 基因并表征 它的生化特性。我们已经大规模表达并纯化了全长且有活性的 EXO1 酶， 开发了一种强大的基于荧光的酶抑制测定，并在我们自己的核心中进行了试点 HTS 设施。因此，与桑福德伯纳姆普雷比医学发现研究所的普雷比中心合作， 我们有能力 1) 通过对精心策划的 HTS 来识别 EXO1 核酸外切酶的抑制剂 约 320,000 个化合物库； 2) 验证命中的效力和选择性； 3）执行“构效关系” （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； 4）确定模式 经验证的 EXO1i 候选探针的作用 (MOA) 和生物效应。我们所有的目标都是响应和 在 PAR-20-271 的范围内。新型 EXO1is 的开发不仅能让我们提供一个关键工具 （即化学探针）来测试复制修复界面的机制见解，但也支持 开发一种新型化疗药物，可阻断上游 DNA 复制步骤和 DSBRR 通路下游，有可能诱导乳腺癌和其他癌症的临床合成致死率 DSBRR 缺乏的癌症。