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.

摘要我们完全响应PAR-20-271的总体目标是开发一种选择性和有效的 多功能DNA修复酶外切酶1(EXO1)的抑制物，既可用作研究 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 毒品。在化学探针门户网站或其他文献中没有列出EXO1特定的小分子抑制剂。 我们将通过发现研究实现我们的目标，从实施主要的高通量屏幕(HTS) 我们已经开发了，通过二次化验的成熟的“关键路径”来验证命中，以 通过购买商业上可获得的有效化学品类似物来执行早期的Hit-to-Lead优化 脚手架和有限的聚焦药物化学。Exo1代表一个可下药的目标，因为它包含 用于加工的双链断裂反应和修复(DSBRR)的功能必需核酸外切酶活性 停滞不前的复制叉子，这是细胞中和内源性DNA损伤和 复制压力。与正常细胞相比，癌细胞携带的双链负荷明显更高 断裂和复制压力，这为治疗癌症提供了一个治疗窗口。为了利用这一点，目前 治疗方法主要针对作用于修复通路或检查点信号通路的蛋白质 控制维修。许多癌细胞在DSBRR中已经存在缺陷；因此，抑制EXO1将导致癌症 通过一种合成致死机制的细胞特异性细胞死亡。此外，EXO1is将显示更大的 比目前使用的PARP抑制剂更具特异性，因为PARP参与了广泛的其他细胞 进程，而EXO1则不是。本课题组首次克隆了人类EXO1基因并对其进行了鉴定 它的生化特性。我们已经规模化表达和纯化了全长和有活性的EXO1酶， 开发了一种强大的基于荧光的酶抑制分析，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， 我们处于有利地位，可以通过进行精心策划的HTS来鉴定EXO1核酸外切酶的抑制剂 ~32万个化合物文库；2)效价和选择性验证；3)构效关系 (合成孔径雷达)按目录“和有限的重点药物化学和基准吸收，分配，代谢， 和最佳探针的排泄(ADME)/药代动力学(PK)表征；以及4)确定 有效的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的开发不仅将使我们能够提供一个关键工具 (即化学探针)，以测试对复制修复接口的机械性洞察，但也将支持 一种新型化疗药物的开发，该药物既能阻断DNA上游复制步骤，又能阻止 DSBRR下游通路，有可能诱导乳腺癌和其他疾病的临床合成死亡 DSBRR基因缺陷的癌症。