Discovery of small molecule mutant SMAD4-PPI inducers

小分子突变体 SMAD4-PPI 诱导剂的发现

基本信息

批准号：
10665612
负责人：
Xiulei Mo
金额：
$ 35.08万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10665612
关键词：
Address Amino Acid Substitution Amino Acids Biochemical Biological Biological Assay Cancer Biology Case Study Cell Cycle Arrest Cell Nucleus Cell Proliferation Cell Survival Cells Chemicals Collection Colon Carcinoma Complex Data Development Dose Fluorescence Resonance Energy Transfer Future Genetic Transcription Genomic Library Genomics Glues Goals Impairment Intuition Lead MADH3 gene MADH4 gene Malignant Neoplasms Malignant neoplasm of pancreas Mediator Missense Mutation Mission Monitor Mutate Mutation Oncogenic Oncology Pathway interactions Performance Pharmaceutical Chemistry Phase Proliferating Recurrence Research Resolution Scientist Series Signal Pathway Signal Transduction Specificity Subgroup Suppressor Mutations Testing Therapeutic Therapeutic Intervention Time Transforming Growth Factor beta Triage Tumor Suppressor Genes Tumor Suppressor Proteins Validation Work analog anti-cancer anticancer activity assay development cancer cell cell growth cheminformatics colon cancer cell line druggable target experience extracellular gastrointestinal high throughput screening lead optimization loss of function mutant neglect novel precision drugs precision oncology protein protein interaction response scaffold screening small molecule small molecule libraries small molecule therapeutics success tumor

项目摘要

SUMMARY Tumor suppressor genes represent a major class of oncogenic “drivers” and offer robust window for therapeutic intervention. However, direct targeting loss-of-function tumor suppressor genes remains challenging, because that majority of tumor suppressors do not have enzymatic activity and exert their normal function through protein- protein interaction (PPI). Noteworthy, a unique class of tumor suppressor mutations are missense mutations encoding single amino acid substitutions that impair the normal PPI. These tumor suppressor mutations are defined as “loss-of-interaction” mutation. We aim to directly target the “loss-of-interaction” tumor suppressor mutations through discovery of small molecule PPI inducers to restore their anticancer functions. SMAD4 is such a tumor suppressor with “loss-of-interaction” mutations in cancer that disrupt its normal PPI with SMAD3. Using SMAD4 as a proof-of-concept study, we propose to utilize our newly developed TR-FRET SMAD4-SMAD3 PPI screening platform to reveal novel small molecule mutant SMAD4-PPI inducer (MuSMADid) that can induce the mutant SMAD4 PPI with SMAD3 and restore the pathway and cellular response to the tumor suppressive TGF-b signaling. Preliminary studies showed that the SMAD4-SMAD3 TR-FRET assay is robust and scalable in 1536-well uHTS format and is sensitive to monitor the SMAD4-SMAD3 PPI dynamic at single amino acid resolution. From a bioactive chemical library, Ro-31-8220, a bisindolylmaleimide derivative, was identified as potential MuSMADid that induced the mutant SMAD4 PPI with SMAD3 and restored the responsiveness of SMAD4 mutant colon cancer cells to the TGF-b anti-proliferation signaling. Identification of Ro-31-8220 as a potential MuSMADid provides strong evidence for direct targeting “loss-of-interaction” SMAD4 mutations. Together, this preliminary data supports our central premise that novel chemical probes can be discovered as potential MuSMADid by leveraging the established uHTS TR-FRET assay to screen structurally diverse chemical libraries. Based on the stages of discovery research, our proposal will focus on Aim 1 “Primary Screen Implementation” to identify MuSMADid hits with new chemical scaffolds, followed by verification with orthogonal PPI assays, and on Aim 2 “Functional Validation” to prioritize a list of validated novel small molecule anti-tumor MuSMADid for future hit-to-lead optimization phase. We will use the uHTS TR-FRET platform to rapidly identify primary hits and validated hits followed by characterization of their PPI induction and cellular activities in restoring the TGF-b tumor suppressive signaling. Accomplishing the goals of the proposed study is anticipated to generate a list of prioritized and confirmed small molecule MuSMADid compounds that show potent biochemical and biological activities in inducing the mutant SMAD4-SMAD3 PPI and restoring the TGF-b anti-proliferation signaling pathways. Top ranked anti-tumor MuSMADid with the strongest structural and functional evidence will be used as chemical probes to study the mutant SMAD4-dependent cancer biology and as candidates for future hit-to-lead studies towards the development of novel small molecule MuSMADid drug for precision oncology.

概括肿瘤抑制基因代表了一类主要的致癌“驱动因素”，并为治疗提供了强有力的窗口然而，直接靶向功能丧失的肿瘤抑制基因仍然具有挑战性，因为大多数肿瘤抑制因子不具有酶活性并通过蛋白质发挥其正常功能值得注意的是，一类独特的肿瘤抑制突变是错义突变。编码损害正常 PPI 的单个氨基酸替换是这些肿瘤抑制突变。定义为“相互作用丧失”突变，我们的目标是直接针对“相互作用丧失”肿瘤抑制基因。通过发现小分子PPI诱导突变来恢复其抗癌功能的SMAD4就是这样。一种肿瘤抑制因子，在癌症中具有“相互作用丧失”突变，可破坏其与 SMAD3 的正常 PPI。 SMAD4 作为概念验证研究，我们建议利用我们新开发的 TR-FRET SMAD4-SMAD3 PPI 筛选平台揭示新型小分子突变体 SMAD4-PPI 诱导剂（MuSMADid），可诱导突变体 SMAD4 PPI 与 SMAD3 结合，恢复肿瘤抑制的通路和细胞反应 TGF-b 信号转导的初步研究表明，SMAD4-SMAD3 TR-FRET 检测方法稳健且可扩展。 1536 孔 uHTS 格式，可灵敏地监测单个氨基酸的 SMAD4-SMAD3 PPI 动态从生物活性化学库中，Ro-31-8220，一种双吲哚基马来酰亚胺衍生物，被鉴定为潜在的 MuSMADid 诱导突变型 SMAD4 PPI 与 SMAD3 并恢复了 SMAD4 突变结肠癌细胞对 TGF-b 的抗增殖信号传导鉴定为 Ro-31-8220。潜在的 MuSMADid 为直接靶向“相互作用丧失”SMAD4 突变提供了强有力的证据。总之，这些初步数据支持了我们的中心前提，即可以通过以下方式发现新型化学探针：利用已建立的 uHTS TR-FRET 测定来筛选结构多样的化学物质，从而具有潜在的 MuSMADid 潜力根据发现研究的阶段，我们的建议将重点关注目标 1“初级筛选”。 “实施”以识别新化学支架的 MuSMADid 命中，然后用正交验证 PPI 测定，以及目标 2“功能验证”，以优先考虑经过验证的新型小分子抗肿瘤药物列表 MuSMADid 用于未来的 hit-to-lead 优化阶段，我们将使用 uHTS TR-FRET 平台来快速识别。主要命中和验证命中，然后表征其 PPI 诱导和恢复中的细胞活性 TGF-b 肿瘤抑制信号传导有望实现所提出的研究目标。优先考虑和确认的小分子 MuSMADid 化合物列表，这些化合物显示出有效的生化和诱导突变型 SMAD4-SMAD3 PPI 和恢复 TGF-b 抗增殖的生物活性具有最强结构和功能证据的顶级抗肿瘤 MuSMADid 将可用作化学探针来研究突变型 SMAD4 依赖性癌症生物学，并作为未来的候选者旨在开发用于精准肿瘤学的新型小分子 MuSMADid 药物的先导研究。