Discovery of small molecule mutant SMAD4-PPI inducers

摘要 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 干预。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，并通过蛋白质发挥正常功能- 蛋白质相互作用(PPI)。值得注意的是，一类独特的肿瘤抑制基因突变是错义突变 编码单个氨基酸取代，损害正常的PPI。这些肿瘤抑制基因突变是 定义为“失去相互作用”的突变。我们的目标是直接针对“失去相互作用”的肿瘤抑制因子 通过发现突变的小分子PPI诱导剂恢复其抗癌功能。Smad4就是这样的 一种肿瘤抑制基因，在癌症中具有“失去相互作用”的突变，破坏了其与SMAD3的正常PPI。vbl.使用 Smad4作为概念验证研究，我们建议使用我们新开发的tr-fret Smad4-Smad3 PPI 新型小分子突变体Smad4-PPI诱导剂(MuSMADid)的筛选平台 突变型Smad4 PPI与Smad3结合恢复肿瘤抑制的途径和细胞反应 转化生长因子-β信号转导。初步研究表明，Smad4-Smad3 tr-fret方法具有较好的稳定性和可扩展性 1536-well uHTS格式，能灵敏地监测单个氨基酸的Smad4-Smad3 PPI动态 决议。从生物活性化学文库中，Ro-31-8220，一个双吲哚基马来酰亚胺衍生物被鉴定为 潜在的MuSMADid诱导突变的Smad4 PPI与Smad3结合并恢复对Smad4的反应性 Smad4突变的结肠癌细胞对转化生长因子-b的抗增殖信号。Ro-31-8220的鉴定 潜在的MuSMADid为直接靶向“失去相互作用”的Smad4突变提供了强有力的证据。 总之，这些初步数据支持我们的中心前提，即新的化学探测器可以被发现为 通过利用已建立的uHTS tr-fret试验筛选结构多样化的化学物质来潜在的MuSMADid 图书馆。根据发现研究的各个阶段，我们的建议将重点放在目标1的主要屏幕上 用新的化学支架鉴定MuSMADid Hits，然后用Orthogline进行验证 PPI分析，以及目标2的“功能验证”，以确定有效的新小分子抗肿瘤药物的优先顺序 MuSMADid，用于未来的点击到领先优化阶段。我们将使用uHTS tr-fret平台快速识别 一次命中和验证命中及其在修复过程中PPI诱导和细胞活动的特征 转化生长因子-β肿瘤抑制信号。实现拟议研究的目标预计将产生 优先和确认的小分子MuSMADid化合物清单，显示出有效的生化和 诱导突变型Smad4-Smad3 PPI和恢复转化生长因子-β抗增殖作用的生物学活性 信号通路。具有最强结构和功能证据的顶级抗肿瘤药物MuSMADid将 作为化学探针用于研究突变的Smad4依赖的癌症生物学并作为未来的候选者 用于精确肿瘤学的新型小分子MuSMADid药物的Hit-to-Lead研究。

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.