Discovery of small molecule mutant SMAD4-PPI inducers

总结 肿瘤抑制基因代表了一类主要的致癌“驱动因子”，并为肿瘤的治疗提供了强大的窗口。 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，而是通过蛋白质- 蛋白质相互作用（PPI）。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的单个氨基酸取代。这些肿瘤抑制基因突变是 定义为“相互作用丧失”突变。我们的目标是直接靶向“失去相互作用”的肿瘤抑制因子 通过发现小分子PPI诱导剂来恢复其抗癌功能。SMAD 4是这样的 一种在癌症中具有“失去相互作用”突变的肿瘤抑制因子，其通过SMAD 3破坏其正常PPI。使用 SMAD 4作为概念验证研究，我们建议使用我们新开发的TR-FRET SMAD 4-SMAD 3 PPI 筛选平台，以揭示可以诱导新的小分子突变体SMAD 4-PPI诱导剂（MuSMADid）， 突变体SMAD 4 PPI与SMAD 3结合，并恢复对肿瘤抑制的途径和细胞应答。 TGF-β信号传导。初步研究表明，SMAD 4-SMAD 3 TR-FRET测定法是稳健的和可扩展的， 1536-良好的uHTS格式，并且对监测单个氨基酸处的SMAD 4-SMAD 3 PPI动态是敏感的 分辨率从生物活性化学文库中，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SMAD 4 PPI与SMAD 3，并恢复 SMAD 4突变结肠癌细胞对TGF-β的抗增殖信号。Ro-31-8220鉴别为 潜在的MuSMADid为直接靶向“失去相互作用”的SMAD 4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假设，即可以发现新的化学探针， 通过利用已建立的uHTS TR-FRET测定筛选结构多样的化学物质， 图书馆.根据发现研究的阶段，我们的建议将侧重于目标1“初步筛选 实施”，以确定MuSMADid命中与新的化学支架，然后验证与正交 PPI测定，以及目标2“功能验证”，以优先考虑经验证的新型小分子抗肿瘤药物列表 MuSMADid用于未来的点击率到领先优势优化阶段。我们将使用uHTS TR-FRET平台快速识别 主要命中和验证命中，然后表征其PPI诱导和细胞活性， TGF-β肿瘤抑制信号。实现拟议研究的目标预计将产生 列出了优先考虑和确认的小分子MuSMADid化合物，这些化合物显示出有效的生物化学和 诱导突变SMAD 4-SMAD 3 PPI和恢复TGF-β抗增殖的生物活性 信号通路具有最强结构和功能证据的顶级抗肿瘤MuSMADid将 作为化学探针研究突变SMAD 4依赖的癌症生物学，并作为未来的候选人， 针对开发用于精确肿瘤学的新型小分子MuSMADid药物的命中领先研究。

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.