Activity-based regulome profiling for the discovery of covalent transcription factor inhibitors

研究综述 人类基因组编码1600多个转录因子(TF)，以及其他 共同执行调控的辅因子、染色质调节剂和结构蛋白 编码在核DNA中的指令。已知这些蛋白质的功能障碍会导致 多种疾病，如癌症、炎症相关疾病和神经疾病。在癌症中， 这些蛋白质经常被扩增或过度表达以驱动基因表达程序 促进各种类型的白血病、肉瘤和其他疾病的发生和发展 肿瘤。尽管这些蛋白质很重要，但转铁蛋白一直被认为是不可用药的。 在体外模拟它们的活动所面临的挑战。我们已经通过以下方式解决了这些缺点 实现细胞内功能蛋白质组学药物发现平台，量化影响 在多种细胞和组织中结合到基因组上的Tf的丰度上的小分子 类型。该平台基于盐析法提取染色质，与数据相结合 独立分析质谱仪(CHESS-DIA)，这是最近报道的。在这 建议，我们采用这项技术来发现共价小分子抑制剂， 一种化学物质，最近使以前不能下药的KRAS成为靶向 蛋白。首先，我们将比较几种不同的共价筛选方法在 结合CHESS-DIA蛋白质组分析，确定哪种策略最适合 复合发现。还将构建计算工具，以强有力地识别功能 共价撞击化合物，并鉴定化合物：形成于 复合装订。几种表征良好的共价抑制剂将被用于验证 这些方法的准确性。有了经过验证的共价筛选转铁蛋白检测，我们将优化 一种使用完整蛋白质来验证HIT化合物可以标记目标蛋白质的二次实验 化学计量学，具体方法。有了这些工具后，我们将执行试运行屏幕，以 使用商业共价物的子集，在发现环境中证明该分析的实用性 复合库。这些化合物含有各种各样的反应性弹头，这将 使我们能够了解优化后的检测的各种性能指标。此数据包 将使我们能够对以Talus的TF为目标的化合物进行全面的内部筛选 兴趣，并为与生物技术公司的业务发展讨论提供基础 以及对转铁蛋白抑制感兴趣的制药公司。

RESEARCH SUMMARY The human genome encodes more than 1,600 transcription factors (TFs), along with additional cofactors, chromatin regulators, and structural proteins that collectively execute the regulatory instructions encoded within the nuclear DNA. Dysfunctions of these proteins are known to drive multiple diseases such as cancer, inflammation-related, and neurological conditions. In cancer, these proteins are frequently amplified or overexpressed to drive a gene expression program that facilitates the initiation and progression of various types of leukemia, sarcoma and other tumors. Despite the importance of these proteins, TFs have been considered undruggable due to challenges in modeling their activity in vitro. We have solved these shortcomings by implementing an in-cell functional proteomics drug discovery platform that quantifies the effects of small-molecules on the abundance of TF bound to the genome in a diversity of cell and tissue types. The platform is based on Chromatin Extraction by Salt Separation, coupled to Data Independent Analysis mass spectrometry (ChESS-DIA), which was recently reported. In this proposal, we adapt this technology to enable the discovery of covalent small molecule inhibitors, a type of chemistry that has recently enabled targeting of the previously undruggable KRAS protein. First, we will compare the efficacy of several different covalent screening approaches in combination with ChESS-DIA proteome analysis, determining which strategy is best for compound discovery. Computational tools will also be built to robustly identify functional covalent hit compounds, and to identify the compound:protein adducts that are formed upon compound binding. Several well-characterized covalent inhibitors will be used to validate the accuracy of these methods. With a validated covalent screening TF assay, we will then optimize a secondary assay using intact protein to verify that hit compounds can label target proteins in a stoichiometric, specific way. With these tools in hand, we will then perform a pilot screen to prove the assay’s utility in a discovery setting, using a subset of a commercial covalent compound library. These compounds contain a diverse array of reactive warheads, and this will allow us to understand various performance metrics of the optimized assay. This data package will enable us to perform full-scale internal screens for compounds that target Talus’ TFs of interest, as well as provide the foundation for business development discussions with biotech and pharmaceutical companies interested in TF inhibition.