Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
J Am Chem Soc. 2013 Feb 20;135(7):2497-500. doi: 10.1021/ja400427e. Epub 2013 Feb 8.
Small-molecule inhibitors can accelerate the functional annotation and validate the therapeutic potential of proteins implicated in disease. Phenotypic screens provide an effective platform to identify such pharmacological agents but are often hindered by challenges associated with target identification. For many protein targets, these bottlenecks can be overcome by incorporating electrophiles into small molecules to covalently trap interactions in vivo and by employing bioorthogonal handles to enrich the protein targets directly from a complex proteome. Here we present the trifunctionalized 1,3,5-triazine as an ideal modular scaffold for generating libraries of irreversible inhibitors with diverse target specificities. A divergent synthetic scheme was developed to derivatize the triazine with an electrophile for covalent modification of target proteins, an alkyne as a click-chemistry handle for target identification, and a diversity element to direct the compounds toward distinct subsets of the proteome. We specifically targeted our initial library toward cysteine-mediated protein activities through incorporation of thiol-specific electrophiles. From this initial screen we identified two compounds, RB-2-cb and RB-11-ca, which are cell permeable and highly selective covalent modifiers for Cys239 of β-tubulin (TUBB) and Cys53 of protein disulfide isomerase (PDI) respectively. These compounds demonstrate in vitro and cellular potencies that are comparable to currently available modulators of tubulin polymerization and PDI activity. Our studies demonstrate the versatility of the triazine as a modular scaffold to generate potent and selective covalent modifiers of diverse protein families for chemical genetics applications.
小分子抑制剂可以加速与疾病相关的蛋白质的功能注释和验证治疗潜力。表型筛选为鉴定此类药理试剂提供了有效的平台，但通常受到与靶标识别相关的挑战的阻碍。对于许多蛋白质靶标，可以通过将亲电试剂引入小分子中来克服这些瓶颈，从而在体内共价捕获相互作用，并通过使用生物正交手柄直接从复杂的蛋白质组中富集蛋白质靶标。在这里，我们提出了三功能化的 1,3,5-三嗪作为一种理想的模块化支架，用于生成具有不同靶标特异性的不可逆抑制剂文库。开发了一种发散的合成方案，使三嗪与亲电试剂衍生化，用于共价修饰靶蛋白，炔烃作为点击化学手柄用于靶标鉴定，以及多样性元素用于将化合物引导至蛋白质组的不同亚群。我们特别通过将硫醇特异性亲电试剂掺入其中，针对半胱氨酸介导的蛋白质活性来靶向我们的初始文库。从这个初始筛选中，我们鉴定出了两种化合物，RB-2-cb 和 RB-11-ca，它们分别是细胞渗透的和针对微管蛋白 (TUBB) 的 Cys239 和蛋白二硫键异构酶 (PDI) 的 Cys53 的高度选择性共价修饰剂。这些化合物在体外和细胞中的效力与目前可用的微管蛋白聚合和 PDI 活性调节剂相当。我们的研究表明三嗪作为一种模块化支架具有多功能性，可用于生成针对不同蛋白质家族的有效和选择性共价修饰剂，用于化学遗传学应用。
