A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

与假激酶的生化和细胞分析相关的一个主要挑战是缺乏用于探测功能的经过靶标验证的小分子化合物。 Tribbles 2 (TRIB2) 是一种与癌症相关的假激酶，具有多种相互作用组，包括典型的 AKT 信号模块。有大量证据表明，人 TRIB2 可促进实体瘤和血癌的存活和耐药性，因此作为治疗靶点很有意义。不寻常的 TRIB2 假激酶结构域包含独特的富含半胱氨酸的 C 螺旋，并与其自身羧基末端尾部的保守肽基序相互作用，这也支持其与 E3 泛素连接酶的相互作用。我们发现 TRIB2 是先前描述的小分子蛋白激酶抑制剂的靶标，这些抑制剂最初旨在抑制表皮生长因子受体酪氨酸激酶家族成员的典型激酶结构域。通过热位移测定，我们在已发布的激酶抑制剂组 (PKIS) 中发现了 TRIB2 结合化合物，并使用药物再利用方法对体外稳定或不稳定 TRIB2 的化合物进行分类。 TRIB2 不稳定剂，包括共价药物阿法替尼，导致人类 AML 癌细胞中 TRIB2 快速降解，从而对信号传导和生存产生易于处理的影响。我们的数据揭示了开发 TRIB2 降解化合物的新药物先导化合物，这对于揭示基于 TRIB2 的信号传导的细胞机制也具有不可估量的价值。我们的研究强调，通过药物“脱靶”引起的小分子诱导的蛋白质下调可能与偶然靶向假激酶的其他抑制剂有关。