Cyclin-dependent kinases (CDKs) have become potential targets for treating various diseases, especially cancer. Compound iCDK9 is an excellent and selective CDK9 inhibitor, but its major limitation is the potential toxicity and poor understanding of the underlying mechanism. The PROTAC (proteolysis targeting chimera) degraders of bioactive molecules can significantly induce in vitro and in vivo degradation of their target protein with high selectivity and effectively reduce the dose-limiting toxicity of small molecule drugs. Therefore, we designed and synthesized the bifunctional PROTAC molecules of iCDK9, being used for identifying its previously unknown target and revealing the underlying pharmacological mechanism. The PROTAC bifunctional molecule CD-5 could selectively and significantly degrade CDK9 with low cell toxicity. Therefore, we selected CD-5 as a chemical prober in the SILAC quantitative proteomic analysis, which disclosed that CD-5 could enormously lessen the lysine acetyltransferase KAT6A. Furthermore, KAT6A degradation induced by CD-5 repressed the levels of H3K14Ac and H3K23Ac. Lastly, the streptavidin immunoprecipitation (IP) assay confirmed a direct interaction between KAT6A and iCDK9. Collectively, our results uncover that KAT6A is a potential non-kinase target of iCDK9. Notably, this study also demonstrates that the PROTAC-SILAC strategy is an alternative approach for cellular target identification of bioactive molecules.

细胞周期蛋白依赖性激酶 (CDK) 已成为治疗各种疾病，尤其是癌症的潜在靶点。化合物 iCDK9 是一种优秀的选择性 CDK9 抑制剂，但其主要局限性在于潜在的毒性和对潜在机制的了解不足。生物活性分子的 PROTAC（蛋白水解靶向嵌合体）降解剂可在体外和体内显着诱导高选择性降解其靶蛋白，有效降低小分子药物的剂量限制性毒性。因此，我们设计并合成了 iCDK9 的双功能 PROTAC 分子，用于识别其先前未知的靶标并揭示潜在的药理机制。PROTAC 双功能分子 CD-5 可以选择性显着降解 CDK9，细胞毒性低。因此，我们选择 CD-5 作为 SILAC 定量蛋白质组学分析的化学探针，表明 CD-5 可以极大地降低赖氨酸乙酰转移酶 KAT6A。此外，CD-5 诱导的 KAT6A 降解抑制了 H3K14Ac 和 H3K23Ac 的水平。最后，链霉亲和素免疫沉淀 (IP) 测定证实了 KAT6A 和 iCDK9 之间的直接相互作用。总的来说，我们的结果表明 KAT6A 是 iCDK9 的潜在非激酶靶标。值得注意的是，这项研究还表明，PROTAC-SILAC 策略是生物活性分子的细胞靶标识别的另一种方法。