Enhanced Suppression of a Protein–Protein Interaction in Cells Using Small-Molecule Covalent Inhibitors Based on an N‑Acyl‑N‑alkyl Sulfonamide Warhead

Protein–protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N-acyl-N-alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.

蛋白质-蛋白质相互作用（Protein–protein interactions, PPIs）紧密调控诸多生物学过程与疾病状态，因此被认定为小分子药物研发极具吸引力的治疗靶点。然而，针对PPIs的高效抑制剂研发被证实极具挑战性，临床成功案例寥寥无几。在此，我们报道了人类双微体2（human double minute 2, HDM2）/p53这一PPI的不可逆抑制剂，该类抑制剂以活性N-酰基-N-烷基磺酰胺（N-acyl-N-alkyl sulfonamide, NASA）基团作为弹头。基于质谱的分析成功揭示了该共价抑制的动力学过程，同时确定HDM2上的修饰位点为N端α-氨基与酪氨酸67位（Tyr67），这两类位点在传统共价抑制剂中均较为罕见。最后，相较于非共价抑制剂，我们证实该类抑制剂可延长p53通路的激活时长，并更高效地诱导p53介导的细胞死亡。本研究凸显了NASA弹头作为一类通用亲电试剂用于PPI共价抑制的潜力，同时为高效共价PPI抑制剂的合理设计开辟了全新路径。