Design, Synthesis, and Biological Evaluation of Novel Activators of Human Caseinolytic Protease P with a Pyrazololactam Scaffold

Based
on D9, a previously reported small-molecule
agonist of hClpP, a class of novel hClpP activators with a pyrazololactam
scaffold was designed and synthesized. Detailed structure–activity
relationship studies (SAR) for this class of compounds led to the
identification of compound 24, which potently activated
recombinant hClpP in a proteolysis assay with submicromolar potency
and effectively inhibited cell growth in a broad panel of cancer cell
lines with IC50 values of 0.1–1 μM. Mechanism
studies indicated that compound 24 can potently bind
to cellular hClpP, effectively promote the formation of the hClpP
tetradecamer, efficiently induce the degradation of hClpP substrates,
robustly upregulate the expression of ATF4, and strongly induce apoptosis
in Molm13 and MDA-MB-231 cells. More importantly, compound 24 has promising PK and safety profiles, and showed potent antitumor
activity in a murine MDA-MB-231 xenograft model.

本研究以已有报道的人源ClpP（hClpP）小分子激动剂D9为基础，设计并合成了一类具有吡唑内酰胺（pyrazololactam）骨架的新型hClpP激活剂。通过对该类化合物开展详细的构效关系（structure–activity relationship, SAR）研究，最终筛选得到化合物24：该化合物在蛋白水解实验中可高效激活重组人源ClpP，活性达亚微摩尔级别；同时能广谱抑制多种癌细胞系的细胞增殖，半数抑制浓度（IC50）为0.1~1 μM。机制研究表明，化合物24可强效结合细胞内的hClpP，有效促进hClpP十四聚体的形成，高效诱导hClpP底物的降解，显著上调激活转录因子4（ATF4）的表达，并可强烈诱导Molm13与MDA-MB-231细胞发生凋亡。更为重要的是，化合物24具备良好的药代动力学（pharmacokinetics, PK）与安全性特征，在小鼠MDA-MB-231异种移植瘤模型中展现出强效的抗肿瘤活性。