Chou Tsui-Fen, Li Kelin, Nordin Brian E., Porubsky Patrick, Frankowski Kevin, Patricelli Mathew P., Aubé Jeffrey, Schoenen Frank J., Deshaies Raymond
Division of Biology, California Institute of Technology, Pasadena, CA 91125
University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, KS 66047
The AAA ATPase p97 is a critical factor in maintaining protein homeostasis in eukaryotic cells, through its roles in promoting degradation of ubiquinated proteins by the proteasome and in maturation of autophagosomes. Starting from a hit obtained by high-throughput screening (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), the team developed probe compounds ML240 and ML241 that both inhibit p97 ATPase activity with an IC of approximately 100 nM, and block degradation of p97-dependent proteasome substrate with an IC of approximately 900 nM and 3500 nM, respectively. By contrast, these probe compounds were far less potent in blocking degradation of the p97-independent substrate oxygen-dependent degradation domain (ODD)-luciferase (IC > 28 μM), underscoring their specificity towards p97. The two probe compounds exhibited markedly different potencies for activating executioner caspases and blocking cell growth. ML240 rapidly induces caspases 3 and 7 in HCT15 and SW403 cells and potently blocked proliferation of these cells, whereas ML241 was >10-fold less efficacious in both assays.
AAA 型 ATP 酶 p97 在维持真核细胞蛋白质稳态方面是一个关键因素，它通过促进蛋白酶体对泛素化蛋白质的降解以及自噬体的成熟发挥作用。该团队从美国国立卫生研究院分子文库小分子储存库（MLSMR）的高通量筛选（HTS）得到的一个活性命中物出发，开发了探针化合物 ML240 和 ML241，它们都能抑制 p97 ATP 酶活性，IC50 约为 100 nM，并且分别以约 900 nM 和 3500 nM 的 IC50 阻断 p97 依赖性蛋白酶体底物的降解。相比之下，这些探针化合物在阻断 p97 非依赖性底物氧依赖性降解结构域（ODD）-荧光素酶的降解方面效力要低得多（IC50>28 μM），这突出了它们对 p97 的特异性。这两种探针化合物在激活执行性半胱天冬酶和阻断细胞生长方面表现出明显不同的效力。ML240 在 HCT15 和 SW403 细胞中迅速诱导半胱天冬酶 3 和 7，并有效阻断这些细胞的增殖，而 ML241 在这两种测定中效力要低 10 倍以上。
