Chemical Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Biochemistry and Molecular Biology Program, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA.
Pfizer Worldwide Research and Development, Neuroscience Medicinal Chemistry, Groton, CT 06340, USA.
Cell Chem Biol. 2017 Jan 19;24(1):3-8. doi: 10.1016/j.chembiol.2016.12.006. Epub 2017 Jan 5.
γ-Secretase, a four-subunit transmembrane aspartic proteinase, is a highly valued drug target in Alzheimer's disease and cancer. Despite significant progress in structural studies, the respective molecular mechanisms and binding modes of γ-secretase inhibitors (GSIs) and modulators (GSMs) remain uncertain. Here, we developed biotinylated cleavable-linker photoprobes based on the BMS-708163 GSI to study its interaction with γ-secretase. Comparison of four cleavable linkers indicated that the hydrazine-labile N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker was cleaved most efficiently to release photolabeled and affinity-captured presenilin-1 (PS1), the catalytic subunit of γ-secretase. Peptide mapping showed that the BMS-708163-based probe photoinserted at L282 of PS1. This insertion site was consistent with the results of molecular dynamics simulations of the γ-secretase complex with inhibitor. Taken together, this work reveals the binding site of a GSI and offers insights into the mechanism of action of this class of inhibitors.
γ-分泌酶，一种由四个亚基组成的跨膜天冬氨酸蛋白酶，是阿尔茨海默病和癌症的一个极具价值的药物靶点。尽管在结构研究方面取得了重大进展，但 γ-分泌酶抑制剂（GSIs）和调节剂（GSMs）的各自分子机制和结合模式仍不确定。在这里，我们基于 BMS-708163 GSI 开发了生物素化可切割连接体光探针，以研究其与 γ-分泌酶的相互作用。对四种可切割连接体的比较表明，肼不稳定的 N-1-(4,4-二甲基-2,6-二氧环己基亚甲基)乙基（Dde）连接体的切割效率最高，可释放光标记和亲和捕获的 γ-分泌酶的催化亚基早老素-1（PS1）。肽图谱显示，基于 BMS-708163 的探针在 PS1 的 L282 处光插入。该插入位点与含有抑制剂的 γ-分泌酶复合物的分子动力学模拟结果一致。总之，这项工作揭示了 GSI 的结合位点，并为这类抑制剂的作用机制提供了深入的了解。
