Kojima Hiroyuki, Yanagi Ryota, Higuchi Eri, Yoshizawa Mami, Shimodaira Tomoyuki, Kumagai Misaki, Kyoya Tatsuhiro, Sekine Miyu, Egawa Daichi, Ohashi Nami, Ishida Hiroaki, Yamamoto Keiko, Itoh Toshimasa
Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan.
J Med Chem. 2023 Apr 13;66(7):4827-4839. doi: 10.1021/acs.jmedchem.2c01986. Epub 2023 Mar 30.
Covalent ligands are generally filtered out of chemical libraries used for high-throughput screening, because electrophilic functional groups are considered to be pan-assay interference compounds (PAINS). Therefore, screening strategies that can distinguish true covalent ligands from PAINS are required. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for evaluating protein stability. Here, we report a covalent modifier screening approach using HDX-MS. In this study, HDX-MS was used to classify peroxisome proliferator-activated receptor γ (PPARγ) and vitamin D receptor ligands. HDX-MS could discriminate the strength of ligand-protein interactions. Our HDX-MS screening method identified LT175 and nTZDpa, which can bind concurrently to the PPARγ ligand-binding domain (PPARγ-LBD) with synergistic activation. Furthermore, iodoacetic acid was identified as a novel covalent modifier that stabilizes the PPARγ-LBD.
共价配体通常会从用于高通量筛选的化学文库中被筛选掉，因为亲电官能团被认为是泛分析干扰化合物（PAINS）。因此，需要能够区分真正的共价配体与PAINS的筛选策略。氢/氘交换质谱（HDX-MS）是评估蛋白质稳定性的强大工具。在此，我们报告一种使用HDX-MS的共价修饰剂筛选方法。在本研究中，HDX-MS被用于对过氧化物酶体增殖物激活受体γ（PPARγ）和维生素D受体配体进行分类。HDX-MS能够区分配体与蛋白质相互作用的强度。我们的HDX-MS筛选方法鉴定出LT175和nTZDpa，它们能够协同激活并同时结合到PPARγ配体结合域（PPARγ-LBD）。此外，碘乙酸被鉴定为一种可稳定PPARγ-LBD的新型共价修饰剂。
