Vlahakis Niko W, Flowers Cameron W, Liu Mengting, Agdanowski Matthew P, Johnson Samuel, Summers Jacob A, Jacobs Lian M C, Keyser Catherine, Russell Phoebe, Rose Samuel L, Orlans Julien, Adhami Nima, Chen Yu, Sawaya Michael R, Basu Shibom, de Sanctis Daniele, Chen Yu, Wakatsuki Soichi, Nelson Hosea M, Loo Joseph A, Tang Yi, Rodriguez Jose A
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095.
Department of Energy Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095.
Proc Natl Acad Sci U S A. 2025 Aug 5;122(31):e2503780122. doi: 10.1073/pnas.2503780122. Epub 2025 Jul 28.
With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach, which we term electron diffraction with native mass spectrometry (ED-MS), allows assignment of protein target structures bound to ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors and crude biosynthetic reactions. This extends to libraries of printed ligands dispensed directly onto TEM grids for later soaking with microcrystal slurries, and complexes with noncovalent ligands. ED-MS resolves structures of the natural product, epoxide-based cysteine protease inhibitor E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. It further identifies papain binding to its preferred natural products, by showing that two analogs of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analog from crude biosynthetic reactions, without purification. ED-MS also resolves binding of the CTX-M-14 β-lactamase, a target of active drug development, to the non-β-lactam inhibitor, avibactam, alone or in a cocktail of unrelated compounds. These results illustrate the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets, promising utility for drug discovery.
为了加速小分子 - 蛋白质复合物的发现，我们利用快速、低剂量、基于事件的电子计数微晶电子衍射（MicroED）数据采集和天然质谱技术。我们将这种方法称为带天然质谱的电子衍射（ED-MS），它能够通过从用已知抑制剂混合物和粗生物合成反应浸泡过的晶体浆液中获得的数据，确定与配体结合的蛋白质靶标结构。这一方法还扩展到直接分配到透射电子显微镜（TEM）网格上的印刷配体库，以便随后用微晶浆液浸泡，以及与非共价配体形成的复合物。ED-MS解析了天然产物环氧基半胱氨酸蛋白酶抑制剂E-64及其与模型半胱氨酸蛋白酶木瓜蛋白酶结合的生物合成类似物的结构。它还通过表明E-64的两种类似物在与木瓜蛋白酶晶体结合方面比其他类似物更具竞争力，以及检测到木瓜蛋白酶与E-64和来自粗生物合成反应的一种类似物结合（无需纯化），进一步确定了木瓜蛋白酶与其优选天然产物的结合。ED-MS还解析了活性药物开发的靶点CTX-M-14β-内酰胺酶与非β-内酰胺抑制剂阿维巴坦单独或在无关化合物混合物中的结合情况。这些结果说明了ED-MS在天然产物配体发现以及基于结构筛选小分子与大分子靶标结合物方面的实用性，有望在药物发现中发挥作用。
