Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, VIC, Australia.
Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, 5651 GG Eindhoven, the Netherlands.
Structure. 2021 Sep 2;29(9):963-974.e6. doi: 10.1016/j.str.2021.04.008. Epub 2021 May 5.
G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in stabilization of GPCR:transducer complexes, together with improvements in cryoelectron microscopy (cryo-EM) have recently been applied to structure-assisted drug design for GPCR agonists. Nonetheless, limitations in the commercial application of these approaches, including the use of nanobody 35 (Nb35) to aid complex stabilization and the high cost of 300 kV imaging, have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated the formation of stable complexes with a modified Gs protein in the absence of Nb35. In parallel, we compare 200 versus 300 kV image acquisition using a Falcon 4 or K3 direct electron detector. Moreover, the 200 kV Glacios-Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.
G 蛋白偶联受体（GPCRs）是细胞表面药物靶点中最大的一类。GPCR：转导复合物的稳定化技术的进步，加上冷冻电镜（cryo-EM）的改进，最近已被应用于 GPCR 激动剂的基于结构的药物设计。尽管如此，这些方法在商业应用中的局限性，包括使用纳米抗体 35（Nb35）来辅助复合物稳定化以及 300kV 成像的高成本，限制了 cryo-EM 在药物发现中的广泛应用。在这里，我们使用 PF 06882961 结合的 GLP-1R 作为范例，验证了在没有 Nb35 的情况下与修饰的 Gs 蛋白形成稳定复合物的形成。同时，我们比较了使用 Falcon 4 或 K3 直接电子探测器进行 200kV 与 300kV 的图像采集。此外，200kV 的 Glacios-Falcon 4 获得了一个 3.2Å 的图谱，其中包含结合药物和多个结构有序的水分子的清晰密度。我们的工作为 cryo-EM 在 GPCR 药物发现中的更广泛商业应用铺平了道路。
