Evolving cryo-EM structural approaches for GPCR drug discovery

Abstract

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted 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.

Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations

“…Moreover, these proteins were originally designed to stabilize GPCRs in their active state and thus to enable the elucidation of GPCR–G protein complexes by X-ray and cryo-electron microscopy (cryo-EM), which has been achieved for the adenosine A 2A (A 2A R) [ 53 ], the dopamine D 1 (D 1 R) [ 54 ], the GPR52 [ 55 ], the serotonin 5-HT 1B (5-HT 1B R) [ 56 ], and 5-HT 2A (5-HT 2A R) [ 57 ] receptors so far. Recent years have seen remarkable advances in the structural determination of GPCR–G protein complexes [ 58 , 59 ]. In 2020 alone, new structures of 34 GPCR–Gs complexes, 19 GPCR-Gi/o complexes, and one GPCR-Gq/11 complex were published ( , access date 8 October 2021 [ 58 ]), providing valuable atomic-level insights into the binding interface of GPCRs with G proteins from different major families.…”

Thermo Scientific™ Glacios Cryo-TEM: A Versatile 200 kV Tool for Structure-Based Drug Discovery

“…A two-day data collection using the Falcon 4 Detector operated in Electron Event Representation mode yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters (Figure 2). Excitingly, while the global resolution was 0.2-0.4 Å lower compared to structures obtained from KriosCryo-TEM, the binding pocket density was effectively identical to that obtained from 300 kV data, demonstrating the potential utility of 200 kV cryo-EM for drug target structure determination [6].…”