Abstract
Here we report an efficient method to generate multiple co-structures of the A2A G protein-coupled receptor (GPCR) with small-molecules from a single preparation of a thermostabilised receptor crystallised in Lipidic Cubic Phase (LCP). Receptor crystallisation is achieved following purification using a low affinity “carrier” ligand (theophylline) and crystals are then soaked in solutions containing the desired (higher affinity) compounds. Complete datasets to high resolution can then be collected from single crystals and seven structures are reported here of which three are novel. The method significantly improves structural throughput for ligand screening using stabilised GPCRs, thereby actively driving Structure-Based Drug Discovery (SBDD).
Highlights
G protein-coupled receptor (GPCR) classes and are transformative from a pharmaceutical perspective, with several drug candidates generated by structure-based drug design (SBDD) techniques[5,6]
Co-crystal structures are obtainable even with low affinity compounds and fragments[17] identified in early stages of discovery projects. This provides a unique opportunity to apply soaking techniques, successfully utilised for soluble targets, to GPCR crystals grown in meso by lipidic cubic phase crystallisation (LCP)
We report an in meso crystal soaking method developed to improve the crystallographic throughput for our work with the adenosine A2A receptor (A2AR), including drug discovery activities
Summary
Protein preparations intended for soaking experiments were carried out in the presence of theophylline whereas the bespoke preparation of A2A-StaR2-bRIL562 in complex with Compound 4e was done in the presence of 5 μM ligand. X-ray diffraction data were measured on a Pilatus 6 M detector at beamline I24 (Diamond Light Source) using a 6 × 9 μm beam size of for crystals of A2A-StaR2-bRIL562 in complex with Compound 4e, Tozadenant or LUAA47070. Diffraction data for the A2A-StaR2-bRIL562-Vipadenant complex were acquired from 3 different crystals on an Eiger 16 M detector at beamline X06SA (Swiss Light Source) at a wavelength of 1 Å, using 10% beam transmission and 0.1° oscillation per frame, with an exposure of 1 second per degree of oscillation. Co-ordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 5OM1, 5OM4, 5OLZ, 5OLV, 5OLO, 5OLH and 5OLG
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