Objective

Structural and biophysical analyses of GPCRs are essential for modern drug discovery but have been limited by access to high quality purified receptor. Contemporary techniques that rely upon detergent solubilisation of membrane proteins are greatly hindered by the challenges, time and costs associated with the bespoke method development required for purification each receptor subtype. Domainex are exploring novel detergent-free strategies for GPCR purification to largely by-pass these problems and aim to develop a generic platform for biophysical GPCR fragment screening using microscale thermophoresis (MST) with purified receptor. Here we describe our strategy to apply styrene-maleic acid polymer (SMA) technology to solubilise and purify intact GPCRs in their native lipid environment, and present our data collect so far to optimise this system for fragment-based drug discovery. The wildtype human neurotensin receptor 1 (NTS1R) was used as a model system, a receptor with multiple neuroscience indications including Alzheimer’s disease and schizophrenia. SNAP/his dual-tagged NTS1R was expressed in mammalian HEK293 cells, and cell membranes solubilised with SMA polymer. Receptor was then purified using sequential nickel affinity purification and size exclusion chromatography, resulting in approximately 100 µg quantities of SMA-NTS1R with good purity. A mass spectrometry binding assay was used to validate binding of the SMA solubilised receptor to the known antagonist SR-142948, which confirmed purification of correctly folded and pharmacologically intact receptor. Our next steps are to investigate the application of MST technology for detecting compound binding against SMA purified NTS1R, and ultimately to run a fragment screen at this target, and others. In summary, we have begun to develop and validate a novel GPCR fragment screening technology by coupling SMA solubilised receptor to MST. We aim to expand this to develop a generic platform-based approach for GPCR biophysical screening, which is anticipated to greatly facilitate GPCR drug discovery.