Abstract

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 needed for
purification each receptor subtype. Domainex are developing a platform approach
to GPCR purification and biophysical screening, enabled by novel detergent-free
strategies that alleviate these issues. Polymers such as styrene maleic acid
(SMA) have been used to this end. These solubilise cellular membranes and
encapsulate membrane proteins that continually remain in their original native
lipid environment, forming polymer lipid particles (PoLiPas). Thus, the
receptor is presented in its folded state in solution and can be purified using
conventional techniques. Here, we showcase our system for rapid and generic
preparation of high quality correctly folded PoLiPa-GPCR proteins, using the
human neurotensin 1 (NTR1) and β2-adrenergic (β2AR) expressed in human cells as
case studies. We also describe the development of a novel ligand observed liquid
chromatography mass spectrometry (LC-MS) assay to detect and quantify binders
at the SMALP-GPCRs, and apply traditional pharmacological analyses. Saturation
binding studies demonstrated high affinity specific binding with low
non-specific binding to small molecule antagonists of the receptors, thus
pharmacologically validating the purified protein. Furthermore, a panel of
agonists and antagonists were shown to competitive displace the tracer ligands,
further confirming known receptor pharmacology. In summary, we have optimised systems
that can rapidly generate detergent-free purified GPCRs, and have developed
tools to pharmacologically validate these. This will open up many exciting
opportunities for GPCR drug discovery.