Development and Application of a High-Content Virion Display Human GPCR Array

Abstract

G protein-coupled receptors (GPCRs) comprise the largest membrane protein family in humans and can respond to a wide variety of ligands and stimuli. Like other multi-pass membrane proteins, the biochemical properties of GPCRs are notoriously difficult to study because they must be embedded in lipid bilayers to maintain their native conformation and function. To enable an unbiased, high-throughput platform to profile biochemical activities of GPCRs in native conformation, we individually displayed 315 human non-odorant GPCRs (>85% coverage) in the envelope of human herpes simplex virus-1 and immobilized on glass to form a high-content Virion Display (VirD) array. Using this array, we found that 50% of the tested commercial anti-GPCR antibodies (mAbs) is ultra-specific, and that the vast majority of those VirD-GPCRs, which failed to be recognized by the commercial mAbs, could bind to their canonical ligands, indicating that they were folded correctly. Next, we used the VirD-GPCR arrays to examine binding specificity of two known peptide ligands and recovered expected interactions, as well as new off-target interactions, three of which were confirmed with real-time kinetics measurements. Finally, we explored the possibility of discovering novel pathogen targets by probing VirD-GPCR arrays with live group B Streptococcus (GBS), a commonGram-positive bacterium causing neonatal meningitis. Using cell invasion assays and a mouse model of hematogenous meningitis, we showed that inhibition of one of the five newly identified GPCRs, CysLTR1, greatly reduced GBS penetration in brain-derived endothelial cells and in mouse brains. Therefore, our work demonstrated that the VirD-GPCR array holds great potential for high-throughput, unbiased screening for small molecule drugs, affinity reagents, and deorphanization.

Therapeutic development of group B Streptococcus meningitis by targeting a host cell signaling network involving EGFR

“…Administration of montelukast (5 mg/kg, a dose which exhibits CysLT1 antagonist activity in mice) (Zhu et al, 2010) intraperitoneally 1 h before intravenous injection of GBS significantly inhibited GBS penetration of the brain of BALB/c mice, as shown by significantly decreased bacterial counts recovered from the brains of the recipients of montelukast compared to those of vehicle control ( Fig EV3E). This finding is consistent with that of a previous report demonstrating that montelukast inhibited GBS penetration into the brain (Syu et al, 2019). The contribution of CysLT1 to GBS penetration into the brain was verified in CysLT1 knockout animals, as the bacterial counts recovered from the brain were significantly less in CysLT1 À/À mice than in the wild-type animals ( Fig 3F).…”