Development and application of a high-content virion display human GPCR array

Abstract

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli. However, GPCRs rely on membrane for proper folding, making their biochemical properties difficult to study. By displaying GPCRs in viral envelopes, we fabricated a Virion Display (VirD) array containing 315 non-olfactory human GPCRs for functional characterization. Using this array, we found that 10 of 20 anti-GPCR mAbs were ultra-specific. We further demonstrated that those failed in the mAb assays could recognize their canonical ligands, suggesting proper folding. Next, using two peptide ligands on the VirD-GPCR array, we identified expected interactions and novel interactions. Finally, we screened the array with group B Streptococcus , a major cause of neonatal meningitis, and demonstrated that inhibition of a newly identified target, CysLTR1, reduced bacterial penetration both in vitro and in vivo. We believe that the VirD-GPCR array holds great potential for high-throughput screening for small molecule drugs, affinity reagents, and ligand deorphanization.

Future perspective: high-throughput construction of new ultrasensitive cytokine and virion liquid chips for high-throughput screening (HTS) of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases

“…Because the molecular drug-receptor interactions on these recombinant virions will trigger charge distribution and electron transition to produce electrochemical signaling, IFNAR2 and IL10RA membrane proteins can be applied with detection of electrical signals for HTS of anti-inflammatory molecular drugs (Fig. 3) [1,6,14,20,21]. Different shear forces such as pulsating (PS) and oscillatory shear force (OS) can also be loaded to gain variable binding kinetics to characterize the drug activity (Fig.…”

“…Because the single-transmembrane protein IFNAR2 presents a very long extracellular domain (27-243aa), which is similar to CD4 and IFNAR2, Spike (14-1195aa) of 2019-nCoV and ACE2 (18-740aa) of alveolar epithelial cells II, they can be very easily incorporated into the envelope of HSV-1 under the glycoprotein B promotor of the HSV-1 genome to make the IFNAR2/ACE2/Spike-HSV-1 and GPCR recombinant virions (www.uniprot.org) [1,2,20,21,26]. Therefore, the IFNAR2/ACE2/Spike and GPCRrecombinant herpes virions (IFNAR2/ACE2/Spike-HSV-1 and GPCR-HSV-1) can be created through high-throughput construction to obtain a new virion solution electrochemical sensor for probing drug activity.…”

“…The applied electrochemical sensor is very sensitive for monitoring electron transition of IFNAR2/IL10RA/ACE2/Spike/GPCR-molecular drug interaction in a small area of the virion solution, which can be compared with the electrical signal of the negative control virions (wild-type virions without IFNAR2/IL10RA/ACE2/ Spike or GPCR incorporation) to gain electrical signaling of clinical diagnosis and to identify effective drugs (Fig. 6b) [2,9,10,21,[26][27][28][29][30][31][32].…”

“…In previous studies, recombinant purified virions were arrayed in a 384-well plate and spotted on FAST slides (Whatman) in a 4 × 4 pattern to fabricate the virion display (VirD) array in order to profile functional membrane proteins using the Cy5-labeled antibody, ligand and lectin to detect immunofluorescence intensity for characterization of their structure and activity ( Fig. 1) [2,20,21]. Next, virion nano-oscillators were made to allow us to measure mobility and charge changes associated with molecular binding strength on each recombinant virion, characterized by the variation in binding kinetics and amplitude (Fig.…”

“…[2,21]. Among the 1400 FDA-approved drugs, 475 (34%) drugs targeting 108 non-odorant GPCRs have been identified.…”