Development of a Novel SPR Assay to Study CXCR4-Ligand Interactions.

Anneleen Hout,Kalyan Das,Tom Loy,Dominique Schols,Sam Noppen,Arnaud Boonen,Abhimanyu Singh

doi:10.3390/bios10100150

Anneleen Hout, Kalyan Das + Show 5 more

Open Access

https://doi.org/10.3390/bios10100150

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Journal: Biosensors	Publication Date: Oct 21, 2020
Citations: 8	License type: CC BY 4.0

Affiliation: Rega Institute for Medical Research, KU Leuven

Abstract

G protein-coupled receptors (GPCRs) are involved in a plethora of different diseases. Consequently, these proteins are considered as an important class of drug targets. Measuring detailed kinetic information on these types of proteins has been challenging. Surface plasmon resonance (SPR) can provide this information, however, the use of SPR on GPCRs remains a complex issue. Here, we report an SPR assay to investigate the interactions between the full-length chemokine receptor CXCR4 and nanobody-Fc (Nb-Fc) ligands. Nb-Fcs consist of two monovalent VHH domains fused with an Fc domain of a human IgG molecule. The CXCR4 protein used in this assay was produced with a C-terminal 10x-histidine tag and was immobilized on a nitrilotriacetic acid chip. In order to verify the sensitivity and effectiveness of this assay, the results were compared to data obtained from cellular assays as well as from another SPR assay using CXCR4 virus-like particles (VLPs). CXCR4 remained intact and stable for at least 12 h, and the kinetic results correlated well with both the cellular assays and the VLP SPR assay results. Apart from determining the binding kinetics of Nb-Fc with CXCR4, our results contributed to understanding CXCR4 interaction dynamics. In conclusion, this assay provides a viable experimental platform that has high potential to be expanded for studying other molecules as well as other histidine-tagged GPCRs.

Highlights

G protein-coupled receptors (GPCRs) are cell membrane receptors representing a very important class of drug targets
About 34% of FDA-approved drugs target GPCRs [1]. Due to their therapeutic significance, GPCR research is a major subject in medical research and drug discovery
The CXC chemokine receptor 4 (CXCR4) is a GPCR that interacts with the CXC motif chemokine

Summary

Introduction

G protein-coupled receptors (GPCRs) are cell membrane receptors representing a very important class of drug targets. About 34% of FDA-approved drugs target GPCRs [1]. Due to their therapeutic significance, GPCR research is a major subject in medical research and drug discovery. The CXC chemokine receptor 4 (CXCR4) is a GPCR that interacts with the CXC motif chemokine. Several isoforms of CXCR4 exist, which differ in the amino acid composition of their N-termini [2,3]. CXCR4 has a calculated molecular weight of ~40 kDa, which can vary due to different glycosylation patterns and post-translational modifications [4]. Studies with genetic knockouts of both CXCR4 and CXCL12 underscored the vital role of this signaling axis during embryogenesis [6,7,8]

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