Using NanoBit complementation and TR FRET to Study the Mechanism of Action of Small‐molecule Modulators of CXCR2
Authors: Nesheva Desislava Nikolaeva , Charlton Steven , Mistry Sheilesh , Holliday Nicholas 
Published: April 2020

The chemokine receptor CXCR2 is involved in inflammatory pathologies including asthma and COPD as well as cancer cell migration and growth
1. Blocking the chemokine‐binding site at the receptor is challenging due to the multi‐step binding of orthosteric ligands, therefore CXCR2 has been targeted by small‐molecule antagonists which bind at an intracellular binding pocket topographically close to the site of G protein coupling
2. Here we investigate the mechanism of action of structurally distinct intracellular CXCR2 modulators using NanoBit complementation in whole cells and time‐resolved Fluorescence resonance energy transfer (TR FRET) in membrane preparations.The N‐terminal SNAP‐tagged human CXCR2 cDNA was fused to the LgBit NanoLuc fragment at the C terminus. The SmBiT114 peptide was used as the N terminal tag for human β‐Arrestin2 or synthetic MiniGoA proteins and HEK293T cells were stably co‐transfected with both cDNAs in pcDNA3.1 (HEK CXCR2 114β‐Arrestin2 / 114Mini Go). HEK CXCR2 114β‐Arrestin2/114MiniGo cells grown to 70% confluence in 96‐well plates were pre‐treated with the relevant modulator at 37°C for 5/60 minutes prior to furimazine substrate loading. Luminescence was recorded every 2 minutes before and after agonist (CXCL8) additions using a BMG Pherastar plate reader. TR FRET experiments were performed in membrane preparations from HEK293T cells stably transfected with N‐terminal SNAP tag and C‐terminal histidine tag labelled with Lumi4‐Tb for 60 minutes at 37°C in LABMED buffer. Experiments were performed in 384‐well plates in duplicate by pre‐treating receptors with a range of competitor concentrations for 30 minutes following the treatment with fluorescently labelled tracer (AF647CXCL8 10 nM). Binding was measured at 1 – 5 h endpoints using a BMG Pherastar plate reader and HTRF settings.NanoBit complementation detected basal and CXCL8‐stimulated effector recruitment by CXCR2. A number of intracellular modulators tested produced mixed inhibition (rightward shifts of CXCL8 response and reduction of the maximal response) consistent with a non‐competitive mechanism of action with receptor reserve (figure
1). A fluorescent chemokine (AF647CXCL8) was identified as a CXCR2 agonist in whole cell assays, and its binding was also detected by TR FRET in membrane preparations (pKd=7.57 ± 0.31 in cells vs. 7.93 ± 0.14 in membranes, n≥3, error=S.E.M.). Unlabelled CXCL8 fully competed for AF647CXCL8 binding, but only partial inhibition of this binding was observed with the intracellular modulators tested.NanoBit complementation provides real‐time measure of modulator inhibition of CXCR2‐effector engagement. Combination with TR FRET analysis allows the correlation of these effects with the action of the modulators on chemokine binding affinity.Support or Funding InformationBiotechnology and Biological Sciences Research CouncilThe effect of SB265610 and AZ10397767 modulators on the basal and CXCL8 stimulated mini Go (top panel) and β‐Arrestin2 (bottom panel) recruitment by CXCR2 (n=5; Error bars = S.E.M.).Figure 1