Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein–Coupled Receptors

Abstract

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein–coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography–mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P–NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P–binding receptor via liquid chromatography–tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

Identification of binding sites for ivacaftor on the cystic fibrosis transmembrane conductance regulator

“…For example, binding sites for ligands for GABA A receptors ( Hall et al., 2010 ), GPCRs, the A2A receptor and NK1 receptor have recently been identified using the photoaffinity label approach. As in the current study, attached affinity (i.e., biotin) tags confirmed binding and facilitate isolation of labeled proteins ( Muranaka et al., 2017 ; Muskens et al., 2019 ).…”

“…Multiple allosteric binding sites for modulatory ligands have been identified for GPCR membrane proteins ( Muranaka et al., 2017 ; Muskens et al., 2019 ). In these cases, primary and secondary binding events have been shown to modulate long range conformational changes.…”

Structure–function relationships in aryl diazirines reveal optimal design features to maximize C–H insertion

“…Trifluoromethyl aryl diazirines are now used in a host of biological target identification experiments (Fig. 1c) 3,[21][22][23][24][25] where they are found to outperform alternative labeling groups like nitrenes or benzophenones, 26,27 and the TPD motif has been genetically encoded into protein structures using expanded genome techniques. 28 TPD has also been used to map the localized protein environment on T cells using an innovative µmapping technique facilitated by Dexter energy transfer, 13 and trifluoromethyl aryl diazirines have been designed into bioadhesives to expedite wound closure.…”

How Physiologic Targets Can Be Distinguished from Drug-Binding Proteins

“…Three stages may be described for this challenging task. First, drug-binding proteins can be identified using one of many strategies including affinity chromatography and photoaffinity labeling (Campos et al, 1996;Das, 2019;Muskens et al, 2019;Tulloch et al, 2017;Wang et al, 2012; Xiao and Li, 2020). Second, molecular effects of a drug can be documented in molecular studies after perturbing cells with drug concentrations that are not toxic (CEC 25 concentration, for example (Bachovchin et al, 2019)).…”