Design, synthesis and evaluation of a diazirine photoaffinity probe for ligand-based receptor capture targeting G protein-coupled receptors

Müskens, F. M., Ward, R. J., Herkt, D., van de Langemheen, H., Tobin, A. B. , Liskamp, R. M.J. and Milligan, G. (2019) Design, synthesis and evaluation of a diazirine photoaffinity probe for ligand-based receptor capture targeting G protein-coupled receptors. Molecular Pharmacology, 95(2), pp. 196-209. (doi: 10.1124/mol.118.114249) (PMID:30514721) (PMCID:PMC6324650)

Abstract

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity however, identifying trans-membrane receptors remains challenging. A new tri-functional probe to aid the non-biased identification of such receptors was developed and synthesized using a convenient 7-step synthesis. This probe contained three functional groups: (1) an NHS-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 NK1 receptor, expressed in an inducible manner using the Flp-InTM T-RExTM system, and the peptidic ligand substance P was employed 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 LC-MS/MS, resulting in the successful identification only of the NK1 receptor. This provides proof-of-concept towards general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

• Item Type: Articles
• Additional Information: F.M.M. thanks the University of Glasgow for a Lord Kelvin-Adam Smith studentship.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Liskamp, Professor Robert and Ward, Dr Richard and Muskens, Frederike and Milligan, Professor Graeme and Tobin, Andrew and Herkt, Dominik and Van De Langemheen, Mr Helmus
• Authors: Müskens, F. M., Ward, R. J., Herkt, D., van de Langemheen, H., Tobin, A. B., Liskamp, R. M.J., and Milligan, G.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences; College of Science and Engineering > School of Chemistry
• Journal Name: Molecular Pharmacology
• Publisher: American Society for Pharmacology and Experimental Therapeutics
• ISSN: 0026-895X
• ISSN (Online): 1521-0111
• Copyright Holders: Copyright © 2019 The Authors
• First Published: First published in Molecular Pharmacology 95:196-209
• Publisher Policy: Reproduced under a Creative Commons License