title: Design, synthesis and evaluation of a molecular probe for ligand-based receptor capture targeting membrane receptors
Creators: name: Müskens, Frederike Maximiliane
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Molecular probe, GPCR, photoaffinity.

description: Membrane proteins are vital to drug discovery, being targeted by some 60% of the currently marketed therapeutic medicines, with more than half of those targeting transmembrane receptors. Identification of transmembrane receptor targets of poorly characterised ligands can provide new starting points for drug innovation, provide valuable information about off-target effects, and enhance mechanistic understanding of molecular pathways. Whereas, over the years, various methods for target identification have been developed, due to unfavourable characteristics, such as hydrophobicity, low abundance and transient ligand-interactions, identification of transmembrane proteins remains a challenge. Described herein is the design, synthesis and evaluation of four universal, trifunctional probes specifically developed to allow the covalent capture of transmembrane receptors in a process called ligand-based receptor capture (LRC). These probes contain three functional groups: (1) a ligand-coupling moiety; (2) a receptor-capturing moiety; (3) and an affinity tag. In an LRC experiment these probes would be coupled to the ligand of interest, after which the adduct would be added to cells believed to express the target receptor(s) to allow receptor-capturing. After affinity purification, captured receptors would be identified using mass spectrometry. All four probes contained an N-hydroxysuccinimide (NHS)-ester to allow ligand-coupling through free amines. For receptor capture, both a protected hydrazine moiety and the photoreactive groups benzophenone and diazirine were investigated. Protected hydrazine moieties will couple to aldehydes, present on sugar tails of glycosylated receptors after mild oxidation, whereas photoreactive groups will form covalent bonds with molecules in close proximity upon activation with UV-light. For affinity purification, probes either contained a biotin group, for purification using streptavidin, or an alkyne moiety, which would allow coupling to any reporter or affinity tag bearing an azide group using the copper-catalyzed azide-alkyne cycloaddition. The interactions between the two peptidic ligands, orexin A and substance P (SP), and their respective G-protein coupled receptors orexin 1 and neurokinin 1 (NK1), expressed in an inducible manner using the Flp-InTM T-RExTM system, were employed as test systems. Initially, these systems were used to investigate individual steps in the LRC protocol, including ligand-coupling, potential interference of the probes on the ligand-receptor interaction, and ability of the probes to covalently couple to the receptor. Only for the probe containing an NHS-ester, a diazirine moiety and a biotin group, could capture of the target receptor be demonstrated. This probe was then coupled to SP and used in a full LRC experiment to successfully identify NK1 as the only SP-binding receptor. This provides a proof of concept, demonstrating that this novel probe could be used as a general tool to help identify target receptors for a variety of ligands in the near future.