Development of a GPCR-binding ligand screening method using real-time interaction analysis on live cells.

G protein-coupled receptors (GPCRs) affect many physiological processes and frequently used as drug targets and biomarkers. The present study focuses on the neurokinin 1 receptor (NK1-R) which is over-expressed on neural tumours with the aim to develop high affinity binders to NK1-R for theranostic purposes. 60 Ligands were synthesized, all containing iodine enabling isotopic exchange with radioactive iodine for imaging or radiotherapeutic applications. Embedded in the cellular membrane, GPCRs adopt various conformational states that can change upon interaction with ligands. Therefore, a time-resolved, live cell assay was developed to evaluate competition with fluorescently labelled endogenous NK1-R ligand, Substance P (FAM-SP). The time resolved assay enabled quantification of the efficiency in which the ligands compete for the rapid and slow releasing NK1-R binding modes of FAM-SP. Of the 60 ligands, 25 were able to displace FAM-SP faster than the anti-nausea drug aprepitant. Two of these ligands displaced the rapid releasing FAM-SP fraction faster than Substance P (SP) and one of them also replaced the slow releasing fraction faster than aprepitant. To validate the assay, the affinity of two ligands for several NK1-R expressing cell lines was further quantified; one that had similar competition characteristics as SP and one that behaved as aprepitant. The ligand with similar competition characteristics as SP displayed a low nM to sub-pM affinity with a stronger binding correlating with higher receptor expression levels. This high affinity makes it a highly promising candidate for further development into a theranostic agent to address neural tumours.