Expanding the apelin receptor pharmacological toolbox using novel fluorescent ligands

Abstract

IntroductionThe apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on [Pyr1]apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647).MethodsFluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue.ResultsAll four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex.DiscussionOur data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.

Expression of the apelin receptor, a novel potential therapeutic target, and its endogenous ligands in diverse stem cell populations in human glioblastoma

“…For imaging and analysis, a protocol similar to that described in Williams et al, 2023 was used. Briefly, automated, high-resolution fluorescent or brightfield images (16-bit, 0.325 × 0.325 μm scaling per pixel) of immunohistochemically or H&E prepared tissue sections were acquired using an Axio Scan.Z1 slide scanner (ZEISS) with a Plan-Apochromat 20x/NA0.8 M27 objective lens, connected to a Hamamatsu Orca Flash camera.…”

Structural and functional determination of peptide versus small molecule ligand binding at the apelin receptor

“…Colours indicate the missense (V/L38 1.42 , blue; T/M89 2.64 , red; R/H168 4.64 , magenta) or frameshift (G/X45 1.49 , T/X227 To characterise apelin receptor variants in vitro, CHO-K1 cells were transiently transfected with constructs encoding variant or wild-type receptor tagged with an enhanced green uorescent protein (eGFP) reporter at the C-terminus. High content screening was used to qualitatively and quantitatively assess expression and uorescent ligand binding in whole cells using the recently characterised apelin647 and ELA647 uorescently labelled peptides 37 . Radioligand binding using [ 125 I]-apelin-13 was also performed to empirically determine speci c binding at receptor variants.…”

“…CHO-K1 cells transiently transfected with wild-type or variant apelin receptor construct tagged C-terminally with eGFP were plated at ~ 10,000/well in CellCarrier-96 Ultra Plates (PerkinElmer). On the day of experimentation, cells were washed with HBSS before treatment with well-validated apelin647 (300 nM) or ELA647 (1 µM) uorescent ligand 37 for 90 mins at room temperature, until equilibrium was reached. Non-speci c binding was determined in the presence of a saturating 10 µM concentration of unlabelled [Pyr 1 ]apelin-13.…”

“…Following acquisition, uorescence was quanti ed using in-built Harmony High Content Imaging and Analysis Software (PerkinElmer), as described previously 37 . Only cells that were positive for eGFP (i.e.…”

“…Radioligand binding studies were performed using [ 125 I]-apelin-13 ([Glp 65 ,Nle 75 ,Tyr 77 ][ 125 I]-apelin-13, PerkinElmer), using a protocol adapted from previously described protocols 10,30,37,57 . The radiolabel has a speci c activity of 2200 Ci/mmol.…”