Abstract
Fluorescent and luminescent probes are essential to both in vitro molecular assays and in vivo imaging techniques, and have been extensively used to measure biological function. However, little is known about the biological activity, thus potential interferences with the assay results, of these probe molecules. Here we show that D-luciferin, one of the most widely used bioluminescence substrates, is a partial agonist for G protein-coupled receptor-35 (GPR35). Label-free phenotypic profiling using dynamic mass redistribution (DMR) assays showed that D-luciferin led to a DMR signal in native HT-29 cells, whose characteristics are similar to those induced by known GPR35 agonists including zaprinast and pamoic acid. DMR assays further showed that D-luciferin is a partial agonist competitive to several known GPR35 agonists and antagonists. D-luciferin was found to cause the phosphorylation of ERK that was suppressed by known GPR35 antagonists, and also result in β-arrestin translocation signal but with low efficacy. These results not only suggest that D-luciferin is a partial agonist of GPR35, but also will evoke careful interpretation of biological data obtained using molecular and in vivo imaging assays when these probe molecules are used.
Highlights
With the advances in detection technology have expanded the applications of fluorescent and luminescent probe molecules for measuring a myriad of biological functions [1]
Multiple dynamic mass redistribution (DMR) assays were employed to characterize the pharmacology of D-luciferin in HT29 cells, a native cell line endogenously expressing G protein-coupled receptor-35 (GPR35) [18]
DMR antagonist assay was used to determine the specificity of the D-luciferin-induced DMR to the activation of endogenous GPR35 using SPB05142, a known GPR35 antagonist with moderate potency [19,20]
Summary
With the advances in detection technology have expanded the applications of fluorescent and luminescent probe molecules for measuring a myriad of biological functions [1]. Dominated in the target-centric basic research and drug discovery are molecular assays that measure a specific signaling pathway and/ or molecule to infer the functional consequences of drugs and molecules [2,3]. These molecular assays often use artificial systems and fluorescent and luminescent molecules, and are limited to a predetermined mechanism of action (MoA) by measuring a single signaling molecular species one at a time [4,5]. Label-free assays offer an integrated functional cellular response, so it is possible to cover a diverse range of pathways downstream a receptor [11], and to detect ligands of diverse MoAs for a target receptor [12]
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