Label-free LC-MS based assay to characterize small molecule compound binding to cells.

Study of small molecule binding to live cells provides important information on the characterization of ligands pharmacologically. Here we developed and validated a label-free, liquid chromatography-mass spectrometry (LC-MS) based cell binding assay, using centrifugation to separate binders from non-binders. This assay was applied to various target classes, with particular emphasis on those for which protein-based binding assay can be difficult to achieve. In one example, to study a G protein coupled receptor (GPCR), we used one antagonist as probe and multiple other antagonists as competitor ligands. Binding of the probe was confirmed to be specific and saturable, reaching a fast equilibrium. Competition binding analysis by titration of five known ligands suggested a good correlation with their inhibition potency. In another example, this assay was applied to an ion channel target with its agonists, of which the determined binding affinity was consistent with functional assays. This versatile method allows quantitative characterization of ligand binding to cell surface expressed targets in a physiologically relevant environment.

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Déclaration de conflit d'intérêts

Declaration of Conflicting Interests The authors have declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All authors are, or were, employed by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and their research and authorship of this article was completed within the scope of their employment. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.