Label-free drug screening assay multiplexed with an orthogonal time-resolved fluorescence labeled assay

Cell-based assays against cell surface receptor targets are essential in vitro models of target-based drug discovery. At the lead generation phase large-scale functional screening assays monitoring individual cellular readouts detect interactions between the compounds and the predefined pathways but might lack sufficient sensitivity owing to the complexity of downstream signaling pathways. Cellular label-free assays offer advantages over labeled detection approaches as they reflect whole-cell responses without the prerequisite of detecting only a single cellular analyte and introducing additional genetic manipulations in favor of the chosen detection method. The combination of a label-free assay and labeled assays might integrate the advantageous characteristics of both approaches with regards to added pharmacological information and a bigger pool of chemical starting material. Here we report multiplexing of dynamic mass redistribution label-free technology with HTRF-based cAMP detection on an alpha2c adrenergic receptor expressing cell line. Besides describing the challenging assay development work associated with the set goal, a pilot screening campaign on ca. 1600 compounds is also presented. The combined assay demonstrated the ability to detect relevant activities in both readouts. Interpretation of the results as well as an outlook for further possible opportunities and applications are also discussed. •Label-free DMR assay can be miniaturized.•HTRF cAMP assay is feasible on adherent cells in special label-free DMR plates.•Label-free DMR and the HTRF assay can be combined in a miniaturized duplex HTS assay.•This assay enables to detect the broadest repertoire of signaling events.•No need for genetic manipulation facilitating duplex HTS on more native cell systems.