Time-resolved fluorescence resonance energy transfer as a versatile tool in the development of homogeneous cellular kinase assays.

Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

Optimization of a novel kinase inhibitor scaffold for the dual inhibition of JAK2 and FAK kinases.

The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.

Successful identification of glycine transporter inhibitors using an adaptation of a functional cell-based assay.

Glycine transporter (GlyT1) function is typically measured by radiolabeled glycine uptake using lysis methods or scintillation proximity assays (SPAs), which have limited throughput. This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of the 384-well format, standard laboratory automation, and cryopreserved CHO-K1 cells stably overexpressing human GlyT1a transporter (CHO-K1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, K(m), V(max), Z' factor analysis, and IC(50) value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 microM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater than 40% inhibition at 10 microM were considered as confirmed positives, yielding a 68% confirmation rate from the original screen. To eliminate compounds that nonspecifically inhibited glycine uptake, IC(50) values were determined in both GlyT1 and GlyT2 assays, and those compounds that inhibited GlyT2 were removed from consideration. The screening campaign identified 300 small molecules as selective GlyT1 inhibitors for lead optimization, demonstrating the utility of this cost-effective method.