A High-Throughput BRET Cellular Target Engagement Assay Links Biochemical to Cellular Activity for Bruton's Tyrosine Kinase.

Protein kinases are intensely studied mediators of cellular signaling. While traditional biochemical screens are capable of identifying compounds that modulate kinase activity, these assays are limited in their capability of predicting compound behavior in a cellular environment. Here, we aim to bridge target engagement and compound-cellular phenotypic behavior by utilizing a bioluminescence resonance energy transfer (BRET) assay to characterize target occupancy within living cells for Bruton's tyrosine kinase (BTK). Using a diverse chemical set of BTK inhibitors, we determine intracellular engagement affinity profiles and successfully correlate these measurements with BTK cellular functional readouts. In addition, we leveraged the kinetic capability of this technology to gain insight into in-cell target residence time and the duration of target engagement, and to explore a structural hypothesis.

A scintillation proximity assay for rna detection.

A homogeneous scintillation proximity assay (SPA) for detection of RNA transcripts is described. 3H-labeled RNA transcripts are hybridized in solution to biotinylated oligodeoxynucleotides (ODNs), which are then bound by streptavidin-coated, scintillant-embedded beads. Only bound 3H-labeled RNA transcripts are brought in close enough proximity to stimulate light emission from the beads. The results from this novel homogeneous assay correlated well with those obtained using the traditional filter-binding methods to measure RNA polymerase activity. The assay has been miniaturized to a 384-well format compatible with automated high-throughput screening. This SPA method has also been successfully used to probe RNA-accessible sites to hybridization, and thus should provide a useful tool for selecting effective antisense ODNs in antisense research.

Selective inhibition of bacterial dihydroorotate dehydrogenases by thiadiazolidinediones.

Dihydroorotate dehydrogenase is a critical enzyme of de novo pyrimidine biosynthesis in prokaryotic and eukaryotic cells. Differences in the primary structure of the enzymes from Gram-positive and -negative bacteria and from mammals indicate significant structural divergence among these enzymes. We have identified a class of small molecules, the thiadiazolidinediones, that inhibit prototypical enzymes from Gram-positive and -negative bacteria, but are inactive against the human enzyme. The most potent compound in our collection functioned as a time-dependent irreversible inactivator of the bacterial enzymes with k(inact)/K(i) values of 48 and 500 M(-1) sec(-1) for the enzymes from Escherichia coli and Enterococcus faecalis, respectively. The data presented here indicate that it is possible to inhibit prokaryotic dihydroorotate dehydrogenases selectively while sparing the mammalian enzyme. Thus, this enzyme may represent a valuable target for the development of novel antibiotic compounds.

Quantitative, high-throughput cell-based assays for inhibitors of trkA receptor.

Two quantitative, high-throughput cell-based assays for evaluating inhibitors of NGF-stimulated trkA phosphorylation in trkA-transfected NIH3T3 cells have been established. Both assays involve capture of the trkA receptor from cell lysates in microtiter plates coated with an anti-trk antibody. The amount of trkA phosphorylation is then measured using either an anti-phosphotyrosine antibody with a colorimetric readout or a lanthanide (europium)-labeled anti-phosphotyrosine antibody with a fluorometric detection. The two assay formats exhibited at least a fivefold increase in phosphorylated trkA signal in trkA-transfected cells compared to vector control. Inhibition plots generated for trkA kinase inhibitors using the two detection systems yielded comparable IC(50) values. Overall, the two assays represent a marked improvement over the standard gel-based/western blot method in terms of throughput, quantitation, and amenability to automation.