Quantifying Target Occupancy of Small Molecules Within Living Cells.

The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

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 new principle for rapid immunoassay of proteins based on in situ precipitate-enhanced ellipsometry.

A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10-20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.

Effect of clofibric acid on the turnover of the fatty acid-binding protein identified in cultured endothelial cells from bovine aorta.

Several types of fatty acid-binding proteins are found in mammalian cells. Cultured endothelial cells from bovine aorta were shown to contain exclusively the cardiac-type fatty acid-binding protein (cFABP) with a mean concentration of 90 ng cFABP/mg extract protein. Only small variations were observed from passage to passage. In pulse-chase labeling experiments with L-[35S]methionine, a half-life of 4.0 d was measured for cFABP which is about two times longer than the average half-life of the extracted proteins. These data imply that in aortic endothelial cells cFABP is not subject to short-term regulation. However, addition of clofibric acid to the culture medium led to a shortening of the half-life of cFABP, which was compensated for by an increase in its biosynthesis. The turnover of the bulk of extract proteins remained unchanged when the cells were challenged with clofibric acid.