Assay Development and Screening of a Serine/Threonine Kinase in an On-Chip Mode Using Caliper Nanofluidics Technology

Kinases are key targets for drug discovery. In the field of screening in general and especially in the kinase area, because of considerations of efficiency and cost, radioactivity-based assays tend to be replaced by alternative, mostly fluorescence-based, assays. Today, the limiting factor is rarely the number of data points that can be obtained but rather the quality of the data, enzyme availability, and cost. In this article, the authors describe the development of an assay for a kinase screen based on the electrophoretic separation of fluorescent product and substrate using a Caliper-based nanofluidics environment in on-chip incubation mode. The authors present the results of screening a focused set of 32,000 compounds together with confirmation data obtained in a filtration assay. In addition, they have made a small-scale comparison between the on-chip and off-chip nanofluidics screening modes. In their hands, the screen in on-chip mode is characterized by high precision most likely due to the absence of liquid pipetting; an excellent confirmation rate (62%) in an independent assay format, namely, filtration; and good sensitivity. This study led to the identification of 4 novel chemical series of inhibitors.