SBIR-STTR Award

The analysis of protein function has conventionally been done by studying, in depth, a single protein. Recently several groups have initiated efforts to display proteins in two-dimensional array suitable for performing various types of binding studies or assays. In particular, Dr. Snyder's laboratory has developed a microwell format for protein display that has versatility and a range of applications that compare favorably with other formats. This format has been used to create arrays of essentially all yeast kinases and to study their specificity. In the present Phase I application we propose to extend this work by creating an array of 96 human protein kinase catalytic domains. To do this we will express domains in a baculovirus system suitable for multiplexing production, and demonstrate the utility of the microwell format by testing the activity and specificity of the kinases. We will also further decrease the size and increase the density of microwells in arrays, in preparation for preparation of large-scale protein arrays.

Thesaurus Terms:
catalyst, microarray technology, protein kinase, technology /technique development Baculoviridae, chronic myelogenous leukemia

Protein kinases are central regulators of intracellular responses and constitute the second largest drug target class. We have developed a microwell format for displaying immobilized proteins in an aqueous medium such that separate isolated reactions can be carried out with many different proteins, substrates, or inhibitors, or binding agents in low volumes on a single microscope slide. In Phase I, we produced significant numbers of active kinases and demonstrated 20 nanoliter volume kinase assays in these microwells. In this Phase II application, we propose to extend this procedure to prepare microwell arrays of essentially the entire complement of human protein kinases and protein kinase catalytic domains, and to develop these arrays for quantitative and comprehensive inhibition assays. We will demonstrate the utility of the arrays for inhibitor specificity profiling by testing the array against a panel of known kinase inhibitors, and will perform functional characterizations of each kinase present in the array. Comprehensive kinase inhibition profiling will significantly enhance the effectiveness and efficiency of kinase drug development.

Thesaurus Terms:
microarray technology, protein kinase, technology /technique development catalyst, drug design /synthesis /production, protein kinase inhibitor