MAP Kinase Kinase Kinase Assay Development for High Throughput Screening

Abstract

The goal of this work was to develop a novel mitogen-activated protein kinase (MAP K) cascade assay suitable for high-throughput screening (HTS). Three major steps were necessary for development and transfer of the assay. Firstly, the assay was developed and optimized at BASF Bioresearch Corporation, Worcester, USA. Within this step the kinase cascade, consisting of three kinases in a row, MAP KKK ® MAP KK ® MAP K, was established and optimized. The MAP KKK phosphorylates and activate the MAP KK, which in turn phosphorylates and activate the MAP K. As method for detecting the stage of phosphorylation homogeneous time resolved fluorescence (HTRF, Packard, USA) was chosen. Secondly, the screening campaign was organized, and the assay was transferred to the HTS group at Knoll AG, Ludwigshafen, Germany. Thirdly, the assay method was adapted to the conditions in the German laboratory and miniaturized from the 96-well to the 384-well format. To introduce the assigned biology-lab-technician, as well as the staff of the group in Germany which would later run the assay, a seminar was given. This seminar covered the theory as well as the practical applications of the assay. A further part of the work was to develop two different follow up assay systems. Follow up assays, has to determine whether a compound active in the primary screen is true or not. False negative and false positive compounds need to be sorted out by these assays. First, a scintillation proximity assay (SPA) setup was tested, and second, a splitted MAP kinase cascade was used. The splitted cascade was optimized and could be used for determination of the enzyme in the cascade which was actually inhibited.