ASSAY and Drug Development Technologies Volume 2, Number 4, 2004 © Mary Ann Liebert, Inc. Technology Review Rapid Structure-Activity and Selectivity Analysis of Kinase Inhibitors by BioMAP Analysis in Complex Human Primary Cell-Based Models Eric J. Kunkel, 1 Ivan Plavec, 1 Dat Nguyen, 1 Jennifer Melrose, 1 Elen S. Rosler, 1 Leon T. Kao, 1 Yuker Wang, 1 Evangelos Hytopoulos, 1 Anthony C. Bishop, 2 Raynard Bateman, 2 Kevan M. Shokat, 2 Eugene C. Butcher, 3, * and Ellen L. Berg 1, * Abstract: Rapid, quantitative methods for characterizing the biological activities of kinase inhibitors in complex human cell systems could allow the biological consequences of differential target selectivity to be monitored early in development, improving the selection of drug candidates. We have previously shown that Biologically Multiplexed Activity Profiling (BioMAP) permits rapid characterization of drug function based on statistical analysis of protein expression data sets from complex primary human cell- based models of disease biology. Here, using four such model systems containing primary human endothelial cells and peripheral blood mononuclear cells in which multiple signaling pathways relevant to inflammation and immune responses are simultaneously activated, we demonstrate that BioMAP analysis can detect and distinguish a wide range of inhibitors directed against different kinase targets. Using a panel of p38 mitogen-activated protein kinase antagonists as a test set, we show further that related compounds can be distinguished by unique features of the biological responses they induce in complex systems, and can be classified according to their induction of shared (on-target) and secondary activities. Statistical comparisons of quantitative BioMAP profiles and analysis of profile features allow correlation of induced biological effects with chemical structure and mapping of biological responses to chemical series or substituents on a common scaffold. Integration of automated BioMAP analysis for prioritization of hits and for structure-activity relationship studies may improve and accelerate the design and selection of optimal therapeutic candidates. 431 Introduction P ROTEIN KINASES ARE ATTRACTIVE DRUG TARGETS for a number of disease indications. 1 Protein phosphory- lation by kinases plays a central role in the control and regulation of signaling pathways in all cells, suggesting that selective targeting of key kinases that regulate dis- ease-specific mechanisms may offer safe and effective therapies. However, the development of protein kinase inhibitors remains challenging because most inhibitors 1 BioSeek, Inc., Burlingame, CA. 2 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA. 3 Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stan- ford, CA. *These authors are co-senior authors. ABBREVIATIONS: BioMAP, Biologically Multiplexed Activity Profiling; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; Cdk, cyclin- dependent kinase; CK2, casein kinase 2; DAF, decay accelerating factor; DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole; ELISA, enzyme-linked immunosorbent assay; FDR, false detection rate; GSK-3, glycogen synthase kinase 3; HMG-CoA, 3-hydroxy-3-methyl-glutaryl coenzyme A; Hsp90, heat shock protein 90; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1; IFN-, interferon-; IL, inter- leukin; IMPDH, inosine 5'-monophosphate dehydrogenase; JAK, Janus kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NF-B, nuclear factor-B; PBMC, peripheral blood mononuclear cells; PI-3K, phosphatidylinositol 3- kinase; PKA, protein kinase A; PPAR, peroxisome proliferation-activated receptor; PTK, protein tyrosine kinase; SAG, superantigen; SAR, structure- activity relationship; siRNA, small interfering RNA; TBB, 4,5,6,7-tetrabromo-2-aza-benzimidazole; TNF-, tumor necrosis factor-; uPAR, uroki- nase-type plasminogen activator receptor; VCAM, vascular cell adhesion molecule; VEGFR, vascular endothelial growth factor receptor. • •