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This methodology provides a powerful tool for unraveling the cellular biology and …

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- An efficient rapid system for profiling the cellular activities of molecular libraries

Jonathan S. Melnick*,, Jeff Janes,, Sungjoon Kim,,, Jim Y. Chang, Daniel G. Sipes, Drew Gunderson, Laura Jarnes, Jason T. Matzen, Michael E. Garcia, Tami L. Hood, Ronak Beigi, Gang Xia, Richard A. Harig, Hayk Asatryan, S. Frank Yan, Yingyao Zhou, Xiang-Ju Gu, Alham Saadat, Vicki Zhou, Frederick J. King, Christopher M. Shaw, Andrew I. Su, Robert Downs, Nathanael S. Gray, Peter G. Schultz*,, Markus Warmuth,, and Jeremy S. Caldwell,

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121; and *The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Peter G. Schultz, December 29, 2005



Rapid quantitative methods for characterizing small molecules,peptides, proteins, or RNAs in a broad array of cellular assayswould allow one to discover new biological activities associatedwith these molecules and also provide a more comprehensive profileof drug candidates early in the drug development process. Herewe describe a robotic system, termed the automated compoundprofiler, capable of both propagating a large number of celllines in parallel and assaying large collections of moleculessimultaneously against a matrix of cellular assays in a highlyreproducible manner. To illustrate its utility, we have characterizeda set of 1,400 kinase inhibitors in a panel of 35 activatedtyrosine-kinase-dependent cellular assays in dose–responseformat in a single experiment. Analysis of the resulting multidimensionaldataset revealed subclusters of both inhibitors and kinaseswith closely correlated activities. The approach also identifiedactivities for the p38 inhibitor BIRB796 and the dual src/ablinhibitor BMS-354825 and exposed the expected side activitiesfor Glivec/STI571, including cellular inhibition of c-kit andplatelet-derived growth factor receptor. This methodology providesa powerful tool for unraveling the cellular biology and molecularpharmacology of both naturally occurring and synthetic chemicaldiversity.

drug discovery | high-throughput screening | tyrosine kinase

PNAS | February 28, 2006 | vol. 103 | no. 9 | 3153-3158. OPEN ACCESS ARTICLE.



The ability to simultaneously interrogate the activities ofa library of molecules against a large panel of cellular assayswould provide a rapid efficient means to begin to characterizeand correlate the biological properties of both natural andsynthetic chemical diversity. For example, libraries of noncodingRNAs, mutant growth factors, small molecule kinase inhibitors,or even existing drugs could be assayed for their potency andselectivity in pathway-based or receptor screens or toxicityand metabolic stability in diverse cell types to discover anew biological activity or optimize the pharmacological propertiesof a molecule (13). Although whole-cell systems representan attractive milieu to characterize gene and small-moleculefunction, no robust and systematic method exists to correlatechemical structure and biological activity across a large numberof molecules and cellular assays. To address this problem, wehave developed an approach that affords rapid cost-effectivebroad-based cellular profiling in parallel against molecularlibraries. An industrial-scale automated compound profiling(ACP) system has been designed, which consists of an automatedtissue culture system for propagating cell lines, integratedwith a system for automatically performing miniaturized cell-basedassays in 384- or 1,536-well microplates. The ACP can rapidlytest thousands of arrayed molecules, in replicates, in dose–responseformat against hundreds of unique cellular assays in a singleexperiment.

To demonstrate this capability, we focused on the problem ofidentifying selective small-molecule inhibitors of protein tyrosinekinases. Tyrosine kinases play a key role in many cellular processes,including development, differentiation, and proliferation; misregulationof tyrosine kinase expression and activity leads to a numberof disorders, most notably cancer (4, 5). For example, spontaneoustranslocations in which tyrosine kinase domains become fusedto other genes (such as Bcr, Tel, and NPM) have been identifiedas the etiological basis for multiple B cell lymphomas, includingthe Philadelphia chromosome Bcr-Abl and chronic myelogenousleukemia (6, 7). It remains a significant challenge to developselective inhibitors for tyrosine kinases given their homologyand potential structural plasticity. To this end, we have establisheda panel of tyrosine-kinase-dependent cellular assays by creatingstably expressed Tel-tyrosine kinase fusions representing eachbranch of the tyrosine kinase phylogeny. A library of >1,000kinase-directed heterocycles was then profiled against thispanel of cellular assays. The resulting dataset was examinedfor global structure–activity relationships (SARs), potencyand selectivity correlations, and opportunistic side activities.

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