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Imagine a technology that can test thousands of properties of cells for faster discovery of better drugs ... a n d i t ’s a v a i l a b l e n o w There is a p r o b l e m in drug discovery: The economics and efficiency of the drug development process are more important now than ever before, while at the same time that process is undergoing radical change. With the advent of gene sequencing, gene expression analysis, and combinatorial chemistry, a new problem has arisen. Now there are too many unqualified targets and leads. A critical need has developed for technologies that allow researchers to quickly, efficiently, and accurately characterize and sort through these targets and leads. Ta r ge t Va l i d a t i o n is a key step in the drug discovery process. Advances in genomics have created an intensely competitive environment in which thousands of genes are being mapped, proposed as important new targets, and patented. Selecting the best target genes is difficult because the function of most of these genes is unknown. Functional Genomics technologies will provide the means to gain understanding of the function of these important genes. With such information one can greatly speed up the selection or elimination of a particular gene as a drug target. Therefore, rapid access to gene function information provides a competitive advantage in t a rg e t v a l i d a t i o n . It is also essential in gaining a strong patent position. L e a d Va l i d a t i o n follows the process of target validation. Companies now have ready access to large chemical libraries either from natural product sources or from With high-throughput robotic assays they can test these libraries against their target gene/protein of interest. But these technologies provide only part of the picture – major hurdles remain. Knowing that a chemical is active against its target is the first of many steps. The ability to rapidly gain information on the specificity of these drug leads is also of great value. Technology that provides insight at the cellular level about the interaction of a lead candidate with the thousands of other cellular proteins, would offer significant benefits. Optimal drug candidates will have specific effects on the target without causing undesired side effects. NOW t h e r e i s a s o l u t i o n combinatorial synthesis. PHENOTYPE MICROARRAYS™ information at the cellular sets of arrays. Each well of the A MAJOR NEW TECHNOLOGY level ... and much more. array is designed to test a P different phenotype. henotype MicroArrays Phenotype MicroArrays are (PMs) represent a breakthrough comprehensive cellular profiles Cells are tested after a genetic in technology, alongside DNA that can be used to identify change or exposure to a drug Microarrays and Proteomic Technologies (Figure 1), to complete the information RNA DNA PROTEIN PHENOTYPE O’Farrel, 1975 Biolog, 2000 needed in the genomic era of drug development. Affymetrix, 1993 Just as DNA Microarrays and Proteomic Technologies have made it possible to assay the expression level of thousands Molecular Analyses of genes or proteins all at once, Phenotype MicroArrays make Cellular Analysis Figure 1 it possible to measure, quantitatively, thousands of gene function, validate targets, lead, and the researcher can cellular phenotypes all at once. and assist lead validation and directly evaluate cellular optimization. response to that change. The DNA Microarrays and scientist simply inoculates a Proteomic Technologies allow HOW THE TECHNOLOGY standardized cell suspension scientists to detect genes or WORKS into the MicroArray panels, proteins that are coregulated Phenotype MicroArray thereby testing thousands of and whose patterns of change technology is an integrated phenotypes at once. The correlate with something system of cellular assays, MicroArray is then incubated, important such as a disease instrumentation, and typically for 24 to 48 hours. state. However, there is no bioinformatic software. The assurance that these changes testing process is diagrammed PMs use Biolog’s patented are really significant to the cell. in Figure 2. redox chemistry, employing Phenotype MicroArrays are a cell respiration as a universal complementary technology Biolog preconfigures a wide reporter. If the phenotype is providing the needed range of phenotypic tests into strongly “positive” in a well, Add Cells Add Cells a. PM Pattern b. OmniLog PM System c. PM Kinetic Result d. Comparison Figure 2 the cells respire actively, phenotypes are monitored reducing a tetrazolium dye and simultaneously by the forming a strong color (Figure OmniLog and more than 2a). If it is weakly positive or 450,000 data points can be nitrogen, phosphorus, negative, respiration is slowed generated in one 24-hour run. and sulfur or stopped, and less color or no • cell surface binding and transport functions • catabolism of carbon, • biosynthesis of small color is formed. The redox To compare the phenotypes of assay provides for both two cell lines, one is recorded amplification and precise as a red tracing and one as a quantitation of phenotypes. green tracing (Figure 2c). These graphs can then be molecules • biosynthesis of polymeric macromolecules • formation of cellular structures Incubation and recording of overlaid by the bioinformatics • cellular respiratory functions phenotypic data is performed software to detect differences. • stress and repair functions by the patented OmniLog PM Areas of overlap (i.e., no • other cellular properties instrument (Figure 2b). The change) are colored yellow, Currently, PMs are available OmniLog captures a digital whereas differences are for use with bacterial and fun- image of the MicroArray color highlighted as patches of red or gal cells. They are being devel- change several times each hour, green (Figure 2d). oped also for mammalian cells. values into computer files. The Phenotype MicroArrays can TARGET VALIDATION: computer files can be displayed monitor, either directly Determining Gene Function to the scientist in the form of or indirectly, most aspects of In many cases, researchers have kinetic graphs. Thousands of cell function. The range of already determined genes they ™ and stores the quantitative phenotypes includes: believe may be good drug TARGET IDENTIFICATION: LEAD VALIDATION: targets. As diagrammed in Establishing New Drug Targets Assaying Drug Candidates Figure 2, a direct cell-to-cell PMs have many other uses in PM technology provides a comparison can be made cellular studies for revolutionary new tool for between a normal cell and a characterizing and defining evaluating drug leads. In this cell with the gene of interest phenotypic properties of cells. application, testing is again inactivated by a genetic mutation (i.e., knocked out). performed in a comparative For example, PM cell line-to- mode. A model cell line is cell line comparisons can be tested without drug exposure Genetic changes result in performed to identify new drug (as a control) and then tested phenotypic changes. Any gene targets. Researchers may want against a collection of drug of unknown function can be to find phenotypes that can be leads of interest. The testing procedure is the same, except arginine that the drug leads are added to the cell suspension just before ornithine the cells are distributed into the MicroArray wells (Figure 4a). Here again, the OmniLog instrument (Figure 4b) MG1655 ynjB::Tn10 monitors, records and compares the assays automatically. Figure 3 The PM pattern that forms in knocked out and the knockout targeted by drugs toxic to: the absence of drug is different cells can be assayed for • pathogenic bacteria, but not than patterns formed with phenotypic changes. An their non-pathogenic relatives various drugs added. From this example is shown in Figure 3 • bacteria and fungi that infect comprehensive cellular assay, comparing the nitrogen plants and animals, but not data from the MicroArrays is metabolism of a normal E. coli the plant and animal hosts analyzed by pattern recognition strain and a mutant that has lost • cancerous cells, but not their and clustering software (Figure function of a pathway for non-cancerous precursors metabolizing arginine and • virus-infected cells, but not ornithine as nitrogen sources. PMs provide a direct cellular assay of gene function. uninfected cells • diseased cells but not their non-diseased neighbors 4c) to sort drugs based on mode of action. Add Cells Add Cells +Drug a. PM Pattern b. OmniLog PM System c. Bioinformatics Analysis Figure 4 PM technology allows drug on non-target sites. mammalian cell PM assays, it researchers to analyze chemical These effects can be critically will be possible to assay drug libraries in a rapid, high- important if they result in candidates against a battery of throughput method with several unacceptable side effects. cell lines that represent the major organs and tissues of the unique and significant Third, one can use PMs body. This will permit rapid containing other drugs as part and cost effective screening to First, it measures the effect of of the overall phenotypic help eliminate unsatisfactory drugs on cells under thousands analysis. This aspect of PM drug candidates earlier in of physiological states. When technology allows you to test development. the cell changes its the drug candidate’s interaction physiological state it also with other drugs. Normally, The PM approach to assaying changes its surface receptors this would be done inefficiently drug leads is unique and and internal protein make-up, as a separate study. Discovering powerful. It provides a highly in a sense becoming a different an unexpected synergy or efficient means to assay the cell. Current assay technologies antagonism with a known drug effect of a drug on thousands of using cells grown under one early on can change the entire cellular targets under thousands condition give a very limited, course of drug development. of physiological states of advantages. the cell. incomplete view of the range of biological possibilities. Soon, PM technology will also be used as a basis for detailed Second, PM technology allows scientists to see the effect of a toxicological studies. With Benefits in Drug Discovery Phenotype MicroArrays: Complement DNA microarrays. DNA microarrays allow scientists to measure the expression of thousands of genes of a cell under one growth condition, whereas Phenotype MicroArrays allow scientists to measure the effect of one gene in the cell under thousands of growth conditions. Provide rapid answers to critical questions about cellular properties in a simple, rapid, efficient, and cost-effective process. Provide rapid answers to how a gene of unknown function affects a cell at a physiological level. Provide cell-to-cell comparisons to find novel drug targets based on in vivo physiological differences. Provide a means to assay the effect of a drug on thousands of cellular targets under thousands of physiological states of the cell. Provide rapid answers to questions about how a new lead affects a cell at a biochemical level. Provide information on how new drug leads compare to and interact with existing drugs. Reduce the time and cost of slow and cumbersome animal, plant, or human studies in late development. Eliminate unproductive leads and projects, saving substantial R&D budget dollars that would be wasted in later development. 3938 Trust Way Hayward, CA 94545 Telephone: 510-785-2564 Fax: 510-782-4639 www.biolog.com 04/01