Assay Systems For Addressing New Targets

Assays can be developed for orphan GPCRs, proteases, kinases, and phosphatases without knowing the cellular function of the new protein. For other classes of proteins, especially proteins with unknown activity, alternative technologies are being developed. Figure 1 summarizes how new gene targets and protein technologies are being coupled with combinatorial chemistry, microarrays for gene expression and in vivo studies to develop drugs.

The yeast two-hybrid system is being used extensively to develop screens to find inhibitors of protein-protein interactions [67]. Microbes have served as surrogate systems to study new mammalian genes in a cellular milieu. An advantage of microbial systems is that microbes can be arrayed on microchips. A method to array yeast cells by coating them on microscope slides mixed with silicon dioxide has been described. As the spread dries the yeast cells are displayed in hexagonal arrays in the porous silica support that allows molecules to

Figure 1 The path from genomics to drugs.

penetrate readily and can be used as biosensors. A fluorescent DNA probe that penetrated the silicon layer was used to show the practicality of this array approach for studying heterologously expressed genes in yeast to examine the function of relevant human targets [68].

Methods to identify the activation of whole pathways are being developed. In one method, the signaling pathways resulting from activation of T-lymphocytes was determined by using promoterless P-lactamase reporter fusion to a number of genes [69]. A fluorogenic substrate is used to detect the activation or repression of individual reporter tagged genes in the T-cells. This is a powerful method for detecting functional pathways that respond to external stimuli. Proteins in pathways can also be identified by activation of the receptor involved with the pathway of interest, separation of proteins using 2-D gel electrophoresis and then detection of phosphorylated proteins using phosphotyrosine and phos-phoserine antibodies [70]. This is a reliable, but tedious, means of identifying the pathways in which new proteins are involved. Alternatively, multiple fluorescent-labeled cellular proteins can be used to screen for inhibitors that act at specific pathways and targets [71]. Compounds that act in these systems are expected to have high value as they show selectivity to specific pathways and work in cellular systems.

In addition to cell-based technologies, many technologies are available to identify small molecules that bind with high affinity to new proteins [72]. These methods utilize classical affinity techniques coupled to mass spectrometry, NMR, scanning calorimery and other physical methods to identify the bound molecules; those that are identified must later be tested in cellular systems to show that they do have functional activity. Structural approaches can be used to design drugs to block protein-protein interactions. The limiting factor to the use of NMR is the inability to get concentrations of 0.1 mM of protein in solution. Recently, an NMR technique called transverse relaxation optimized spectroscopy (TROSY) has been described. TROSY can be a powerful tool in studying protein-protein interactions and for studying the structure of proteins larger than 100 kDa. Transverse relaxation optimized spectroscopy does not use the traditional 2-D NMR spectra, but focuses on one of the four component lines and uses it as the correlation peak [73,74]. Microarrays have been used to identify DNA-binding proteins that interact with oligonucleotides. In this method, 40 mer-long double stranded oligonucleotides are immobilized by their 3' end in arrays. The DNA is labeled during the hybridization and priming steps and was used to bind methylases and restriction enzymes [75].

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