The determination of intracellular accumulation and target selectivity/specificity is an essential and challengingaspect of modern probe development. Although controversial 'rules' have been proposed to account for thebioavailability of orally administered drugs, techniques to determine target selectivity and intracellularcompartmentalization of drugs within living cells have not been adequately developed.A lead compound identified from high-throughput screening is generally not suitable for in vivo biological useuntil a systematic evaluation of the small molecule in cells has taken place. This step is required because highthroughputbiochemical and cellular assays are not representative of the multiple different physicochemicalenvironments that exist within living organisms. For example, upon exposure to a small molecule, numerousorganelles and compartments within the cell can sequester compounds and prevent association with thedesired biological target. Correspondingly, the development of small molecule enzyme inhibitors and receptormodulators is often limited by an inability to target specific proteins. Molecular probes derived from biologicallyactive small molecules have the potential to shed light on these issues. However, many efforts to developoptimal molecular probes suffer the following pitfalls: (1) the inability to accurately monitor where biologicallyactive compounds accumulate within cells, (2) the inability to accurately know which biological targets are trulyaffected upon exposure of cells to small molecules, and (3) the inability to accurately determine the relativeselectivity of compounds for multiple protein targets in their native environment. Previous attempts to addressthese issues have often used linked fluorophores and other labels that can intrinsically promote differentialaccumulation of the small molecule away from the site of biological relevance. Consequently, a generalsystematic protocol is needed to efficiently evaluate the intracellular accumulation and target selectivity ofmolecular probes.To circumvent these bottlenecks, we propose to develop two complementary approaches to identify proteinsthat bind small molecules. The first approach will utilize fluorescent probes that readily diffuse through cellularmembranes, but do not localize in any compartment. These fluorescent probes will then be used in pulsechaseexperiments to react with protein-bound inhibitors and monitored via confocal microscopy to elucidatethe extent of intracellular sequestration of the fluorescent small molecule. In addition, these probes will beutilized to determine target selectivity by modification of a tether to allow covalent attachment to bound proteinswithin the cell. Immunopurification and subsequent LCMS will be used to isolate the proteins linked to thesefluorophores and determine their identity. A second approach for isolating targets will employ a novel yeast 3-hybrid screen to identify proteins encoded by cDNA libraries that activate transcription by binding to chemicalinducers of dimerization (CIDs). To accomplish these objectives, we propose the following specific aims:1. Develop methodology for visualizing and identifying intracellular protein targets using fluorescentmolecular probes. We propose to develop optimal cell-permeable fluorophores, multifunctional tethers,approaches for elucidation of localization of probes, and methods for target identification (ID) usingpulse-chase affinity labeling and applications of selective antibodies.2. Develop yeast three-hybrid (Y3H) systems as tools for the identification of protein targets of smallmolecules. We propose to combine yeast genetic screening with fluorescence-activated cell sorting(FACS), develop methods to facilitate the penetration of small molecules into living yeast cells, andimprove the utility of yeast genetic systems for screening of small molecules and target ID.This proposal is innovative because it aims to address some of the most difficult problems associated withdelivering probe molecules identified through high-throughput screening to researchers engaged in real-worldbiomedical science. Two novel approaches are presented that have the potential to identify and ultimatelyeliminate undesired cellular trafficking issues and properties that could interfere with targeting of a particularprobe. By associating this project with a Specialized Chemistry Center, we also intend to develop generaltagging techniques that could be employed as needed in collaboration with other MLPCN groups.
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