The determination of intracellular accumulation and target selectivity/specificity is an essential and challenging aspect of modern probe development. Although controversial """"""""rules"""""""" have been proposed to account for the bioavailability of orally administered drugs, techniques to determine target selectivity and intracellular compartmentalization 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 use until a systematic evaluation of the small molecule in cells has taken place. This step is required because highthroughput biochemical and cellular assays are not representative of the multiple different physicochemical environments that exist within living organisms. For example, upon exposure to a small molecule, numerous organelles and compartments within the cell can sequester compounds and prevent association with the desired biological target. Correspondingly, the development of small molecule enzyme inhibitors and receptor modulators is often limited by an inability to target specific proteins. Molecular probes derived from biologically active small molecules have the potential to shed light on these issues. However, many efforts to develop optimal molecular probes suffer the following pitfalls: (1) the inability to accurately monitor where biologically active compounds accumulate within cells, (2) the inability to accurately know which biological targets are truly affected upon exposure of cells to small molecules, and (3) the inability to accurately determine the relative selectivity of compounds for multiple protein targets in their native environment. Previous attempts to address these issues have often used linked fluorophores and other labels that can intrinsically promote differential accumulation of the small molecule away from the site of biological relevance. Consequently, a general systematic protocol is needed to efficiently evaluate the intracellular accumulation and target selectivity of molecular probes. To circumvent these bottlenecks, we propose to develop two complementary approaches to identify proteins that bind small molecules. The first approach will utilize fluorescent probes that readily diffuse through cellular membranes, but do not localize in any compartment. These fluorescent probes will then be used in pulsechase experiments to react with protein-bound inhibitors and monitored via confocal microscopy to elucidate the extent of intracellular sequestration of the fluorescent small molecule. In addition, these probes will be utilized to determine target selectivity by modification of a tether to allow covalent attachment to bound proteins within the cell. Immunopurification and subsequent LCMS will be used to isolate the proteins linked to these fluorophores 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 chemical inducers of dimerization (CIDs). To accomplish these objectives, we propose the following specific aims: 1. Develop methodology for visualizing and identifying intracellular protein targets using fluorescent molecular probes. We propose to develop optimal cell-permeable fluorophores, multifunctional tethers, approaches for elucidation of localization of probes, and methods for target identification (ID) using pulse-chase affinity labeling and applications of selective antibodies. 2. Develop yeast three-hybrid (Y3H) systems as tools for the identification of protein targets of small molecules. 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, and improve 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 with delivering probe molecules identified through high-throughput screening to researchers engaged in real-world biomedical science. Two novel approaches are presented that have the potential to identify and ultimately eliminate undesired cellular trafficking issues and properties that could interfere with targeting of a particular probe. By associating this project with a Specialized Chemistry Center, we also intend to develop general tagging techniques that could be employed as needed in collaboration with other MLPCN groups.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54HG005031-06
Application #
8528669
Study Section
Special Emphasis Panel (ZRG1-IFCN-K)
Project Start
Project End
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
6
Fiscal Year
2013
Total Cost
$404,007
Indirect Cost
$125,198
Name
University of Kansas Lawrence
Department
Type
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Aflaki, Elma; Borger, Daniel K; Moaven, Nima et al. (2016) A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism. J Neurosci 36:7441-52
Gold, Ben; Smith, Robert; Nguyen, Quyen et al. (2016) Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis. J Med Chem 59:6027-44
Chattopadhyay, Debasish; Swingle, Mark R; Salter, Edward A et al. (2016) Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C. Biochem Pharmacol 109:14-26
Schroeder, Chad E; Neuenswander, Sarah A; Yao, Tuanli et al. (2016) One-pot, regiospecific assembly of (E)-benzamidines from δ- and γ-amino acids via an intramolecular aminoquinazolinone rearrangement. Org Biomol Chem 14:3950-5
Roy, Sudeshna; Å ileikytÄ—, Justina; Neuenswander, Benjamin et al. (2016) N-Phenylbenzamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore. ChemMedChem 11:283-8
Skarsfeldt, Mark A; Jepps, Thomas A; Bomholtz, Sofia H et al. (2016) pH-dependent inhibition of Kâ‚‚P3.1 prolongs atrial refractoriness in whole hearts. Pflugers Arch 468:643-54
He, Shanshan; Jain, Prashi; Lin, Billy et al. (2015) High-Throughput Screening, Discovery, and Optimization To Develop a Benzofuran Class of Hepatitis C Virus Inhibitors. ACS Comb Sci 17:641-52
Hong, Lin; Guo, Yuna; BasuRay, Soumik et al. (2015) A Pan-GTPase Inhibitor as a Molecular Probe. PLoS One 10:e0134317
Fang, Chen-Jie; Gui, Lin; Zhang, Xiaoyi et al. (2015) Evaluating p97 inhibitor analogues for their domain selectivity and potency against the p97-p47 complex. ChemMedChem 10:52-6
Ndjomou, Jean; Corby, M Josie; Sweeney, Noreena L et al. (2015) Simultaneously Targeting the NS3 Protease and Helicase Activities for More Effective Hepatitis C Virus Therapy. ACS Chem Biol 10:1887-96

Showing the most recent 10 out of 41 publications