The overall goals of the proposed research is to identify small molecule regulators of critical cellular processes in high-throughput screens of synthetic chemical libraries and to develop new methods to rapidly identify the cellular targets of active compounds from phenotypic screens. Chemical genomic approaches to identify the cellular functions of proteins have recently be shown to have great promise as a new tool for functional genomics and for the identification of new lead compounds and cellular targets for drug development. The proposed research aims to address two current deficiencies in the Chemical Genomic approach: modulating protein-protein interactions as targets of small molecules; identifying the cellular targets of bioactive small molecules identified in phenotype-directed screens.
The specific aims of the proposed research are (1) Develop synthetic chemistry to achieve a versatile high-yield, convergent solid-phase synthesis of the streptogramin B analogs. (2) Devise novel linkers for attachment of library members to solid phase synthesis resins such that linker cleavage generates an electrophilic reactive site (Michael acceptor) on each member of the library. (3) Synthesis and evaluation of a 'target identification probe' (TIP) reagent to enable the rapid identification of the cellular targets of bioactive compounds from combinatorial libraries. (4) Identification of biologically active library members and determine the cellular targets of bioactive library members using target identification probe reagents. Our integrated approach to chemical genomics includes new methods for the synthesis of electrophilic libraries, novel biological assays and a novel method for target identification as a platform for biological discovery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065406-04
Application #
7006662
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Fabian, Miles
Project Start
2003-01-01
Project End
2006-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
4
Fiscal Year
2006
Total Cost
$240,078
Indirect Cost
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Krusemark, Casey J; Frey, Brian L; Belshaw, Peter J et al. (2009) Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization. J Am Soc Mass Spectrom 20:1617-25
Miller, Russell A (2009) Ligand-regulated peptide aptamers. Methods Mol Biol 535:315-31
Krusemark, Casey J; Ferguson, Jonathan T; Wenger, Craig D et al. (2008) Global amine and acid functional group modification of proteins. Anal Chem 80:713-20
Frey, Brian L; Krusemark, Casey J; Ledvina, Aaron R et al. (2008) Ion-Ion Reactions with Fixed-Charge Modified Proteins to Produce Ions in a Single, Very High Charge State. Int J Mass Spectrom 276:136-143
Lamos, Shane M; Shortreed, Michael R; Frey, Brian L et al. (2007) Relative quantification of carboxylic acid metabolites by liquid chromatography-mass spectrometry using isotopic variants of cholamine. Anal Chem 79:5143-9
Krusemark, Casey J; Belshaw, Peter J (2007) Covalent labelling of fusion proteins in live cells via an engineered receptor-ligand pair. Org Biomol Chem 5:2201-4
Miller, Russell A; Binkowski, Brock F; Belshaw, Peter J (2007) Ligand-regulated peptide aptamers that inhibit the 5'-AMP-activated protein kinase. J Mol Biol 365:945-57
Lamos, Shane M; Krusemark, Casey J; McGee, Christopher J et al. (2006) Mixed isotope photoaffinity reagents for identification of small-molecule targets by mass spectrometry. Angew Chem Int Ed Engl 45:4329-33
Shortreed, Michael R; Lamos, Shane M; Frey, Brian L et al. (2006) Ionizable isotopic labeling reagent for relative quantification of amine metabolites by mass spectrometry. Anal Chem 78:6398-403
Levitsky, Konstantin; Boersma, Melissa D; Ciolli, Christopher J et al. (2005) Exo-mechanism proximity-accelerated alkylations: investigations of linkers, electrophiles and surface mutations in engineered cyclophilin-cyclosporin systems. Chembiochem 6:890-9

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