trichostatin-a and monorden

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.

Topics: Adaptor Proteins, Signal Transducing; Antifungal Agents; Cell-Free System; Chromatography, Liquid; Dopamine Agonists; Escherichia coli; Fungal Proteins; GTP-Binding Proteins; HeLa Cells; Humans; Hydroxamic Acids; Indenes; Macrolides; Mass Spectrometry; Methotrexate; Methyltransferases; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Proteomics; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.

Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Animals; Benzoquinones; Binding Sites; Cattle; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Lactones; Macrolides; Macromolecular Substances; Precipitin Tests; Protein Binding; Quinones; Substrate Specificity; Tandem Repeat Sequences; Two-Hybrid System Techniques; Yeasts