osip-339391 and monorden

Identification of drug targets is a key step in the development of novel pharmaceuticals. To this end, chemical probes or affinity matrices are often used, requiring substantial structure-activity relationship (SAR) studies. Here we report on the development of a novel technique for drug-target identification from total cellular lysate conducted independently of SAR information. This technique relies on binding of a drug to its target inducing a conformational change in target protein, thereby altering its susceptibility to proteolysis and resulting in specific degradation in some cases or in protection of target protein in others. As proof of concept, three drugs with identified targets were used. First, incubation of cellular lysates with okadaic acid elicited a specific protective effect on its target, protein phosphatase 2A catalytic subunit. Second, specific protection from exogenous protease of FKBP12 by FK506 and Hsp90 fragments by radicicol were observed. We then used the method to validate the targets of UCS15A, an Src signaling inhibitor. UCS15A induced proteolysis of a number of proteins, one of which was identified as Sam68. These studies suggest that the technology may be generally useful for identification and validation of drug targets.

Topics: Benzaldehydes; Cells; Drug Delivery Systems; Macrolides; Okadaic Acid; Peptide Hydrolases; Pharmaceutical Preparations; Protease Inhibitors; Protein Conformation; Protein Phosphatase 2; Proteins

Src tyrosine kinase plays key roles in signal transduction following growth factor stimulation and integrin-mediated cell-substrate adhesion. Since src-signal transduction defects are implicated in a multitude of human diseases, we have sought to develop new ways to identify small molecule inhibitors using a yeast-based, activated-src over-expression system. In the present study, we describe the identification of a unique src-signal transduction inhibitor, UCS15A. UCS15A was found to inhibit the src specific tyrosine phosphorylation of numerous proteins in v-src-transformed cells. Two of these phosphoproteins were identified as bona-fide src substrates, cortactin and Sam68. UCS15A differed from conventional src-inhibitors in that it did not inhibit the tyrosine kinase activity of src. In addition, UCS15A appeared to differ from src-destabilizing agents such as herbimycin and radicicol that destabilize src by interfering with Hsp90. Our studies suggest that UCS15A exerted its src-inhibitory effects by a novel mechanism that involved disruption of protein-protein interactions mediated by src. One of the biological consequences of src-inhibition by UCS15A was its ability to inhibit the bone resorption activity of osteoclasts in vitro. These data suggest that UCS15A may inhibit the bone resorption activity of osteoclasts, not by inhibiting src tyrosine kinase activity, but by disrupting the interaction of proteins associated with src, thereby modulating downstream events in the src signal transduction pathway.

Topics: 3T3 Cells; Adaptor Proteins, Signal Transducing; Animals; Anti-Bacterial Agents; Benzaldehydes; Benzoquinones; Bone Resorption; Cell Transformation, Neoplastic; Cortactin; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Genes, src; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Male; Mice; Microfilament Proteins; Organ Culture Techniques; Osteoclasts; Phosphorylation; Quinones; Rats; Rifabutin; RNA-Binding Proteins; Signal Transduction; src-Family Kinases; Tyrosine