HALDIMANN A, SPERISEN P, MUKHIJA S, SILHAVY T, ISLAM K; Interscience Conference on Antimicrobial Agents and Chemotherapy (42nd : 2002 : San Diego, Calif.).
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2002 Sep 27-30; 42: abstract no. F-725.
ARPIDA, Munchenstein, Switzerland.
BACKGROUND: Two-component systems (TCSs) constitute a family of widespread protein pairs mediating signal transduction in bacteria and thereby providing a central regulatory function. A typical TCS consists of a sensor kinase and its cognate response regulator, interacting by transferring a phosphoryl group from a conserved histidine residue to a conserved aspartate residue. CpxAR TCS of Escherichia coli, a system known to be important for pathogenesis, was used to search for inhibitors of TCSs. METHODS: A high throughput assay based on the scintillation proximity technology was developed: Test compound, N-terminally His-tagged CpxR, and radioactively pre-phosphorylated MBP-CpxA101 were successively added to a Ni[2+]-NTA-Flashplate (NEN). Phosphotransfer was quantified by determining the radiolabelled CpxR. Secondary assays included reporter gene assays on other TCSs, e.g., ArcBA or PhoRB. RESULTS: The high throughput screen of 30,000 compounds yielded several reconfirmed positives (IC[50]s of 5 to 200 microM). The compounds were grouped into several classes based on their chemical structures and were profiled in secondary assays and in unrelated assays (e.g. various bacterial and eukaryotic kinases) to determine their specificity for CpxR. By this approach one third of the molecules were found to be non-specific. Selectivity for CpxAR was determined by using reporter gene assays which showed that the majority of inhibitors did not inhibit other TCSs in spite of the high sequence and structural relationship among this family of protein pairs. Finally, we determined antimicrobial activities on representative Gram-positive and Gram-negative bacterial strains. None of the compounds showed any significant antibacterial activity as predicted for CpxAR TCS inhibitors since it is non-essential for growth of E. coli cells under ordinary laboratory conditions. Future studies aim to determine the activity of these compounds in animal models of infection.
Publication Types:
Keywords:
- Anti-Bacterial Agents
- Aspartic Acid
- Bacteria
- Escherichia coli
- Escherichia coli Proteins
- Histidine
- Phosphotransferases
- Signal Transduction
- antagonists & inhibitors
Other ID:
UI: 102267886
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