PINKO C; 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-727.

Quorex Pharmaceuticals, Carlsbad, CA

BACKGROUND: Two-component signaling pathways mediate the majority of signal transduction in bacteria. This transduction is generally mediated by a membrane spanning receptor histidine kinase (HK) and a cognate DNA binding response regulator. Ligand induced activation of the kinase domain results in ATP hydrolysis and phosphate transfer to a conserved histidine residue in the H-box domain. Phosphoryl group transfer to the response regulator occurs through this phosphohistidine intermediate. Although the deletion of individual histidine kinases in Strep pneumo does not attenuate virulence (Throup et al., Mo Micro (2000) 35 (3), 566-576), we believe that the inhibition of multiple HKs may result in a lethal phenotype. Given the high degree of homology in the active site of these HKs, it may be possible to design a generic "histidine kinase" inhibitor that is active against multiple HKs from many different pathogens. METHODS: We have undertaken an exhaustive protein sequence analysis of Type I HK domains from Staph aureus, Strep pneumonia, Pseudomonas aeruginosa and Enterococcus faecalis. An examination of the > 450HK domains resulted in the selection of 30 HKs that have a high degree on amino acid conservation in the ATP binding pocket. Soluble kinase domains have been tested for high-affinity binding to fluorescent nucleotides. RESULTS: High-throughput screening (HTS) has been initiated using the displacement of fluorescent nucleotide analogues to screen libraries of molecules likely to be competitive with ATP. In a complementary series of experiments, genetic knockouts of selected have been generated. Analysis of these knockouts has identified distinct phenotypes that may be useful as secondary cell-based screens for our HK inhibitors. CONCLUSIONS: Our HTS screen is continuing and has an observed hit rate of 0.5%. A [32]P-ATP based secondary screen using conserved HKs known to be important in virulence factor secretion will be initiated to confirm hits.

Publication Types:

• Meeting Abstracts

Keywords:

• Anti-Infective Agents
• Bacteria
• Binding Sites
• Histidine
• Human Development
• Phosphotransferases
• Protein Kinases
• Sequence Analysis, Protein
• Signal Transduction
• growth & development
• phosphohistidine
• protein-histidine kinase

Other ID:

• GWAIDS0028254

UI: 102267878

From Meeting Abstracts