High-throughput microplate phosphorylation assays based on DevR-DevS/Rv2027c 2-component signal transduction pathway to screen for novel antitubercular compounds.
DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [gamma-(32)P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [gamma-(32)P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.
Saini
Deepak Kumar
DK
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110-029, India.
Tyagi
Jaya Sivaswami
JS
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D000071677:Histidine Kinase; D008832:Microchemistry / Q000379:methods; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014157:Transcription Factors / Q000037:antagonists & inhibitors
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D014157:Transcription Factors; D011494:Protein Kinases; D000071677:Histidine Kinase