Development of a simple assay protocol for high-throughput screening of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors.
Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Km of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 microM, 5 microM, and 25 microM, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-microl volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.
Singh
Upasana
U
Combichem Bioresource Center, National Chemical Laboratory, Dr. Homi Bhabha Rd., Pune 411008, India.
Panchanadikar
Vinita
V
Sarkar
Dhiman
D
D004121:Dimethyl Sulfoxide; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000302:isolation & purification / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000494:pharmacology; D009169:Mycobacterium tuberculosis / Q000201:enzymology*
D004791:Enzyme Inhibitors; D008717:Methionine Sulfoximine; D005974:Glutamate-Ammonia Ligase; D004121:Dimethyl Sulfoxide