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Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

Vinita Panchanadikar

Dhiman Sarkar

Combi Chem-Bio Resource Center, National Chemical Laboratory, Dr. Homi Bhabha Rd., Pune 411008, Indiadsarkar{at}dalton.ncl.res.in

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 K_mof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The K_m for ADP, arsenate, and Mn²⁺ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their K_m values, the activity remained constant for at least 3 h at both 25° C and 37° 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-[.proportional]l 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.

Key Words: glutamine synthetase • high-throughput screening • Mycobacterium tuberculosis • Beckman Coulter

This version was published on October 1, 2005

Journal of Biomolecular Screening, Vol. 10, No. 7, 725-729 (2005)
DOI: 10.1177/1087057105278013


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				U. Singh and D. Sarkar Development of a Simple High-Throughput Screening Protocol Based on Biosynthetic Activity of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors J Biomol Screen, December 1, 2006; 11(8): 1035 - 1042. [Abstract] [PDF]