02295nas a2200217 4500008004100000022001400041245016700055210006900222260007600291300001200367490000700379520140400386653002001790653002501810653003001835653003101865100001301896700002101909700001901930856012801949 2005 eng d a1087-057100aDevelopment of a simple assay protocol for high-throughput screening of mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors0 aDevelopment of a simple assay protocol for highthroughput screen a2455 TELLER RD, THOUSAND OAKS, CA 91320 USAbSAGE PUBLICATIONS INCcOCT a725-7290 v103 a

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-m of 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 mu M, 5 mu M, and 25 mu 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 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-mu 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.

10aBeckman Coulter10aGlutamine synthetase10ahigh-throughput screening10aMycobacterium tuberculosis1 aSingh, U1 aPanchanadikar, V1 aSarkar, Dhiman uhttps://library.ncl.res.in/content/development-simple-assay-protocol-high-throughput-screening-mycobacterium-tuberculosis-002437nas a2200217 4500008004100000022001400041245018800055210006900243260007600312300001400388490000700402520151700409653002001926653002601946653002501972653003001997653003102027100001902058700001902077856012302096 2006 eng d a1087-057100aDevelopment of a simple high-throughput screening protocol based on biosynthetic activity of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors0 aDevelopment of a simple highthroughput screening protocol based  a2455 TELLER RD, THOUSAND OAKS, CA 91320 USAbSAGE PUBLICATIONS INCcDEC a1035-10420 v113 a

A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The K., values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH4Cl, 3.25 mM for MgCl2, and 2.5 mM for adenosine triphosphate. The K-m value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25 degrees C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z' factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 mu g/ml concentration. The IC50 values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 mu M and 30 pM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z' and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.

10aBeckman Coulter10abiosynthetic activity10aGlutamine synthetase10ahigh-throughput screening10aMycobacterium tuberculosis1 aSingh, Upasana1 aSarkar, Dhiman uhttps://library.ncl.res.in/content/development-simple-high-throughput-screening-protocol-based-biosynthetic-activity-002448nas a2200241 4500008004100000022001400041245008500055210006900140260008900209300001000298490000700308520157200315653002001887653002501907653003101932653002801963653002501991100001702016700001902033700002102052700001902073856011402092 2008 eng d a0882-401000aPresence of a functional nitrate assimilation pathway in mycobacterium smegmatis0 aPresence of a functional nitrate assimilation pathway in mycobac a24-28 OVAL RD, LONDON NW1 7DX, ENGLANDbACADEMIC PRESS LTD ELSEVIER SCIENCE LTDcJAN a71-770 v443 a

Ability of Mycobacterium smegmatis to assimilate nitrate was evaluated in its active and dormant phase. Nitrate (10 mM), nitrite (0.5 mM) and ammonia (10 mM)allowed growth of M. smegmatis concomitant with their complete depletion from the culture in 144, 120 and 96 h, respectively, when used as sole nitrogen Source. Azide (50 mu M) stopped the growth of M. smegmatis when nitrate was used as sole nitrogen source. L-methionine-S-sulfoximine (L-MSO), which is a well-known inhibitor of glutamine synthetase, an enzyme also involved in nitrogen metabolic pathway, when applied at 10 mu g/ml concentration, completely inhibited the growth of the organism when nitrate or nitrite was used as sole nitrogen source. There was no effect of either azide or L-MSO at above concentrations on the growth of the organism when asparagine or ammonia was used as sole nitrogen source. More significantly, utilization of nitrate, nitrite and ammonia continued even in oxygen depletion induced dormant culture at the rates of 289, 25 and 354 mu M/day, respectively. These rates were 5-8 times slower than the rates of 1966, 127 and 2890 mu M/day, respectively, in active replicating phase. In the presence of azide (50 mu M) and L-MSO (10 mu g/ml), 2.1 and 1.51 logs reduction in viability of dormant M. smegmatis was observed using nitrate and nitrite, respectively, as sole nitrogen source. Altogether, the results indicated the presence of nitrate assimilation pathway operating in both active and dormant stage of M. smegmatis. (C) 2007 Elsevier Ltd. All rights reserved.

10aDormant bacilli10aGlutamine synthetase10aL-methionine-S-sulfoximine10aMycobacterium smegmatis10aNitrate assimilation1 aKhan, Arshad1 aAkhtar, Shamim1 aAhmad, Jawid, N.1 aSarkar, Dhiman uhttps://library.ncl.res.in/content/presence-functional-nitrate-assimilation-pathway-mycobacterium-smegmatis-0