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-002740nas a2200229 4500008004100000022001400041245013200055210006900187260007300256300001200329490000700341520187900348653001402227653003102241653002102272653000902293100001902302700001902321700002102340700001902361856013002380 2011 eng d a0167-701200aNovel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents0 aNovel screening method based on menadione mediated rapid reducti aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcFEB a202-2070 v843 a

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 mu M of XTT and 60 mu M of menadione in 250 mu l of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG. M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were > 0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether. XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli. (C) 2010 Elsevier B.V. All rights reserved.

10aMenadione10aMycobacterium tuberculosis10aTetrazolium salt10aXRMA1 aSingh, Upasana1 aAkhtar, Shamim1 aMishra, Abhishek1 aSarkar, Dhiman uhttps://library.ncl.res.in/content/novel-screening-method-based-menadione-mediated-rapid-reduction-tetrazolium-salt-testing-001774nas a2200253 4500008004100000022001400041245005400055210005400109260009200163300001400255490000700269520092500276653002401201653001401225653003101239653002901270100002501299700002101324700002301345700001901368700002601387700002101413856008601434 2012 eng d a1028-602000aPhyllocladane diterpenes from anisomeles heyneana0 aPhyllocladane diterpenes from anisomeles heyneana a4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLANDbTAYLOR & FRANCIS LTDcNOV a1162-11680 v143 a

New phyllocladane diterpene, phyllocladan-16 alpha,17-dihydroxy-19-oic acid (1), together with known phyllocladane diterpene, phyllocladan-16 alpha,19-diol (2), cembrane diterpene ovatodiolide (3), sitosteryl-3-O-beta-D-glucoside (4), and verbascoside (5), were isolated from aerial parts of Anisomeles heyneana. The structure of compound 1 was elucidated by 1D and 2D NMR analyses which included HSQC, HMBC, and nuclear overhauser effect spectroscopy (NOESY) experiments as well as X-ray crystallography. This is the first report of phyllocladane diterpenes from genus Anisomeles. Compounds 1, 3, 4, and 5 were evaluated for inhibition of Mycobacterium tuberculosis and 3 was found to exhibit anti-mycobacterial activity with IC90 6.53 mu g/ml. Compounds 1, 3, and 5, at 100 mu g/ml, were also evaluated for inhibition of Thp-1 cell lines, and compounds 1 and 3 showed 59.02% and 96.4% inhibitions, respectively.

10aAnisomeles heyneana10aLamiaceae10aMycobacterium tuberculosis10aphyllocladane diterpenes1 aKulkarni, Roshan, R.1 aShurpali, Ketaki1 aGawade, Rupesh, L.1 aSarkar, Dhiman1 aPuranik, Vedavati, G.1 aJoshi, Swati, P. uhttps://library.ncl.res.in/content/phyllocladane-diterpenes-anisomeles-heyneana-002223nas a2200217 4500008004100000022001400041245016600055210006900221260007600290300001200366490000700378520134900385653003001734653001401764653003101778653002201809653000901831100001801840700001901858856012801877 2012 eng d a1087-057100aPotential use of nitrate reductase as a biomarker for the identification of active and dormant inhibitors of mycobacterium tuberculosis in a THP1 infection model0 aPotential use of nitrate reductase as a biomarker for the identi a2455 TELLER RD, THOUSAND OAKS, CA 91320 USAbSAGE PUBLICATIONS INCcAUG a966-9730 v173 a

The development of a macrophage-based, antitubercular high-throughput screening system could expedite discovery programs for identifying novel inhibitors. In this study, the kinetics of nitrate reduction (NR) by Mycobacterium tuberculosis during growth in Thp1 macrophages was found to be almost parallel to viable bacilli count. NR in the culture medium containing 50 mM of nitrate was found to be optimum on the fifth day after infection with M. tuberculosis. The signal-to-noise (S/N) ratio and Z-factor obtained from this macrophage-based assay were 5.4 and 0.965, respectively, which confirms the robustness of the assay protocol. The protocol was further validated by using standard antitubercular inhibitors such as rifampicin, isoniazid, streptomycin, ethambutol, and pyrazinamide, added at their IC90 value, on the day of infection. These inhibitors were not able to kill the bacilli when added to the culture on the fifth day after infection. Interestingly, pentachlorophenol and rifampicin killed the bacilli immediately after addition on the fifth day of infection. Altogether, this assay protocol using M. tuberculosis-infected Thp-1 macrophages provides a novel, cost-efficient, robust, and easy-to-perform screening platform for the identification of both active and hypoxic stage-specific inhibitors against tuberculosis.

10ahigh-throughput screening10ainhibitor10aMycobacterium tuberculosis10anitrate reductase10aTHP11 aSarkar, Sampa1 aSarkar, Dhiman uhttps://library.ncl.res.in/content/potential-use-nitrate-reductase-biomarker-identification-active-and-dormant-inhibitors-001879nas a2200325 4500008004100000022001400041245012300055210006900178260007100247300001400318490000600332520075200338653002101090653002801111653001701139653002301156653003101179653001601210653001201226100002401238700001801262700002401280700001601304700002101320700002401341700002601365700001901391700002301410856012001433 2015 eng d a1948-587500aDesign and synthesis of a focused library of diamino triazines as potential mycobacterium tuberculosis DHFR inhibitors0 aDesign and synthesis of a focused library of diamino triazines a a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcNOV a1140-11440 v63 a

We report design of a series of 2,4-diamino triazines as Mycobacterium tuberculosis (Mtb) dihydrofolate reductase inhibitors. The synthesized compounds were evaluated against Mtb (H(37)Rv and Dormant stage H37Ra), their cytotoxicity was assessed (HepG2 and A549 cell lines), and selectivity toward Mtb was evaluated by testing against other bacterial strains. Some derivatives showed promising activity along with low cytotoxicity. The most potent compound in the whole cell assay (MIC 0.325 mu M against H(37)Rv) showed selectivity in the enzyme assay and exhibited synergy with second line anti-TB agent p-amino salicylic acid. This study therefore provides promising molecules for further development as antituberculosis DHFR inhibitors.

10aDiamino triazine10adihydrofolate reductase10aenzyme assay10amolecular modeling10aMycobacterium tuberculosis10aselectivity10asynergy1 aLele, Arundhati, C.1 aRaju, Archana1 aKhambete, Mihir, P.1 aRay, M., K.1 aRajan, M., G. R.1 aArkile, Manisha, A.1 aJadhav, Nandadeep, J.1 aSarkar, Dhiman1 aDegani, Mariam, S. uhttps://library.ncl.res.in/content/design-and-synthesis-focused-library-diamino-triazines-potential-mycobacterium-002695nas a2200265 4500008004100000022001400041245015600055210006900211260000800280300001200288490000700300520175900307653001502066653001502081653003102096653002202127653002702149653002702176653001802203653001702221653002202238100002702260700001902287856012302306 2021 eng d a0739-110200aPharmacophore modeling of pretomanid (PA-824) derivatives for antitubercular potency against replicating and non-replicating Mycobacterium tuberculosis0 aPharmacophore modeling of pretomanid PA824 derivatives for antit cFEB a889-9000 v393 a

Pretomanid (PA-824) is the recently (2019) approved drug for the treatment of extensively drug-resistant (XDR) TB and the multidrug-resistant (MDR) TB by US FDA. The experimental data of antitubercular activity of 543 pretomanid derivatives (total 6 datasets) against replicating (active) and non-replicating (dormant) forms of Mycobacterium tuberculosis (strain H37Rv) are available in the literature. Such vast experimental data of pretomanid derivatives against both of these endpoints, and recent approval of pretomanid molecule as a drug encouraged us to utilize this existing experimental information for the development of the 3D-pharmacophore models. The developed model (Hypo-1, MABA) showed the three physicochemical features namely, the oxygen atom of nitro group (HBA_1), fused pyran ring of imidazopyran heterocycle (HYAl_2) and the 4-fluorophenyl moiety (HYAr_3) are crucial for the antitubercular activity against replicating M. tb. Subsequently, the pharmacophore model (Hypo-1, LORA) developed against the non-replicating form of M. tb also showed the contribution of three physicochemical features namely, the 4-tri-fluoromethyl group (HYAl_2) and both the phenyl groups (HYAr_3, HYAr_4) of biaryl moiety in increasing the antitubercular activity. Both the pharmacophoric classifier models showed the classification accuracies of 82.98 and 74.42% for the training set compounds, and 63.91 and 61.60% for the test set compounds respectively, for labelling the compounds into higher and lower active classes. Both the models were also found to be retaining the higher active compounds in top 1.00% of the total number of compounds (decoys and actives), after performing the decoy set screening. Communicated by Ramaswamy H. Sarma

10aLORA assay10aMABA assay10aMycobacterium tuberculosis10anon-replicating M10apharmacophore modeling10apretomanid derivatives10areplicating M10atuberculosis10avirtual screening1 aAher, Rahul, Balasaheb1 aSarkar, Dhiman uhttps://library.ncl.res.in/content/pharmacophore-modeling-pretomanid-pa-824-derivatives-antitubercular-potency-against02099nas a2200253 4500008004100000022001400041245010600055210006900161260000800230300001100238490000800249520125600257653001701513653003101530653001301561653002201574100002301596700002001619700001901639700001901658700001901677700002301696856012601719 2021 eng d a0045-206800aSynthesis of new 2-(thiazol-4-yl)thiazolidin-4-one derivatives as potential anti-mycobacterial agents0 aSynthesis of new 2thiazol4ylthiazolidin4one derivatives as poten cOCT a1051920 v1153 aTo search for potent antimycobacterial lead compounds, a new series of 3-substituted phenyl-2-(2-(substituted phenyl)thiazol-4-yl) thiazolidin-4-one (5a-t) derivatives have been synthesized by the condensation of 2 substituted phenyl thiazole-4-carbaldehyde with aromatic amine followed by cyclocondensation with thioglycolic acid. The structure of the newly synthesized 2-(thiazol-4-yl)thiazolidin-4-one derivatives were characterized by the spectroscopic analysis. The synthesized compounds were screened for antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MTB) (ATCC 25177) and Mycobacterium bovis BCG (BCG, ATCC 35743). Most of the 2-(thiazol-4-yl)thiazolidin-4-one derivatives showed good to excellent antimycobacterial activity against both the Mtb strains. Nine derivatives 5c, 5g, 5j, 5m, 5n, 5o, 5p, 5s, and 5t showed excellent activity against M. bovis BCG with MIC 4.43 to 24.04 mu M were further evaluated for the cytotoxicity activity against HeLa A549, and HCT-116 cell lines and showed no significant cytotoxic activity at the maximum concentration evaluated. The potential antimycobacterial activities enforced that the thiazolyl-thiazolidin-4-one derivatives could lead to compounds that could treat tuberculosis.10aCytotoxicity10aMycobacterium tuberculosis10aThiazole10aThiazolidin-4-one1 aAbhale, Yogita, K.1 aShinde, Abhijit1 aShelke, Monika1 aNawale, Laxman1 aSarkar, Dhiman1 aMhaske, Pravin, C. uhttps://library.ncl.res.in/content/synthesis-new-2-thiazol-4-ylthiazolidin-4-one-derivatives-potential-anti-mycobacterial02277nas a2200337 4500008004100000022001400041245011600055210006900171260000800240300001400248490000700262520120700269653000601476653000601482653002101488653001101509653003001520653002801550653003101578100001801609700001701627700002501644700002401669700001701693700002301710700002401733700002201757700002201779700001901801856011901820 2022 eng d a1381-199100aDetection of a target protein (GroEl2) in Mycobacterium tuberculosis using a derivative of 1,2,4-triazolethiols0 aDetection of a target protein GroEl2 in Mycobacterium tuberculos cOCT a2535-25480 v263 a

Herein, we identified a potent lead compound RRA2, within a series of 54 derivatives of 1,2,4-triazolethiols (exhibit good potency as an anti-mycobacterial agents) against intracellular Mycobacterium tuberculosis (Mtb). Compound RRA2 showed significant mycobactericidal activity against active stage Mycobacterium bovis BCG and Mtb with minimum inhibitory concentration (MIC) values of 2.3 and 2.0 mu g/mL, respectively. At MIC value, RRA2 compound yielded 0.82 log reduction of colony-forming unit (cfu) against non-replicating Mtb. Furthermore, RRA2 compound was selected for further target identification due to the presence of alkyne group, showing higher selectivity index (> 66.66 +/- 0.22, in non-replicating stage). Using ``click'' chemistry, we synthesized the biotin linker-RRA2 conjugate, purified with HPLC method and confirmed the conjugation of biotin linker-RRA2 complex by HR-MS analysis. Furthermore, we successfully pulled down and identified a specific target protein GroEl2, from Mtb whole-cell extract. Furthermore, computational molecular modeling indicated RRA2 could interact with GroEl2, which explains the structure-activity relationship observed in this study.

10a110a210a4-triazolethiols10aGroEl210aMycobactericidal activity10aMycobacterium bovis BCG10aMycobacterium tuberculosis1 aSarkar, Sampa1 aSwami, Sagar1 aSoni, Sarvesh, Kumar1 aHolien, Jessica, K.1 aKhan, Arshad1 aKorwar, Arvind, M.1 aLikhite, Anjali, P.1 aJoshi, Ramesh, A.1 aJoshi, Rohini, R.1 aSarkar, Dhiman uhttps://library.ncl.res.in/content/detection-target-protein-groel2-mycobacterium-tuberculosis-using-derivative-12401555nas a2200253 4500008004100000022001400041245011200055210006900167520065400236653001500890653002700905653001700932653003100949653001400980653001300994100002701007700002301034700002401057700002401081700002301105700001901128700002201147856013201169 2022 eng d a1551-700400aSynthesis and antimycobacterial screening of a novel series of alpha-amino acids containing thiazole linker0 aSynthesis and antimycobacterial screening of a novel series of a3 a

A small focused library of uncommon (S)-2-amino-3-(4-(((4-methyl-2-arylthiazol-5-yl)methyl)amino)phenyl) propanoic acid (5a-e) and (S)-2-amino-3-(4-(((2-arylthiazol-4-yl)methyl)amino)phenyl)propanoic acid (9a-d) derivatives have been efficiently synthesized by employing molecular simplification. The title compounds were screened for inhibitory activity against Mycobacterium tuberculosis H37Ra (MTB) and Mycobacterium bovis (BCG) strains. The cytotoxicity study was conducted against primary Human Umbilical Vein Endothelial Cells (HUVECs), on two different human tumor cells HeLa, and HCT 116 and was observed non-toxic to host cells.

10aAmino acid10aantibacterial activity10aCytotoxicity10aMycobacterium tuberculosis10asynthesis10aThiazole1 aNalawade, Jitendra, L.1 aMhaske, Pravin, C.1 aShinde, Abhijit, D.1 aChavan, Abhijit, P.1 aAbhale, Yogita, K.1 aSarkar, Dhiman1 aBobade, Vivek, D. uhttps://library.ncl.res.in/content/synthesis-and-antimycobacterial-screening-novel-series-alpha-amino-acids-containing-thiazole