scholarly journals Inhibitors of the Salicylate Synthase (MbtI) from Mycobacterium tuberculosis Discovered by High-Throughput Screening

ChemMedChem ◽  
2010 ◽  
Vol 5 (12) ◽  
pp. 2079-2087 ◽  
Author(s):  
Mahalakshmi Vasan ◽  
João Neres ◽  
Jessica Williams ◽  
Daniel J. Wilson ◽  
Aaron M. Teitelbaum ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Salicylate Synthase

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

scholarly journals High throughput screening of a library based on kinase inhibitor scaffolds against Mycobacterium tuberculosis H37Rv

Tuberculosis ◽  
2012 ◽  
Vol 92 (1) ◽  
pp. 72-83 ◽  
Author(s):  
Robert C. Reynolds ◽  
Subramaniam Ananthan ◽  
Ellen Faaleolea ◽  
Judith V. Hobrath ◽  
Cecil D. Kwong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitor ◽  
Tuberculosis H37rv ◽  
Mycobacterium Tuberculosis H37rv ◽  
Inhibitor Scaffolds

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

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
Author(s):  
Upasana Singh ◽  
Vinita Panchanadikar ◽  
Dhiman Sarkar
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Coli Strain ◽  
Compound Library ◽  
Essential Enzyme ◽  
Assay Protocol

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 Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km 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 Km 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.

ChemInform Abstract: Discovery of Novel Inhibitors Targeting the Mycobacterium tuberculosis O-Acetylserine Sulfhydrylase (CysK1) Using Virtual High-Throughput Screening.

ChemInform ◽  
2013 ◽  
Vol 44 (28) ◽  
pp. no-no
Author(s):  
Variam Ullas Jean Kumar ◽  
Oemer Poyraz ◽  
Shalini Saxena ◽  
Robert Schnell ◽  
Perumal Yogeeswari ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals Mycobacterium tuberculosis virulence inhibitors discovered by Mycobacterium marinum high-throughput screening

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hasan Tükenmez ◽  
Isabel Edström ◽  
Ramesh Ummanni ◽  
Stina Berglund Fick ◽  
Charlotta Sundin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mycobacterium Marinum

A High-Throughput Screening Assay for Simultaneous Selection of Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-Xylulose-5-Phosphate Synthase (Dxs) or 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)

2011 ◽  
Vol 16 (3) ◽  
pp. 303-312 ◽  
Author(s):  
Vaishali Humnabadkar ◽  
Ramesh K. Jha ◽  
Nuzhat Ghatnekar ◽  
Sunita M. De Sousa
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Isothermal Calorimetry ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Enzyme Assays ◽  
High Concentration ◽  
High Throughput Screening Assay ◽  
Individual Enzyme ◽  
Phosphate Synthase

1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is involved in the synthesis of isoprenoids by the methylerythritol phosphate pathway. Dxr is essential in Mycobacterium tuberculosis ( Mtu), absent in humans and amenable to structure-aided design. To further assess the druggability of the enzyme, the energetics of binding of fosmidomycin to Mtu Dxr was studied by isothermal calorimetry. Binding was enhanced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and driven by enthalpy (ΔH –10.2 kcal/mol, ΔS 1.1 cal mol−1K−1). This suggests the possibility of finding novel inhibitors that bind enthalpically, making Dxr an attractive target. The cost of the Dxr substrate, 1-deoxy-D-xylulose-5-phosphate, for high-throughput screening (HTS) is prohibitive. Hence, an HTS assay that couples Dxr to the upstream enzyme 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), also a valid target, was developed. A high concentration of NADPH was used to bias it to detect Dxr inhibitors that bind like fosmidomycin. The assay Z′ was 0.75. It was equally sensitive to inhibitors of Dxs and Dxr, that is, fosmidomycin and fluropyruvate inhibited it with IC50s similar to that in the individual enzyme assays (79 vs 54 nM for fosmidomycin). To distinguish inhibitors of Dxs from Dxr, individual enzyme assays and a microplate thermofluor binding assay were developed. The assay simultaneously screens two targets and is cost-effective.

scholarly journals Screening, Identification, and Characterization of Mechanistically Diverse Inhibitors of the Mycobacterium Tuberculosis Enzyme, Pantothenate Kinase (CoaA)

2011 ◽  
Vol 17 (3) ◽  
pp. 293-302 ◽  
Author(s):  
Janani Venkatraman ◽  
Jyothi Bhat ◽  
Suresh M. Solapure ◽  
Jatheendranath Sandesh ◽  
Debasmita Sarkar ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Inhibitors ◽  
Single Experiment ◽  
Pantothenate Kinase ◽  
Ligand Displacement ◽  
High Throughput Assay ◽  
Thermal Melting

The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis ( Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift–based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated Kie and Kies for representative compounds and through nuclear magnetic resonance–based ligand displacement assays.

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