Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

ABSTRACT A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

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Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Genetics of dTDP-Rhamnose Synthetic Enzymes and Development of a Microtiter Plate-Based Screen for Inhibitors of Conversion of dTDP-Glucose to dTDP-Rhamnose

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.5.1407-1416.2001 ◽

2001 ◽

Vol 45 (5) ◽

pp. 1407-1416 ◽

Cited By ~ 101

Author(s):

Yufang Ma ◽

Richard J. Stern ◽

Michael S. Scherman ◽

Varalakshmi D. Vissa ◽

Wenxin Yan ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Enzyme Assay ◽

Enzyme Inhibitors ◽

Microtiter Plate ◽

Structural Motif ◽

Chemical Library ◽

Microtiter Plate Assay ◽

Synthetic Enzymes ◽

Genes Encoding

ABSTRACT An l-rhamnosyl residue plays an essential structural role in the cell wall of Mycobacterium tuberculosis. Therefore, the four enzymes (RmlA to RmlD) that form dTDP-rhamnose from dTTP and glucose-1-phosphate are important targets for the development of new tuberculosis therapeutics. M. tuberculosis genes encoding RmlA, RmlC, and RmlD have been identified and expressed inEscherichia coli. It is shown here that genes for only one isotype each of RmlA to RmlD are present in the M. tuberculosis genome. The gene for RmlB is Rv3464. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose. The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP+ with a concomitant decrease in optical density at 340 nm (OD340). Inhibitor candidates were monitored for their ability to lower the rate of OD340change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 μM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 μg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against wholeM. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth ofM. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 μg/ml.

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A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Analytical Biochemistry ◽

10.1016/j.ab.2016.01.004 ◽

2016 ◽

Vol 498 ◽

pp. 53-58 ◽

Cited By ~ 10

Author(s):

Xiaoxia Shi ◽

Shanshan Sha ◽

Likun Liu ◽

Xin Li ◽

Yufang Ma

Keyword(s):

Mycobacterium Tuberculosis ◽

High Throughput ◽

High Throughput Screening ◽

Microtiter Plate ◽

Microtiter Plate Assay ◽

Plate Assay

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Immobilized Enzyme-Based Microtiter Plate Assay for Glucose in Foods

Journal of Agricultural and Food Chemistry ◽

10.1021/jf960261l ◽

1996 ◽

Vol 44 (12) ◽

pp. 3858-3863 ◽

Cited By ~ 16

Author(s):

Hiroyuki Ukeda ◽

Yoshihiro Fujita ◽

Miki Ohira ◽

Masayoshi Sawamura

Keyword(s):

Immobilized Enzyme ◽

Microtiter Plate ◽

Microtiter Plate Assay ◽

Plate Assay

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A microtiter plate assay for the detection of inhibitors of the Na +, K + -ATpase

Applied Biochemistry and Biotechnology ◽

10.1007/bf02788547 ◽

1994 ◽

Vol 49 (2) ◽

pp. 135-141

Author(s):

Seona E. Macgregor ◽

John M. Walker

Keyword(s):

Microtiter Plate ◽

Microtiter Plate Assay ◽

Plate Assay

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Prediction of Drug-Drug Interactions for AUCoral of High Clearance Drug from In Vitro Data: Utilization of a Microtiter Plate Assay and a Dispersion Model

Current Drug Metabolism ◽

10.2174/138920006775541570 ◽

2006 ◽

Vol 7 (2) ◽

pp. 135-146 ◽

Cited By ~ 2

Author(s):

Takahito Yamamoto ◽

Akio Suzuki ◽

Yoshiro Kohno ◽

Kiyoshi Nagata ◽

Yasushi Yamazoe

Keyword(s):

Drug Interactions ◽

Dispersion Model ◽

Microtiter Plate ◽

Data Utilization ◽

Microtiter Plate Assay ◽

Plate Assay ◽

Vitro Data ◽

In Vitro Data ◽

High Clearance

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Quantitative measurement of antibiotic resistance in Mycobacterium tuberculosis reveals genetic determinants of resistance and susceptibility in a target gene approach

10.1101/2021.09.14.460353 ◽

2021 ◽

Author(s):

◽

Joshua J Carter

Keyword(s):

Mycobacterium Tuberculosis ◽

Quantitative Measurement ◽

Target Gene ◽

Microtiter Plate ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

World Health ◽

Genetic Determinants ◽

Microtiter Plate Assay ◽

Health Organization

AbstractThe World Health Organization goal of universal drug susceptibility testing for patients with tuberculosis is most likely to be achieved through molecular diagnostics; however, to date these have focused largely on first-line drugs, and always on predicting binary susceptibilities. Here, we used whole genome sequencing and a quantitative microtiter plate assay to relate genomic mutations to minimum inhibitory concentration in 15,211 Mycobacterium tuberculosis patient isolates from 27 countries across five continents.This work identifies 449 unique MIC-elevating genetic determinants across thirteen drugs, as well as 91 mutations resulting in hypersensitivity for eleven drugs. Our results provide a guide for further implementation of personalized medicine for the treatment of tuberculosis using genetics-based diagnostics and can serve as a training set for novel approaches to predict drug resistance.

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