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Antimicrobial Agents and Chemotherapy, May 2001, p. 1407-1416, Vol. 45, No. 5
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.5.1407-1416.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Yufang Ma,1 Richard J. Stern,1 Michael S. Scherman,1 Varalakshmi D. Vissa,1 Wenxin Yan,1 Victoria Cox Jones,1 Fangqiu Zhang,2 Scott G. Franzblau,2 Walter H. Lewis,3 and Michael R. McNeil1,*

Department of Microbiology, Colorado State University, Fort Collins, Colorado 805231; College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 606122; and Department of Biology, Washington University, St. Louis, Missouri 631303

Received 14 August 2000/Returned for modification 3 October 2000/Accepted 12 February 2001

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 in Escherichia 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 OD340 change. 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 whole M. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth of M. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 µg/ml.

* Corresponding author. Mailing address: Department of Microbiology, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-1784. Fax: (970) 491-1815. E-mail: mmcneil{at}cvmbs.colostate.edu.

Antimicrobial Agents and Chemotherapy, May 2001, p. 1407-1416, Vol. 45, No. 5
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.5.1407-1416.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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