Multi-drug resistant tuberculosis is a major world health problem, especially for HIV infected individuals. There is an urgent need for the development of new tuberculosis drugs that will be active against multi-drug resistant (MDR) and latent tuberculosis infection. The goal of this research is to identify compounds that will block the biosynthesis of mycothiol (MSH). Mycothiol is the major thiol present in actinomycetes and has functions similar to glutathione. MSH biosynthesis has been shown to be essential for growth of Mycobacterium tuberculosis and is hypothesized to be required for its persistence in the dormant state. The goal of this research is to identify compounds that block MSH biosynthesis through development and application of appropriate high-throughput screening (HTS) assays. Such compounds will serve as leads for development of new drugs for MDR TB and will serve as powerful tools for investigation of the biological functions of mycothiol in the many different species of actinomycetes where it is found. The mshA and mshC genes of MSH biosynthesis have been shown to be essential for growth of M. tuberculosis but are not required for growth of M. smegmatis. This makes M. smegmatis a very useful model to identify inhibitors of MSH biosynthesis. In this research, HTS assays will be developed for inhibitors of the two essential enzymes, MshA and MshC, and for inhibitors blocking MSH production in intact M. smegmatis cells.
The specific aims of this research are: (1) to develop and apply a HTS assay for inhibitors of MshC, an ATP- dependent ligase, using pyrophosphatase as a coupling enzyme to convert the product pyrophosphate to phosphate which is measured colorimetrically; (2) to develop and apply a whole cell HTS assay with M. smegmatis for inhibitors of MSH production based upon use of monochlorobimane to fluorescently label mycothiol in cells; (3) to develop a HTS assay for inhibitors of MshA, a glycosyltransferase, using an inositol monophosphatase to release phosphate from the product of the MshA reaction, the phosphate being determined colorimetrically; (4) to develop a whole cell HTS assay with M. smegmatis for inhibitors of MSH production based upon growth in the presence of isoniazid by mycothiol-deficient cells and lack of growth of mycothiol-producing cells. These HTS assays are designed to meet the goals of PA-04-068 and will be proposed for screening of libraries available under the Molecular Libraries Initiative. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI072133-01
Application #
7178780
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Goldman, Robert C
Project Start
2007-04-01
Project End
2010-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
1
Fiscal Year
2007
Total Cost
$320,375
Indirect Cost
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Newton, Gerald L; Fahey, Robert C; Rawat, Mamta (2012) Detoxification of toxins by bacillithiol in Staphylococcus aureus. Microbiology 158:1117-26
Upton, Heather; Newton, Gerald L; Gushiken, Melissa et al. (2012) Characterization of BshA, bacillithiol glycosyltransferase from Staphylococcus aureus and Bacillus subtilis. FEBS Lett 586:1004-8
Hatzios, Stavroula K; Schelle, Michael W; Newton, Gerald L et al. (2011) The Mycobacterium tuberculosis CysQ phosphatase modulates the biosynthesis of sulfated glycolipids and bacterial growth. Bioorg Med Chem Lett 21:4956-9
Newton, Gerald L; Buchmeier, Nancy; La Clair, James J et al. (2011) Evaluation of NTF1836 as an inhibitor of the mycothiol biosynthetic enzyme MshC in growing and non-replicating Mycobacterium tuberculosis. Bioorg Med Chem 19:3956-64
Ta, Philong; Buchmeier, Nancy; Newton, Gerald L et al. (2011) Organic hydroperoxide resistance protein and ergothioneine compensate for loss of mycothiol in Mycobacterium smegmatis mutants. J Bacteriol 193:1981-90
Newton, Gerald L; Leung, Stephan S; Wakabayashi, Judy I et al. (2011) The DinB superfamily includes novel mycothiol, bacillithiol, and glutathione S-transferases. Biochemistry 50:10751-60
Sharma, Sunil V; Jothivasan, Vishnu K; Newton, Gerald L et al. (2011) Chemical and Chemoenzymatic syntheses of bacillithiol: a unique low-molecular-weight thiol amongst low Gýýý+ýýýC Gram-positive bacteria. Angew Chem Int Ed Engl 50:7101-4
Parsonage, Derek; Newton, Gerald L; Holder, Robert C et al. (2010) Characterization of the N-acetyl-ýý-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis . Biochemistry 49:8398-414
Gaballa, Ahmed; Newton, Gerald L; Antelmann, Haike et al. (2010) Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli. Proc Natl Acad Sci U S A 107:6482-6
Newton, Gerald L; Rawat, Mamta; La Clair, James J et al. (2009) Bacillithiol is an antioxidant thiol produced in Bacilli. Nat Chem Biol 5:625-7

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