The overarching goal of this proposal is to better understand key steps in the biosynthesis, utilization and recycling of trehalose in mycobacteria. The disaccharide trehalose is important for multiple aspects of mycobacterial physiology and has been shown to be essential for viability as is the downstream utilization pathway that leads to trehalose monomycolate production and export. Recycling of the trehalose used to build the outer membrane is important for M. tuberculosis virulence and is used to elongate a cytosolic glucan. This research project possesses 3 separate aspects that are all related to trehalose metabolism. First, we will identify structural features of M. tuberculosis GlgE that promote substrate binding and catalysis as a basis for understanding its function in synthesizing the mycobacteria glucan. Since inhibition of GlgE promotes rapid killing of M. tuberculosis, mechanism based inhibitors will be synthesized and used in conjunction with steady-state kinetics to better understand the enzyme mechanism. X-ray crystallographic studies will be performed to characterize interactions with GlgE substrates, which will form the basis for drug-development targeting GlgE. The second portion of this study aims to further characterize the mechanism of mycobacterial killing by the anti-tubercular drug ebselen. We have shown that ebselen strongly inhibits Antigen 85C through a covalent modification that disrupts the enzyme active site and inactivates it. Experiments performed in vivo and ex vivo will identify which mycobacterial proteins are modified by ebselen in a bacterial culture.
The third aim will characterize the structure-function relationship of the enzyme catalyzing the final step in the de novo trehalose biosynthetic pathway, trehalose phosphate phosphatase 2. Steady-state kinetics will be used to study the effects of active site mutations and inhibition in vitro. Inactive mutant will also be used in equilibrium binding studies to better understand substrate selectivity. This information will inform studies performed in vivo and determine if TPP2 is a valid drug target. The results from these studies will be used to advance our knowledge of the metabolic pathways that use trehalose. The biosynthesis of the building blocks used to form the mycomembrane, mycolic acids and trehalose monomycolate, are known targets of first and second-line anti-tubercular drugs. Therefore, it is expected that further defining the biosynthetic pathway leading to trehalose monomycolate and characterizing the enzymes that attach mycolic acids to the mycomembrane will offer new insights for anti-tubercular drug development. It is expected that this study will extend the available Mycobacterium tuberculosis drug targets to include enzymes in the trehalose biosynthetic and utilization pathways.

Public Health Relevance

The World Health Organization estimates that 1.4 million people each year die from tuberculosis. The first-line drugs used to treat tuberculosis have been used for 50 years and strains resistant to these drugs are becoming increasingly common. This research aims to characterize the biosynthesis and use of a simple sugar that is vitally important for the viability of M. tuberculosis extend the research community's ability to develop new therapies to treat this infectious disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI105084-01A1
Application #
8596082
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Lacourciere, Karen A
Project Start
2013-08-20
Project End
2017-07-31
Budget Start
2013-08-20
Budget End
2014-07-31
Support Year
1
Fiscal Year
2013
Total Cost
$346,625
Indirect Cost
$111,625
Name
University of Toledo
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
051623734
City
Toledo
State
OH
Country
United States
Zip Code
43606
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Favrot, Lorenza; Lajiness, Daniel H; Ronning, Donald R (2014) Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors. J Biol Chem 289:25031-40
Veleti, Sri Kumar; Lindenberger, Jared J; Ronning, Donald R et al. (2014) Synthesis of a C-phosphonate mimic of maltose-1-phosphate and inhibition studies on Mycobacterium tuberculosis GlgE. Bioorg Med Chem 22:1404-11
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Favrot, Lorenza; Grzegorzewicz, Anna E; Lajiness, Daniel H et al. (2013) Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen. Nat Commun 4:2748