The three greatest current needs for progress in the treatment of tuberculosis are to (a) develop drugs against drug resistant strains of Mycobacterium tuberculosis, (b) develop effective approaches to treat the latent states of the disease, and (c) shorten the course of therapy, which is related to the second goal. Here we propose to characterize the DevS/DosR and the redundant DosT/DosR two-component regulatory systems of M. tuberculosis. These regulatory systems control a panel of approximately 50 genes that is induced by low oxygen concentrations or NO exposure. The anaerobic state of M. tuberculosis is thought to resemble (but not be identical to) that found in latent infections and serves as model for it. We have found that the DevS and DosT sensor kinases have a heme group in their sensory domain and will investigate the structure of these sensors, their sensitivity to O2, CO, and NO, and the mechanisms that link binding of a gas to the heme group with autophosphorylation of a histidine residue in the kinase. A high-throughput screen will be performed to identify inhibitors of the DevS / DosT kinases, as inhibitors offer a possible route to treatment of the latent disease. Determination of structure-activity profiles and optimization of the inhibitors will be pursued. In a related but distinct effort, we propose to clone, express, and characterize the P450 enzyme complement of M. tuberculosis. Clinically used azole drugs that target P450 enzymes in fungi attenuate mycobacterial growth even in the latent state, but the substrates and biological roles of the twenty M. tuberculosis P450 enzymes are not known. Two of the P450 enzymes have been described in the literature, and we already have three further members of this M. tuberculosis family in hand. Each enzyme will be characterized by spectroscopic and crystallographic methods, and its affinity for azole drugs will be determined. A search for the relevant substrates for each enzyme will be pursued using a variety of techniques. The role of the P450 enzymes will also be investigated by individually disrupting (""""""""knocking out"""""""") each P450 enzyme in M. tuberculosis. The consequences of each P450 enzyme disruption will be analyzed by DNA microarray studies in addition to determination of the associated changes in bacterial growth and virulence. The project thus encompasses two heme- and oxygen-dependent system of M. tuberculosis that offer new avenues to the development of drugs effective against the latent form of the disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI074824-04
Application #
7918919
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Lacourciere, Karen A
Project Start
2007-09-01
Project End
2012-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
4
Fiscal Year
2010
Total Cost
$401,400
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Yanev, Stanislav G; Stoyanova, Tsveta D; Valcheva, Violeta V et al. (2018) Xanthates: Metabolism by Flavoprotein-Containing Monooxygenases and Antimycobacterial Activity. Drug Metab Dispos 46:1091-1095
de Montellano, Paul R Ortiz (2018) 1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology. Med Chem (Los Angeles) 8:
Ortiz de Montellano, Paul R (2018) Potential drug targets in the Mycobacterium tuberculosis cytochrome P450 system. J Inorg Biochem 180:235-245
Basudhar, Debashree; Madrona, Yarrow; Yukl, Erik T et al. (2016) Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS. J Biol Chem 291:16100-11
Madrona, Yarrow; Waddling, Christopher A; Ortiz de Montellano, Paul R (2016) Crystal structures of the CO and NOBound DosS GAF-A domain and implications for DosS signaling in Mycobacterium tuberculosis. Arch Biochem Biophys 612:1-8
Frank, Daniel J; Zhao, Yan; Wong, Siew Hoon et al. (2016) Cholesterol Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors. J Biol Chem 291:7325-33
Frank, Daniel J; Waddling, Christopher A; La, Maggie et al. (2015) Cytochrome P450 125A4, the Third Cholesterol C-26 Hydroxylase from Mycobacterium smegmatis. Biochemistry 54:6909-16
Conner, Kip P; Cruce, Alex A; Krzyaniak, Matthew D et al. (2015) Drug modulation of water-heme interactions in low-spin P450 complexes of CYP2C9d and CYP125A1. Biochemistry 54:1198-207
Frank, Daniel J; Madrona, Yarrow; Ortiz de Montellano, Paul R (2014) Cholesterol ester oxidation by mycobacterial cytochrome P450. J Biol Chem 289:30417-25
Sivaramakrishnan, Santhosh; de Montellano, Paul R Ortiz (2013) The DosS-DosT/DosR Mycobacterial Sensor System. Biosensors (Basel) 3:259-282

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