The three most important research 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. Cholesterol is crucial for infection of macrophages and survival of the mycobacteria in that environment. In the expiring period of support, we have identified three M. tuberculosis cytochrome P450 enzymes able to initiate cholesterol catabolism. Our studies have identified an intermediate, cholest-4-en-3-one that accumulates when these P450 enzymes are knocked out. This intermediate inhibits growth of MTB on several carbon sources. We propose to identify the site and mechanism of action of cholest-4-en-3-one, as it is a potential drug target. Furthermore, we will undertake the development of mechanism-based inhibitors of the two primary enzymes involved in degradation of the cholesterol side-chain, as their inactivation will not only block cholesterol utilization but will lead to accumulation of cholest-4-en-3-one. In a second facet of this project, we will characterize and define the biological roles of two P450 enzymes related to virulence that are proposed to oxidize methyl-branched hydrocarbon chains. In a third facet of our P450 studies, we will advance our work to define the structures, substrates, and roles of the other M. tuberculosis P450 enzymes. Of the twenty P450 enzymes, we now know the substrates and functions of four. We have cloned the remaining 16, partially characterized four, and have the structure of one. The collective evidence indicates that the P450 enzymes in MTB have specific biological roles rather than being involved in xenobiotic metabolism. In a related but distinct effort, we will further characterize the redundant DosS/DosT/DosR two-component regulatory systems of M. tuberculosis. These sensors control a regulon of approximately 50 genes that is induced by hypoxia, NO, or CO. The associated metabolic shift is thought to be similar to that which initiates the dormant state of M. tuberculosis. We have demonstrated that the sensors are heme proteins, defined the differential response to the various gases, and determined the crystal structure of DosT. We now propose to complete definition of the system by analyzing the mechanism by which the identity of the gas binding to the heme iron atom is transferred to the kinase domain of the sensor as either an off- (O2) or on- (NO, CO) signal. We will also perform a high-throughput search for inhibitors of the DosS / DosT kinases that may provide lead compounds for the development of agents effective against the latent states of M. tuberculosis. The proposed work rests on cutting-edge techniques, including proteomic, lipidomic, and metabolomic analysis of M. tuberculosis mutants and knockouts, NMR studies of protein conformational changes with 13C- and 19F-labeled site-specifically incorporated amino acids, and chemical biological studies of the roles of steroids and other factors in the biology of M. tuberculosis.

Public Health Relevance

Tuberculosis is world-wide scourge, as shown by the fact that (a) one-third of the globe's population is infected with Mycobacterium tuberculosis and approximately two million people die of TB each year, (b) each second a new person is infected with TB, and (c) 5-10% of the individuals infected with latent (dormant) TB will become actively sick during their lifetime. The emergence of drug resistant strains, including strains resistant to all available drugs, has refocused attention on this disease. New targets for drug design efforts are urgently needed and the cytochrome P450 enzymes and gas sensors of Mycobacterium tuberculosis have emerged as novel targets for the development of tuberculosis therapeutic agents.

National Institute of Health (NIH)
Research Project (R01)
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Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
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Lacourciere, Karen A
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University of California San Francisco
Schools of Pharmacy
San Francisco
United States
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Frank, Daniel J; Madrona, Yarrow; Ortiz de Montellano, Paul R (2014) Cholesterol ester oxidation by mycobacterial cytochrome P450. J Biol Chem 289:30417-25
Garcia-Fernandez, Esther; Frank, Daniel J; Galan, Beatriz et al. (2013) A highly conserved mycobacterial cholesterol catabolic pathway. Environ Microbiol 15:2342-59
Ouellet, Hugues; Chow, Eric D; Guan, Shenheng et al. (2013) Genetic and mass spectrometric tools for elucidating the physiological function(s) of cytochrome P450 enzymes from Mycobacterium tuberculosis. Methods Mol Biol 987:79-94
Jensen, Kenneth; Johnston, Jonathan B; de Montellano, Paul R Ortiz et al. (2012) Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains. Biotechnol Lett 34:239-45
Sivaramakrishnan, Santhosh; Ouellet, Hugues; Matsumura, Hirotoshi et al. (2012) Proximal ligand electron donation and reactivity of the cytochrome P450 ferric-peroxo anion. J Am Chem Soc 134:6673-84
Vos, Marten H; Bouzhir-Sima, Latifa; Lambry, Jean-Christophe et al. (2012) Ultrafast ligand dynamics in the heme-based GAF sensor domains of the histidine kinases DosS and DosT from Mycobacterium tuberculosis. Biochemistry 51:159-66
Ouellet, Hugues; Johnston, Jonathan B; de Montellano, Paul R Ortiz (2011) Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis. Trends Microbiol 19:530-9
Yukl, Erik T; Ioanoviciu, Alexandra; Sivaramakrishnan, Santhosh et al. (2011) Nitric oxide dioxygenation reaction in DevS and the initial response to nitric oxide in Mycobacterium tuberculosis. Biochemistry 50:1023-8
Ouellet, Hugues; Kells, Petrea M; Ortiz de Montellano, Paul R et al. (2011) Reverse type I inhibitor of Mycobacterium tuberculosis CYP125A1. Bioorg Med Chem Lett 21:332-7
Nishida, Clinton R; Ortiz de Montellano, Paul R (2011) Bioactivation of antituberculosis thioamide and thiourea prodrugs by bacterial and mammalian flavin monooxygenases. Chem Biol Interact 192:21-5

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