Nancy A. Buchmeier

Drug Discovery Using Chemical Systems Biology: Repositioning the Safe Medicine Comtan to Treat Multi-Drug and Extensively Drug Resistant Tuberculosis

The rise of multi-drug resistant (MDR) and extensively drug resistant (XDR) tuberculosis around the world, including in industrialized nations, poses a great threat to human health and defines a need to develop new, effective and inexpensive anti-tubercular agents. Previously we developed a chemical systems biology approach to identify off-targets of major pharmaceuticals on a proteome-wide scale. In this paper we further demonstrate the value of this approach through the discovery that existing commercially available drugs, prescribed for the treatment of Parkinsons disease, have the potential to treat MDR and XDR tuberculosis. These drugs, entacapone and tolcapone, are predicted to bind to the enzyme InhA and directly inhibit substrate binding. The prediction is validated by in vitro and InhA kinetic assays using tablets of Comtan, whose active component is entacapone. The minimal inhibition concentration (MIC99) of entacapone for Mycobacterium tuberculosis (M.tuberculosis) is approximately 260.0 µM, well below the toxicity concentration determined by an in vitro cytotoxicity model using a human neuroblastoma cell line. Moreover, kinetic assays indicate that Comtan inhibits InhA activity by 47.0% at an entacapone concentration of approximately 80 µM. Thus the active component in Comtan represents a promising lead compound for developing a new class of anti-tubercular therapeutics with excellent safety profiles. More generally, the protocol described in this paper can be included in a drug discovery pipeline in an effort to discover novel drug leads with desired safety profiles, and therefore accelerate the development of new drugs.

Cu,Zn Superoxide Dismutase of Mycobacterium tuberculosis Contributes to Survival in Activated Macrophages That Are Generating an Oxidative Burst

ABSTRACT Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence ofMycobacterium tuberculosis. M. tuberculosis has two genes encoding superoxide dismutase proteins, sodA andsodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity ofM. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosis from reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protecting M. tuberculosis from the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in thesodC gene. In this report, we show that theM. tuberculosis sodC mutant is readily killed by superoxide generated externally, while the isogenic parentalM. tuberculosis is unaffected under these conditions. Furthermore, the sodC mutant has enhanced susceptibility to killing by gamma interferon (IFN-γ)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-γ or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance of M. tuberculosisagainst oxidative burst products generated by activated macrophages.

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

SUMMARY Mycothiol (MSH; AcCys-GlcN-Ins) is the major thiol found in Actinobacteria and has many of the functions of glutathione, which is the dominant thiol in other bacteria and eukaryotes but is absent in Actinobacteria. MSH functions as a protected reserve of cysteine and in the detoxification of alkylating agents, reactive oxygen and nitrogen species, and antibiotics. MSH also acts as a thiol buffer which is important in maintaining the highly reducing environment within the cell and protecting against disulfide stress. The pathway of MSH biosynthesis involves production of GlcNAc-Ins-P by MSH glycosyltransferase (MshA), dephosphorylation by the MSH phosphatase MshA2 (not yet identified), deacetylation by MshB to produce GlcN-Ins, linkage to Cys by the MSH ligase MshC, and acetylation by MSH synthase (MshD), yielding MSH. Studies of MSH mutants have shown that the MSH glycosyltransferase MshA and the MSH ligase MshC are required for MSH production, whereas mutants in the MSH deacetylase MshB and the acetyltransferase (MSH synthase) MshD produce some MSH and/or a closely related thiol. Current evidence indicates that MSH biosynthesis is controlled by transcriptional regulation mediated by σB and σR in Streptomyces coelicolor. Identified enzymes of MSH metabolism include mycothione reductase (disulfide reductase; Mtr), the S-nitrosomycothiol reductase MscR, the MSH S-conjugate amidase Mca, and an MSH-dependent maleylpyruvate isomerase. Mca cleaves MSH S-conjugates to generate mercapturic acids (AcCySR), excreted from the cell, and GlcN-Ins, used for resynthesis of MSH. The phenotypes of MSH-deficient mutants indicate the occurrence of one or more MSH-dependent S-transferases, peroxidases, and mycoredoxins, which are important targets for future studies. Current evidence suggests that several MSH biosynthetic and metabolic enzymes are potential targets for drugs against tuberculosis. The functions of MSH in antibiotic-producing streptomycetes and in bioremediation are areas for future study.

Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages

Mutations in the genes recA and recBC were constructed in the virulent Salmonella typhimurium strain 14028s. Both the recA and recBC mutants were attenuated in mice. The mutants were also sensitive to killing by macrophages in vitro. The recombination mutants were no longer macrophage sensitive in a variant line of J774 macrophage‐like cells that fail to generate superoxide. This suggests that repair of DNA damage by Salmonella is necessary for full virulence in vivo and that the oxidative burst of phagocytes is one source of such DNA damage.

DNA repair is more important than catalase for Salmonella virulence in mice.

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify reactive oxygen species, and DNA repair systems which repair damage resulting from oxidative stress. To determine the relative importance of these two potentially protective defense mechanisms against oxidative stress encountered by Salmonella during infection of the host, a Salmonella typhimurium double mutant unable to produce either the HPI or HPII catalase was constructed, and compared with an isogenic recA mutant deficient in DNA repair. The recA mutant was hypersusceptible to H2O2 at low cell densities in vitro, while the catalase mutant was more susceptible to high H2O2 concentrations at high cell densities. The catalase mutant was found to be resistant to macrophages and retained full murine virulence, in contrast to the recA mutant which previously was shown to be macrophage-sensitive and attenuated in mice. These observations suggest that Salmonella is subjected to low concentrations of H2O2 while at relatively low cell density during infection, conditions requiring an intact DNA repair system but not functional catalase activity.

A parallel intraphagosomal survival strategy shared by Mycobacterium tuberculosis and Salmonella enterica

Mycobacterium tuberculosis and Salmonella enterica cause very different diseases and are only distantly related. However, growth within macrophages is crucial for virulence in both of these intracellular pathogens. Here, we demonstrate that in spite of the phylogenetic distance, M. tuberculosis and Salmonella employ a parallel survival strategy for growth within macrophage phagosomes. Previous studies established that the Salmonella mgtC gene is required for growth within macrophages and for virulence in vivo. M. tuberculosis contains an open reading frame exhibiting 38% amino acid identity with the Salmonella MgtC protein. Upon inactivation of mgtC, the resulting M. tuberculosis mutant was attenuated for virulence in cultured human macrophages and impaired for growth in the lungs and spleens of mice. Replication of the mgtC mutant was inhibited in vitro by a combination of low magnesium and mildly acidic pH suggesting that the M. tuberculosis‐containing phagosome has these characteristics. The similar phenotypes displayed by the mgtC mutants of M. tuberculosis and Salmonella suggest that the ability to acquire magnesium is essential for virulence in intracellular pathogens that proliferate within macrophage phagosomes.

Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics

Mycothiol, MSH or 1d‐myo‐inosityl 2‐(N‐acetyl‐l‐cysteinyl)amido‐2‐deoxy‐α‐d‐glucopyranoside, is an unusual conjugate of N‐acetylcysteine (AcCys) with 1d‐myo‐inosityl 2‐acetamido‐2‐deoxy‐α‐d‐glucopyranoside (GlcN‐Ins), and is the major low‐molecular‐mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced ≈ 20% of wild‐type levels of MSH. Levels of the Rv1170 substrate, GlcNAc‐Ins, were elevated, whereas those of the product, GlcN‐Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc‐Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first‐line antituberculosis drugs.

Mycothiol Is Essential for Growth of Mycobacterium tuberculosis Erdman

Mycothiol (MSH) is the major low-molecular-mass thiol in mycobacteria and is associated with the protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. The biosynthesis of MSH is a multistep process, with the enzymatic reaction designated MshC being the ligase step in MSH production. A targeted disruption of the native mshC gene in M. tuberculosis Erdman produced no viable clones possessing either a disrupted mshC gene or reduced levels of MSH. However, when a second copy of the mshC gene was incorporated into the chromosome prior to the targeted disruption, multiple clones having the native gene disrupted and the second copy of mshC intact were obtained. These clones produced normal levels of MSH. These results demonstrate that the mshC gene and, more generally, the production of MSH are essential for the growth of M. tuberculosis Erdman under laboratory conditions.

Growth of Mycobacterium tuberculosis in a Defined Medium Is Very Restricted by Acid pH and Mg2+ Levels

ABSTRACT Mycobacterium tuberculosis grows within the phagocytic vacuoles of macrophages, where it encounters a moderately acidic and possibly nutrient-restricted environment. Other mycobacterial species encounter acidic conditions in soil and aquatic environments. We have evaluated the influence of pH and divalent cation levels on the growth of M. tuberculosis and seven other mycobacterial species. In a defined medium, the growth of M. tuberculosis was very restricted by acidic pH. Higher levels of Mg2+ were required for growth of M. tuberculosis in mildly acidic media (pH 6.0 to 6.5) compared to pH 7.0 medium. The divalent cations Ca2+, Zn2+, or Mn2+ could not replace Mg2+ during growth at pH 6.25, but Ca2+could at least partially substitute for Mg2+ during growth at pH 7.0. Among eight species of mycobacteria tested, there was a diversity of growth rates in media with acidic pH and low Mg2+ levels. M. tuberculosis was the most restricted in growth at pH 6.0, and all of this growth required elevated levels of Mg2+. M. kansasii andM. smegmatis also grew very poorly in acidic media with limiting Mg2+. M. fortuitum, M. marinum, M. scrofulaceum, M. avium, andM. chelonae grew at pH 6.0 in an unrestricted manner. These results demonstrate that M. tuberculosis is unique among the mycobacteria in its extreme sensitivity to acid and indicate thatM. tuberculosis must acquire sufficient Mg2+ in order to grow in a mildly acidic environment such as within the phagosome of macrophages.

A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

Mycothiol (MSH) (acetyl-Cys-GlcN-Ins) is the major low-molecular-mass thiol in Mycobacterium tuberculosis. MSH has antioxidant activity, can detoxify a variety of toxic compounds, and helps to maintain the reducing environment of the cell. The production of MSH provides a potential novel target for tuberculosis treatment. Biosynthesis of MSH requires at least four genes. To determine which of these genes is essential in M. tuberculosis, we have been constructing targeted gene disruptions. Disruption in the mshC gene is lethal to M. tuberculosis, while disruption in the mshB gene results in MSH levels 20 to 100% of those of the wild type. For this study, we have constructed a targeted gene disruption in the mshD gene that encodes mycothiol synthase, the final enzyme in MSH biosynthesis. The mshD mutant produced approximately 1% of normal MSH levels but high levels of the MshD substrate Cys-GlcN-Ins and the novel thiol N-formyl-Cys-GlcN-Ins. Although N-formyl-Cys-GlcN-Ins was maintained in a highly reduced state, Cys-GlcN-Ins was substantially oxidized. In both the wild type and the mshD mutant, cysteine was predominantly oxidized. The M. tuberculosis mshD mutant grew poorly on agar plates lacking catalase and oleic acid and in low-pH media and had heightened sensitivity to hydrogen peroxide. The inability of the mshD mutant to survive and grow in macrophages may be associated with its altered thiol-disulfide status. It appears that N-formyl-Cys-GlcN-Ins serves as a weak surrogate for MSH but is not sufficient to support normal growth of M. tuberculosis under stress conditions such as those found within the macrophage.