The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2'-deoxycytidine for uridine and 2'-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn(2+)-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K(m)=1004 microM and k(cat)=4.8s(-1) for cytidine, and K(m)=1059 microM and k(cat)=3.5s(-1) for 2'-deoxycytidine. The pH dependence of k(cat) and k(cat)/K(M) for cytidine indicate that protonation of a single ionizable group with apparent pK(a) value of 4.3 abolishes activity, and protonation of a group with pK(a) value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 A resolution. Analysis of the crystallographic structure indicated the presence of a Zn(2+) coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.

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Descrevemos a atividade inibitória do IQG607, pentaciano(isoniazida)ferrato(II), frente a cepas de Mycobacterium tuberculosis tanto resistentes quanto sensíveis à isoniazida, assim como a toxicidade oral e a adaptação da síntese química do IQG607 para reatores maiores. O IQG607 representa um potencial agente quimioterápico que inibe um alvo molecular definido.Here we describe the inhibitory activity of IQG607, pentacyano(isoniazid)ferrate(II), on isoniazid-sensitive and isoniazid-resistant strains of Mycobacterium tuberculosis, its oral toxicity, and efforts to adapt IQG607 synthesis to large chemical reactors. IQG607 represents a promising chemotherapeutic agent aiming at the inhibition of a validated and druggable molecular target. Keywords: Mycobacterium tuberculosis, enoyl reductase, toxicology, large-scale synthesis, metallodrug IntroductionTuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. 1 In 2007, there were an estimated 9.27 million cases of TB, and 1.37 million (15%) were also HIV-positive patients, who are more likely to develop active TB. 2 Brazil ranks 14 th in terms of the total number of incident cases amongst the high-burden countries. 2 There were an estimated 0.5 million cases of multidrugresistant TB (MDR-TB), which is caused by strains resistant to, at least, isoniazid and rifampicin. 2 The emergence of extensively drug-resistant (XDR) TB cases, which are found in TB-infected patients whose isolates are MDR and also resistant to a fluoroquinolone and, at least, one second-line injectable agent, 2,3 its widespread distribution, 4 and unprecedented fatality rate, 5 raise the prospect of virtually incurable and deadly TB worldwide. The factors that most influence the emergence of drugresistant strains include inappropriate treatment regimens and patient noncompliance in completing the prescribed courses of therapy due to the lengthy standard "shortcourse" treatment (isoniazid, rifampicin, pyrazinamide, and ethambutol or streptomycin for two months, followed by a combination of isoniazid and rifampicin for additional four months) or when the side effects become unbearable. 6 Moreover, no sustainable control of TB epidemic can be reached in any country without properly addressing this global public health problem, including research as a key component. 7 M. tuberculosis has been considered the world's most successful pathogen, and this is largely due to the ability of the bacillum to persist in host tissues, where drugs that are rapidly bactericidal in vitro require prolonged administration to achieve comparable in vivo effects. 8 Hence, more effective and less toxic anti-tubercular agents are immediately needed to shorten the duration of current treatment, improve the treatment of drug-resistant TB, and to provide effective treatment of latent TB infection.The modern approach in the development of new chemical entities (NCEs) against TB is based on the use of defined molecular targets. This involves (i) the search and identification ...

The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed.Nucleoside monophosphate (NMP) kinases are key enzymes in the metabolism of ribo-and deoxyribonucleoside triphosphates, catalyzing the reversible phosphoryl transfer from a nucleoside triphosphate (usually ATP) to a specific NMP (25). The resulting nucleoside diphosphates are subsequently phosphorylated to generate nucleoside triphosphates, precursors of nucleic acids. Bacterial CMP kinase (CMK), which is part of pyrimidine nucleotide interconversion pathways, catalyzes the transfer of a phosphate group from ATP to either CMP or dCMP to form CDP or dCDP and ADP, respectively (1). In Mycobacterium tuberculosis, the causative agent of human tuberculosis, a putative cmk (Rv1712) gene has been identified in the genome of the H37Rv strain by sequence similarity (8).In addition, it has been proposed that the cmk gene product is essential for the optimal in vitro growth of M. tuberculosis based on transposon site hybridization studies (15). However, this approach is a large-scale screening methodology, and hence, cmk gene manipulation experiments must be carried out to firmly support the hypothesis of its essentiality in M. tuberculosis. The first step should thus be demonstration that the Rv1712 locus indeed encodes a protein having CMK activity in M. tuberculosis. Here, we report heterologous recombinant protein expression, purification to homogeneity, N-terminal amino acid sequencing, electrospray ionization-mass spectrometry (ESI-MS) analysis, and size exclusion chromatography of a functional cmk-encoded M. tuberculosis CMK (MtCMK). Steady-state kinetics showed that MtCMK preferentially phosphorylates CMP and dCMP, and double-reciprocal plots indicated a sequential mechanism for MtCMK with ternary complex formation. The availability of the MtCMK protein in large quantities will allow further functional and structural efforts to be undertaken in order to provide a framework on which to base the design of chemical compounds that inhibit the enzyme activity.A 693-bp fragment consistent with the size expected for the predicted cmk coding sequence was PCR amplified from M. tuberculosis H37Rv genomic DNA (see Fig. S1A in the supplemental material). For directional cloning, a forward primer (5Ј-GGC ATATGAGTCGCCTAAGCGCAGCGGTAGT-3Ј) and a reverse primer (5Ј-GTGGATCCTCACCGCACTGCCTCACTT CGC-3Ј) were designed to contain, respectively, NdeI and BamHI restriction sites (underlined). The PCR fragment was purified and ligated into the pET-23a(ϩ) expression vector (Novagen), and its sequence was determined by automated DNA sequencing. The resulting pET-23a(ϩ)::cmk plasmid was introduced into Escherichia coli BL21(DE3) by electroporation and selected on LB plates containing 50 g/ml ampicillin.

Tuberculosis remains the major cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. The molecular mechanisms of infection and persistence have not been completely elucidated for this pathogen. Studies involving nucleoid-associated proteins (NAPs), which have been related to the control and influence of virulence genes in pathogenic bacteria, can help unveil the virulence process of M. tuberculosis. Here, we describe the initial characterization of an ORF for an M. tuberculosis putative NAP. The Rv3852 gene was cloned and expressed, and its product purified to homogeneity. A qualitative protein-DNA binding assay was carried out by gelretardation and the protein affinity for specific DNA sequences was assessed quantitatively by surface plasmon resonance (SPR). A stoichiometry of 10 molecules of monomeric protein per molecule of DNA was determined. The monophasic apparent dissociation rate constant values increased to a saturable level as a function of protein concentration, yielding two limiting values for the molecular recognition of proU2 DNA. A protein-DNA binding mechanism is proposed. In addition, functional complementation studies with an Escherichia coli hns mutant reinforce the likelihood that the Rv3852 protein represents a novel NAP in M. tuberculosis. INTRODUCTIONTuberculosis (TB) is one of the major causes of death worldwide caused by a single infectious agent, Mycobacterium tuberculosis. TB resurgence in the late 1980s was caused by a combination of several factors, such as HIV co-infection, increased poverty in urban areas and emergence of M. tuberculosis multidrug-resistant strains (MDR-TB) (Raviglione, 2003). According to the 2008 Global TB Control Report of the World Health Organization (WHO, 2008), there were approximately 9.2 million new TB cases in 2006, of which 0.5 million were MDR-TB. Moreover, the emergence of extensively drugresistant (XDR) TB cases (CDC, 2007), defined as cases in persons with TB whose isolates are MDR-TB as well as resistant to any one of the fluoroquinolone drugs and to at least one of the three injectable second-line drugs, amikacin, kanamycin or capreomycin, and their global distribution (Dorman & Chaisson, 2007), raise the prospect of virtually incurable TB worldwide. To compound the problem, it has been estimated that of 9.27 million incident TB cases in 2007, 1.37 million (15 %) were HIV-positive (WHO, 2009).M. tuberculosis has been considered the world's most successful pathogen. It is able to resist macrophage killing and persist in body tissues, thereby establishing a latent infection which can be reactivated when the host immune system wanes (Gomez & McKinney, 2004;Hingley-Wilson et al., 2003). The mechanism by which M. tuberculosis establishes latency and persistence is largely unknown and efforts have been made to address this and other issues, such as virulence (Andersen, 2007;Gandotra et al., 2007;Saunders & Britton, 2007;Schnappinger et al., 2006). Nucleoid-associated proteins (NAPs), also known as histone-like proteins, are a divers...