Synthesis of novel Iron(III) chelators based on triaza macrocycle backbone and 1-hydroxy-2(H)-pyridin-2-one coordinating groups and their evaluation as antimicrobial agents

AbstractSeveral novel chelators based on 1-hydroxy-2(1H)-pyridinone coordinating groups decorating a triaza macrocyclic backbone scaffold were synthesised as potential powerful Fe3+ chelators capable of competing with bacterial siderophores. In particular, a novel chloromethyl derivative of 1-hydroxy-2(1H)-pyridinone exploiting a novel protective group for this family of coordinating groups was developed. These are the first examples of hexadentate chelators based on 1-hydroxy-2(1H)-pyridinone to be shown to have a biostatic activity against a range of pathogenic bacteria. Their efficacy as biostatic agents was assessed revealing that minor variations in the structure of the chelator can affect efficacy profoundly. The minimal inhibitory concentrations of our best tested novel chelators approach or are comparable to those for 1,4,7-tris(3-hydroxy-6-methyl-2-pyridylmethyl)-1,4,7-triazacyclononane, the best Fe3+ chelator known to date. The retarding effect these chelators have on microbial growth suggests that they could have a potential application as a co-active alongside antibiotics in the fight against infections

Sterol 3β-glucosyltransferase biocatalysts with a range of selectivities, including selectivity for testosterone

The main objectives of this work were to characterise a range of purified recombinant sterol 3β-glucosyltransferases and show that rational sampling of the diversity that exists within sterol 3β-glucosyltransferase sequence space can result in a range of enzyme selectivities. In our study the catalytically active domain of the Saccharomyces cerevisiae 3β-glucosyltransferase was used to mine putative sterol 3β-glucosyltransferases from the databases. Selected diverse sequences were expressed in and purified from Escherichia coli and shown to have different selectivities for the 3β-hydroxysteroids ergosterol and cholesterol. Surprisingly, three enzymes were also selective for testosterone, a 17β-hydroxysteroid. This study therefore reports for the first time sterol 3β-glucosyltransferases with selectivity for both 3β- and 17β-hydroxysteroids and is also the first report of recombinant 3β-glucosyltransferases with selectivity for steroids with a hydroxyl group at positions other than C-3. These enzymes could therefore find utility in the pharmaceutical industry for the green synthesis of a range of glycosylated compounds of medicinal interest

Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria

Background. Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings. We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strainMycobacterium bovis BCG, as well as in the related pathogenic speciesMycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified fromdrug-treated mycobacteria showed a significant loss of cyclopropanation in both the a- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance. This is a first report on them echanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment

New drugs and vaccines for drug-resistant Mycobacterium tuberculosis infections

Tuberculosis remains the most common cause of death due to a single infective organism. Despite the availability of a vaccine and chemotherapeutic options, the global disease burden remains relatively unaffected. The ability of the mycobacterial etiological agents to adopt a semidormant, phenotypically drug-resistant state requires that chemotherapy is both complex and lengthy. The emergence of drug resistance has raised the possibility of virtually untreatable tuberculosis. Furthermore, the currently used bacillus Calmette–Guerin vaccine has had mixed success in protecting susceptible populations. Given this backdrop, the need for novel anti-infectives and more effective vaccines is clearly evident. Recent progress, described herein, has seen the development and entry into clinical trials of several new drugs and vaccine candidate

Currrent status and research strategies in tuberculosis drug development

In this Miniperspective we broadly cover the recent developments and strategies that are being adopted globally in the battle to urgently discover and develop new drugs to treat tuberculosis (TB). It highlights the impact and outcomes of recent nongovernment organizations (NGO) and academic research consortia that have made a significant impact in recent years in the field, providing the reader with an insight into the overall status of TB drug discovery and development while illustrating the current strategies and directions that are currently being adopted to find new drugs to control the disease

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K-M value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis

Sequence and Analysis of a Plasmid-Encoded Mercury Resistance Operon from Mycobacterium marinum Identifies MerH, a New Mercuric Ion Transporter▿

In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg2+ transporter. MerH was found to transport mercuric ions in Escherichia coli via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase

Structure of the diaminopimelate epimerase DapF from Mycobacterium tuberculosis

The meso (or d,L) isomer of diaminopimelic acid (DAP), a precursor of L-lysine, is a key component of the pentapeptide linker in bacterial peptidoglycan. While the peptidoglycan incorporated in the highly complex cell wall of the pathogen Mycobacterium tuberculosis structurally resembles that of Escherichia coli, it is unique in that it can contain penicillin-resistant meso-DAP -> meso-DAP linkages. The interconversion of L,L-DAP and meso-DAP is catalysed by the DAP epimerase DapF, a gene product that is essential in M. tuberculosis. Here, the crystal structure of the ligand-free form of M. tuberculosis DapF (MtDapF) refined to a resolution of 2.6 angstrom is reported. MtDapF shows small if distinct deviations in secondary structure from the two-domain alpha/beta-fold of the known structures of Haemophilus influenzae DapF and Bacillus anthracis DapF, which are in line with its low sequence identity (<= 27%) to the former. Modelling the present structure onto that of L,L-aziridino-DAP-bound H. influenzae DapF illustrates that a rigid-body movement of domain II and a rearrangement of the B4-A2 loop (residues 80-90) of domain I are likely to accompany the transition from the present inactive form to a catalytically competent enzyme. Despite a highly conserved active-site architecture, the model indicates that stabilization of the DAP backbone occurs in MtDapF through a tyrosine residue that is specific to mycobacterial DAP epimerases