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1.
Proc Natl Acad Sci U S A ; 120(5): e2212755120, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36693100

RESUMO

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Fosfatidilinositóis/metabolismo , Mycobacterium tuberculosis/metabolismo , Membrana Celular/metabolismo , Tuberculose/microbiologia , Antituberculosos/metabolismo
2.
J Biol Chem ; 296: 100307, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33476646

RESUMO

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Trealose/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Parede Celular/genética , Parede Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Mutação , Mycobacterium tuberculosis/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Trealose/análogos & derivados , Virulência
3.
Org Biomol Chem ; 18(18): 3607-3612, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32350493

RESUMO

The uptake and metabolism of the disaccharide trehalose by Mycobacterium tuberculosis is essential for the virulence of this pathogen. Here we describe the chemoenzymatic synthesis of new azido-functionalised asymmetric trehalose probes that resist degradation by mycobacterial enzymes and are used to probe trehalose processing pathways in mycobacteria.


Assuntos
Dissacarídeos/metabolismo , Mycobacterium tuberculosis/química , Trealose/metabolismo , Configuração de Carboidratos , Dissacarídeos/análise , Microscopia de Fluorescência , Mycobacterium tuberculosis/metabolismo , Trealose/análogos & derivados , Trealose/química
4.
J Biol Chem ; 293(25): 9770-9783, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29728457

RESUMO

The Mycobacterium tuberculosis (Mtb) pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 Å resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/ß binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.


Assuntos
Acetilglucosamina/análogos & derivados , Amidoidrolases/química , Amidoidrolases/metabolismo , Mycobacterium tuberculosis/enzimologia , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Amidoidrolases/genética , Domínio Catalítico , Cristalografia por Raios X , Metais/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica
5.
Org Biomol Chem ; 17(43): 9524-9528, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31659363

RESUMO

Dimeric benzoboroxoles that are covalently linked by a short scaffold enhance selective anti-tubercular activity. These multimeric benzoboroxole compounds are capable of engaging the specific extracellular Mycobacterium tuberculosis glycans, do not lead to the evolution of resistance and bypass the need to cross the impermeable mycobacterial cell envelope barrier.


Assuntos
Antituberculosos/farmacologia , Ácidos Borônicos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Células A549 , Antituberculosos/síntese química , Antituberculosos/química , Ácidos Borônicos/síntese química , Ácidos Borônicos/química , Dimerização , Eritrócitos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular
6.
Biomacromolecules ; 19(1): 256-264, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29195272

RESUMO

Because of the emergence of antimicrobial resistance to traditional small-molecule drugs, cationic antimicrobial polymers are appealing targets. Mycobacterium tuberculosis is a particular problem, with multi- and total drug resistance spreading and more than a billion latent infections globally. This study reports nanoparticles bearing variable densities of poly(dimethylaminoethyl methacrylate) and the unexpected and distinct mechanisms of action this multivalent presentation imparts against Escherichia coli versus Mycobacterium smegmatis (model of M. tuberculosis), leading to killing or growth inhibition, respectively. A convergent "grafting to" synthetic strategy was used to assemble a 50-member nanoparticle library, and using a high-throughput screen identified that only the smallest (2 nm) particles were stable in both saline and complex cell media. Compared with the linear polymers, the nanoparticles displayed two- and eight-fold enhancements in antimicrobial activity against M. smegmatis and E. coli, respectively. Mechanistic studies demonstrated that the antimicrobial particles were bactericidal against E. coli due to rapid disruption of the cell membranes. Conversely, against M. smegmatis the particles did not lyse the cell membrane but rather had a bacteriostatic effect. These results demonstrate that to develop new polymeric antituberculars the widely assumed, broad spectrum, membrane-disrupting mechanism of polycations must be re-evaluated. It is clear that synthetic nanomaterials can engage in more complex interactions with mycobacteria, which we hypothesize is due to the unique cell envelope at the surface of these bacteria.


Assuntos
Antibacterianos/farmacologia , Antituberculosos/farmacologia , Escherichia coli/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Nanopartículas , Polímeros/farmacologia , Antibacterianos/química , Antituberculosos/química , Testes de Sensibilidade Microbiana , Nanopartículas/química
7.
Biomacromolecules ; 18(5): 1592-1599, 2017 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-28365981

RESUMO

Antimicrobial resistance is a global healthcare problem with a dwindling arsenal of usable drugs. Tuberculosis, caused by Mycobacterium tuberculosis, requires long-term combination therapy and multi- and totally drug resistant strains have emerged. This study reports the antibacterial activity of cationic polymers against mycobacteria, which are distinguished from other Gram-positive bacteria by their unique cell wall comprising a covalently linked mycolic acid-arabinogalactan-peptidoglycan complex (mAGP), interspersed with additional complex lipids which helps them persist in their host. The present study finds that poly(dimethylaminoethyl methacrylate) has particularly potent antimycobacterial activity and high selectivity over two Gram-negative strains. Removal of the backbone methyl group (poly(dimethylaminoethyl acrylate)) decreased antimycobacterial activity, and poly(aminoethyl methacrylate) also had no activity against mycobacteria. Hemolysis assays revealed poly(dimethylaminoethyl methacrylate) did not disrupt red blood cell membranes. Interestingly, poly(dimethylaminoethyl methacrylate) was not found to permeabilize mycobacterial membranes, as judged by dye exclusion assays, suggesting the mode of action is not simple membrane disruption, supported by electron microscopy analysis. These results demonstrate that synthetic polycations, with the correctly tuned structure are useful tools against mycobacterial infections, for which new drugs are urgently required.


Assuntos
Antituberculosos/química , Metacrilatos/química , Mycobacterium/efeitos dos fármacos , Nylons/química , Poliaminas/química , Antituberculosos/efeitos adversos , Antituberculosos/farmacologia , Membrana Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemólise , Metacrilatos/farmacologia , Nylons/farmacologia , Poliaminas/efeitos adversos , Poliaminas/farmacologia , Polieletrólitos
8.
Molecules ; 19(10): 16274-90, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25310152

RESUMO

Novel drugs to treat tuberculosis are required and the identification of potential targets is important. Piperidinols have been identified as potential antimycobacterial agents (MIC < 5 µg/mL), which also inhibit mycobacterial arylamine N-acetyltransferase (NAT), an enzyme essential for mycobacterial survival inside macrophages. The NAT inhibition involves a prodrug-like mechanism in which activation leads to the formation of bioactive phenyl vinyl ketone (PVK). The PVK fragment selectively forms an adduct with the cysteine residue in the active site. Time dependent inhibition of the NAT enzyme from Mycobacterium marinum (M. marinum) demonstrates a covalent binding mechanism for all inhibitory piperidinol analogues. The structure activity relationship highlights the importance of halide substitution on the piperidinol benzene ring. The structures of the NAT enzymes from M. marinum and M. tuberculosis, although 74% identical, have different residues in their active site clefts and allow the effects of amino acid substitutions to be assessed in understanding inhibitory potency. In addition, we have used the piperidinol 3-dimensional shape and electrostatic properties to identify two additional distinct chemical scaffolds as inhibitors of NAT. While one of the scaffolds has anti-tubercular activity, both inhibit NAT but through a non-covalent mechanism.


Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/metabolismo , Sítios de Ligação , Humanos , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Ligação Proteica
9.
Chem Sci ; 2024 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-39144457

RESUMO

The sensitive, rapid and accurate diagnosis of Mycobacterium tuberculosis (Mtb) infection is a central challenge in controlling the global tuberculosis (TB) pandemic. Yet the detection of mycobacteria is often made difficult by the low sensitivity of current diagnostic tools, with over 3.6 million TB cases missed each year. To overcome these limitations there is an urgent need for next-generation TB diagnostic technologies. Here we report the use of a discrete panel of native 19F-trehalose (F-Tre) analogues to label and directly visualise Mtb by exploiting the uptake of fluorine-modified trehalose analogues via the mycobacterial trehalose LpqY-SugABC ATP-binding cassette (ABC) importer. We discovered the extent of modified F-Tre uptake correlates with LpqY substrate recognition and characterisation of the interacting sites by saturation transfer difference NMR coupled with molecular dynamics provides a unique glimpse into the molecular basis of fluorine-modified trehalose import in Mtb. Lipid profiling demonstrated that F-Tre analogues modified at positions 2, 3 and 6 are incorporated into mycobacterial cell-surface trehalose-containing glycolipids. This rapid one-step labelling approach facilitates the direct visualisation of F-Tre-labelled Mtb by Focused Ion Beam (FIB) Secondary Ion Mass Spectrometry (SIMS), enabling detection of the Mtb pathogen. Collectively, our findings highlight that F-Tre analogues have potential as tools to probe and unravel Mtb biology and can be exploited to detect and image TB.

10.
11.
Chem Commun (Camb) ; 58(67): 9361-9364, 2022 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-35917119

RESUMO

Dimeric boronic acids kill Mycobacterium tuberculosis (Mtb) by targeting mycobacterial specific extracellular glycans, removing the requirement for a therapeutic agent to permeate the complex cell envelope. Here we report the successful development and use of new 'clickable' boronic acid probes as a powerful method to enable the direct detection and visualisation of this unique class of cell-surface targeting antitubercular agents.


Assuntos
Ácidos Borônicos , Mycobacterium tuberculosis , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Ácidos Borônicos/metabolismo , Ácidos Borônicos/farmacologia , Polímeros/metabolismo , Polissacarídeos/metabolismo
12.
RSC Med Chem ; 13(10): 1225-1233, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36320433

RESUMO

Tuberculosis, caused by Mycobacterium tuberculosis, claims ∼1.5 million lives annually. Effective chemotherapy is essential to control TB, however the emergence of drug-resistant strains of TB have seriously threatened global attempts to control and eradicate this deadly pathogen. Trehalose recycling via the LpqY-SugABC importer is essential for the virulence and survival of Mtb and inhibiting or hijacking this transport system is an attractive approach for the development of novel anti-tubercular and diagnostic agents. Therefore, we interrogated the drug-like compounds in the open-source Medicines for Malaria Pathogen Box and successfully identified seven compounds from the TB, kinetoplastids and reference compound disease sets that recognise LpqY. The molecules have diverse chemical scaffolds, are not specific trehalose analogues, and may be used as novel templates to facilitate the development of therapeutics that kill Mtb with a novel mechanism of action via the mycobacterial trehalose LpqY-SugABC transport system.

13.
Bioorg Med Chem Lett ; 21(4): 1185-90, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21251821

RESUMO

The synthesis and inhibitory potencies of a novel series of ß-amino alcohols, based on the hit-compound 3-[3'-(4''-cyclopent-2'''-en-1'''-ylphenoxy)-2'-hydroxypropyl]-5,5 dimethylimidazolidine-2,4-dione as specific inhibitors of mycobacterial N-acetyltransferase (NAT) enzymes are reported. Effects of synthesised compounds on growth of Mycobacterium tuberculosis have been determined.


Assuntos
Amino Álcoois/química , Antituberculosos/química , Arilamina N-Acetiltransferase/antagonistas & inibidores , Amino Álcoois/síntese química , Amino Álcoois/farmacologia , Antituberculosos/síntese química , Antituberculosos/farmacologia , Arilamina N-Acetiltransferase/metabolismo , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Mycobacterium/efeitos dos fármacos , Mycobacterium/enzimologia , Relação Estrutura-Atividade
14.
Metallomics ; 13(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33693931

RESUMO

The treatment of tuberculosis (TB) poses a major challenge as frontline therapeutic agents become increasingly ineffective with the emergence and spread of drug-resistant strains of Mycobacterium tuberculosis (Mtb). To combat this global health problem, new antitubercular agents with novel modes of action are needed. We have screened a close family of 17 organometallic half-sandwich Os(II) complexes [(arene)Os(phenyl-azo/imino-pyridine)(Cl/I)]+Y- containing various arenes (p-cymene, biphenyl, or terphenyl), and NMe2, F, Cl, or Br phenyl or pyridyl substituents, for activity towards Mtb in comparison with normal human lung cells (MRC5). In general, complexes with a monodentate iodido ligand were more potent than chlorido complexes, and the five most potent iodido complexes (MIC 1.25-2.5 µM) have an electron-donating Me2N or OH substituent on the phenyl ring. As expected, the counter anion Y (PF6-, Cl-, I-) had little effect on the activity. The pattern of potency of the complexes towards Mtb is similar to that towards human cells, perhaps because in both cases intracellular thiols are likely to be involved in their activation and their redox mechanism of action. The most active complex against Mtb is the p-cymene Os(II) NMe2-phenyl-azopyridine iodido complex (2), a relatively inert complex that also exhibits potent activity towards cancer cells. The uptake of Os from complex 2 by Mtb is rapid and peaks after 6 h, with temperature-dependence studies suggesting a major role for active transport. Significance to Metallomics Antimicrobial resistance is a global health problem. New advances are urgently needed in the discovery of new antibiotics with novel mechanisms of action. Half-sandwich organometallic complexes offer a versatile platform for drug design. We show that with an appropriate choice of the arene, an N,N-chelated ligand, and monodentate ligand, half-sandwich organo-osmium(II) complexes can exhibit potent activity towards Mycobacterium tuberculosis (Mtb), the leading cause of death from a single infectious agent. The patterns of activity of the 17 azo- and imino-pyridine complexes studied here towards Mtb and normal lung cells suggest a common redox mechanism of action involving intracellular thiols.


Assuntos
Antineoplásicos/farmacologia , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Compostos Organometálicos/farmacologia , Osmio/química , Tuberculose/tratamento farmacológico , Antineoplásicos/química , Antituberculosos/química , Proliferação de Células , Humanos , Estrutura Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Compostos Organometálicos/química , Tuberculose/microbiologia , Células Tumorais Cultivadas
15.
Cell Surf ; 7: 100052, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34296047

RESUMO

Mycobacterium tuberculosis (Mtb) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: ΔuspC or the entire transport complex: ΔuspABC. Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an Nin-Cin orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and ΔuspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways.

16.
Biochem J ; 418(2): 369-78, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19014350

RESUMO

In Mycobacterium tuberculosis, the genes hsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) and nat (arylamine N-acetyltransferase) are essential for survival inside of host macrophages. These genes act as an operon and have been suggested to be involved in cholesterol metabolism. However, the role of NAT in this catabolic pathway has not been determined. In an effort to better understand the function of these proteins, we have expressed, purified and characterized TBNAT (NAT from M. tuberculosis) and HsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) from M. tuberculosis. Both proteins demonstrated remarkable heat stability with TBNAT and HsaD retaining >95% of their activity after incubation at 60 degrees C for 30 min. The first and second domains of TBNAT were demonstrated to be very important to the heat stability of the protein, as the transfer of these domains caused a dramatic reduction in the heat stability. The specific activity of TBNAT was tested against a broad range of acyl-CoA cofactors using hydralazine as a substrate. TBNAT was found to be able to utilize not just acetyl-CoA, but also n-propionyl-CoA and acetoacetyl-CoA, although at a lower rate. As propionyl-CoA is a product of cholesterol catabolism, we propose that NAT could have a role in the utilization of this important cofactor.


Assuntos
Viabilidade Microbiana , Mycobacterium tuberculosis/fisiologia , Estabilidade Proteica , Temperatura , Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Coenzima A/química , Coenzima A/metabolismo , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Espaço Intracelular/metabolismo , Redes e Vias Metabólicas/genética , Viabilidade Microbiana/genética , Modelos Biológicos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
RSC Med Chem ; 12(1): 43-56, 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34041481

RESUMO

Securing novel, safe, and effective medicines to treat Mycobacterium tuberculosis remains an elusive goal, particularly influenced by the largely impervious Mtb envelope that limits exposure and thus efficacy of inhibitors at their cellular and periplasmic targets. The impact of physicochemical properties on pharmacokinetic parameters that govern oral absorption and exposure at sites of infection is considered alongside how these properties influence penetration of the Mtb envelope, with the likely influence of transporter proteins. The findings are discussed to benchmark current drugs and the emerging pipeline, whilst considering tactics for future rational and targeted design strategies, based around emerging data on Mtb transporters and their structures and functions.

18.
Chem Sci ; 10(23): 5935-5942, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31360399

RESUMO

Innovative chemotherapeutic agents that are active against Mycobacterium tuberculosis (Mtb) are urgently required to control the tuberculosis (TB) epidemic. The Mtb cell envelope has distinct (lipo)polysaccharides and glycolipids that play a critical role in Mtb survival and pathogenesis and disruption of pathways involved in the assembly of the Mtb cell envelope are the primary target of anti-tubercular agents. Here we introduce a previously unexplored approach whereby chemical agents directly target the extracellular glycans within the unique Mtb cell envelope, rather than the intracellular biosynthetic machinery. We designed and synthesised multimeric boronic acids that are selectively lethal to Mtb and function by targeting these structurally unique and essential Mtb cell envelope glycans. By tuning the number of, and distance between, boronic acid units high selectivity to Mtb, low cytotoxicity against mammalian cells and no observable resistance was achieved. This non-conventional approach may prevent the development of drug-resistance and will act as a platform for the design of improved, pathogen-specific, next generation antibiotics.

19.
ACS Chem Biol ; 14(9): 1879-1887, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31433162

RESUMO

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833c) is the substrate binding domain of the UgpABCE transporter that recognizes glycerophosphocholine (GPC), indicating that this transporter has a role in recycling glycerophospholipid metabolites. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography, we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB not only recognizes GPC but is also promiscuous for a broad range of glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis precisely define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC, therefore optimizing the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites via a single transporter during intracellular infection.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Glicerilfosforilcolina/metabolismo , Mycobacterium tuberculosis/química , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato
20.
Curr Drug Metab ; 9(6): 510-9, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18680471

RESUMO

Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure.


Assuntos
Arilamina N-Acetiltransferase/metabolismo , Mycobacterium/enzimologia , Sequência de Aminoácidos , Arilamina N-Acetiltransferase/genética , Conformação Molecular , Dados de Sequência Molecular , Mycobacterium/genética , Polimorfismo Genético
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