Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 16 de 16
Filter
Add more filters










Publication year range
1.
ACS Infect Dis ; 8(11): 2315-2326, 2022 11 11.
Article in English | MEDLINE | ID: mdl-36325756

ABSTRACT

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.


Subject(s)
Mycobacterium tuberculosis , Thioacetazone , Bacterial Proteins/metabolism , Mycolic Acids/chemistry , Thioacetazone/metabolism , Thioacetazone/pharmacology , Hydro-Lyases/chemistry , Hydro-Lyases/metabolism , Hydro-Lyases/pharmacology
2.
Virulence ; 12(1): 1438-1451, 2021 12.
Article in English | MEDLINE | ID: mdl-34107844

ABSTRACT

Mycobacterium ulcerans is the causal agent of Buruli ulcer, a chronic infectious disease and the third most common mycobacterial disease worldwide. Without early treatment, M. ulcerans provokes massive skin ulcers, caused by the mycolactone toxin, its main virulence factor. However, spontaneous healing may occur in Buruli ulcer patients several months or years after the disease onset. We have shown, in an original mouse model, that bacterial load remains high and viable in spontaneously healed tissues, with a switch of M. ulcerans to low levels of mycolactone production, adapting its strategy to survive in such a hostile environment. This original model offers the possibility to investigate the regulation of mycolactone production, by using an RNA-seq strategy to study bacterial adaptation during mouse infection. Pathway analysis and characterization of the tissue environment showed that the bacillus adapted to its new environment by modifying its metabolic activity and switching nutrient sources. Thus, M. ulcerans ensures its survival in healing tissues by reducing its secondary metabolism, leading to an inhibition of mycolactone synthesis. These findings shed new light on mycolactone regulation and pave the way for new therapeutic strategies.


Subject(s)
Buruli Ulcer , Macrolides/metabolism , Mycobacterium Infections , Mycobacterium ulcerans , Adaptation, Biological , Animals , Buruli Ulcer/microbiology , Gene Expression Regulation, Bacterial , Humans , Mice , Mycobacterium Infections/microbiology , Mycobacterium ulcerans/genetics
3.
Article in English | MEDLINE | ID: mdl-33139282

ABSTRACT

Phenotypic screening of inhibitors of the essential Mycobacterium tuberculosis FAS-II dehydratase HadAB led to the identification of GSK3011724A, a compound previously reported to inhibit the condensation step of FAS-II. Whole-cell-based and cell-free assays confirmed the lack of activity of GSK3011724A against the dehydratase despite evidence of cross-resistance between GSK3011724A and HadAB inhibitors. The nature of the resistance mechanisms is suggestive of alterations in the FAS-II interactome reducing access of GSK3011724A to KasA.


Subject(s)
Mycobacterium tuberculosis , Bacterial Proteins/genetics , Fatty Acid Synthase, Type II , Mycolic Acids
4.
ACS Infect Dis ; 6(2): 195-204, 2020 02 14.
Article in English | MEDLINE | ID: mdl-31775512

ABSTRACT

Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs that abolish mycolic acid biosynthesis and kill Mycobacterium tuberculosis (Mtb) through the inhibition of the essential type II fatty acid synthase (FAS-II) dehydratase HadAB. While mutations preventing ISO and TAC either from being converted to their active form or from covalently modifying their target are the most frequent spontaneous mutations associated with high-level resistance to both drugs, the molecular mechanisms underlying the high-level ISO and TAC resistance of Mtb strains harboring missense mutations in the second, nonessential, FAS-II dehydratase HadBC have remained unexplained. Using a combination of genetic, biochemical, and biophysical approaches and molecular dynamics simulation, we here show that all four reported resistance mutations in the HadC subunit of HadBC alter the stability and/or specific activity of the enzyme, allowing it in two cases (HadBCV85I and HadBCK157R) to compensate for a deficiency in HadAB in whole Mtb bacilli. The analysis of the mycolic acid profiles of Mtb strains expressing the mutated forms of HadC further points to alterations in the activity of the mycolic acid biosynthetic complex and suggests an additional contributing resistance mechanism whereby HadC mutations may reduce the accessibility of HadAB to ISO and TAC. Collectively, our results highlight the importance of developing optimized inhibitors of the dehydration step of FAS-II capable of inhibiting both dehydratases simultaneously, a goal that may be achievable given the structural resemblance of the two enzymes and their reliance on the same catalytic subunit HadB.


Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/genetics , Fatty Acid Synthase, Type II/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Bacterial Proteins/genetics , Dehydration , Molecular Dynamics Simulation , Mutation , Mycobacterium tuberculosis/enzymology , Mycolic Acids/analysis
5.
FEMS Microbiol Lett ; 364(4)2017 02 01.
Article in English | MEDLINE | ID: mdl-28130366

ABSTRACT

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis.


Subject(s)
Gene Expression , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/pathogenicity , Pentosyltransferases/genetics , Tuberculosis/microbiology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Disease Models, Animal , Gene Knockout Techniques , Mice , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Pentosyltransferases/metabolism , Uracil/metabolism , Uracil/pharmacology , Virulence
6.
J Biol Chem ; 291(36): 18867-79, 2016 09 02.
Article in English | MEDLINE | ID: mdl-27417139

ABSTRACT

The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development. Despite intensive efforts to identify the ligase(s) responsible for the covalent attachment of the two major heteropolysaccharides of the mycobacterial cell wall, arabinogalactan (AG) and peptidoglycan (PG), the enzyme or enzymes responsible have remained elusive. We here report on the identification of the two enzymes of Mycobacterium tuberculosis, CpsA1 (Rv3267) and CpsA2 (Rv3484), responsible for this function. CpsA1 and CpsA2 belong to the widespread LytR-Cps2A-Psr (LCP) family of enzymes that has been shown to catalyze a variety of glycopolymer transfer reactions in Gram-positive bacteria, including the attachment of wall teichoic acids to PG. Although individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtained, the combined inactivation of both genes appears to be lethal. In the closely related microorganism Corynebacterium glutamicum, the ortholog of cpsA1 is the only gene involved in this function, and its conditional knockdown leads to dramatic changes in the cell wall composition and morphology of the bacteria due to extensive shedding of cell wall material in the culture medium as a result of defective attachment of AG to PG. This work marks an important step in our understanding of the biogenesis of the unique cell envelope of mycobacteria and opens new opportunities for drug development.


Subject(s)
Bacterial Proteins/genetics , Cell Wall/metabolism , Galactans/metabolism , Mycobacterium tuberculosis/metabolism , Peptidoglycan/metabolism , Teichoic Acids/metabolism , Bacterial Proteins/metabolism , Cell Wall/genetics , Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , Galactans/genetics , Mycobacterium tuberculosis/genetics , Peptidoglycan/genetics , Teichoic Acids/genetics
7.
ACS Infect Dis ; 1(2): 91-97, 2015 Feb 13.
Article in English | MEDLINE | ID: mdl-25897434

ABSTRACT

Isoxyl and Thiacetazone are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation and the nature of their activated forms remained unknown. We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity. Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs. Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

8.
Antimicrob Agents Chemother ; 58(11): 6413-23, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25136022

ABSTRACT

MmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis. Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis, including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.


Subject(s)
Adamantane/analogs & derivatives , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Ethylenediamines/pharmacology , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , Adamantane/pharmacology , Anti-Bacterial Agents/pharmacology , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Carrier Proteins/antagonists & inhibitors , Cell Membrane , Cord Factors/metabolism , Drug Resistance, Multiple, Bacterial , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/biosynthesis , Membrane Transport Proteins , Microbial Sensitivity Tests , Mycolic Acids/metabolism , Phenylurea Compounds/pharmacology , Piperazines/pharmacology , Proton Ionophores/pharmacology , Pyrroles/pharmacology , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiology , Valinomycin/pharmacology , Vitamin K 2/metabolism
9.
Nat Commun ; 4: 2748, 2013.
Article in English | MEDLINE | ID: mdl-24193546

ABSTRACT

The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen (EBS) as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that EBS binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of EBS shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antigens, Bacterial/metabolism , Azoles/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Organoselenium Compounds/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Antigens, Bacterial/genetics , Azoles/chemistry , Isoindoles , Membrane Proteins , Models, Molecular , Molecular Structure , Mutation , Mycobacterium tuberculosis/genetics , Organoselenium Compounds/chemistry , Protein Binding , Protein Conformation , Saccharomyces cerevisiae Proteins
10.
Methods Mol Biol ; 966: 309-24, 2013.
Article in English | MEDLINE | ID: mdl-23299743

ABSTRACT

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is the source of carbohydrates of exceptional structure which play essential roles in the physiology of the bacterium and in its interactions with the host during infection. Much of what is known about their biosynthesis was derived from the phenotypic analysis of knockout or conditional knockout mutants of mycobacteria generated by random or specific insertional mutagenesis. Here, we describe the current techniques used to subfractionate M. tuberculosis cells and investigate major quantitative and qualitative changes in their cell envelope (lipo)polysaccharides.


Subject(s)
Cell Wall/metabolism , Lipopolysaccharides/isolation & purification , Mycobacterium tuberculosis/metabolism , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Gas Chromatography-Mass Spectrometry , Lipopolysaccharides/metabolism , Molecular Sequence Data
11.
J Biol Chem ; 287(46): 38434-41, 2012 Nov 09.
Article in English | MEDLINE | ID: mdl-23002234

ABSTRACT

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.


Subject(s)
Mycobacterium bovis/metabolism , Mycobacterium tuberculosis/metabolism , Mycolic Acids/antagonists & inhibitors , Phenylthiourea/analogs & derivatives , Thioacetazone/pharmacology , Alleles , Antitubercular Agents/pharmacology , Cell Wall/metabolism , Chromatography, Liquid/methods , Fatty Acid Synthases/metabolism , Gas Chromatography-Mass Spectrometry/methods , Genome, Bacterial , Lipids/chemistry , Mass Spectrometry/methods , Models, Chemical , Phenylthiourea/pharmacology , Recombinant Proteins/chemistry , Sequence Analysis, DNA , Time Factors
12.
Bioorg Med Chem ; 20(10): 3255-62, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22522007

ABSTRACT

Adamantyl ureas were previously identified as a group of compounds active against Mycobacterium tuberculosis in culture with minimum inhibitor concentrations (MICs) below 0.1 µg/ml. These compounds have been shown to target MmpL3, a protein involved in secretion of trehalose mono-mycolate. They also inhibit both human soluble epoxide hydrolase (hsEH) and M. tuberculosis epoxide hydrolases. However, active compounds to date have high cLogP's and are poorly soluble, leading to low bioavailability and thus limiting any therapeutic application. In this study, a library of 1600 ureas (mostly adamantyl ureas), which were synthesized for the purpose of increasing the bioavailability of inhibitors of hsEH, was screened for activity against M. tuberculosis. 1-Adamantyl-3-phenyl ureas with a polar para substituent were found to retain moderate activity against M. tuberculosis and one of these compounds was shown to be present in serum after oral administration to mice. However, neither it, nor a closely related analog, reduced M. tuberculosis infection in mice. No correlation between in vitro potency against M. tuberculosis and the hsEH inhibition were found supporting the concept that activity against hsEH and M. tuberculosis can be separated. Also there was a lack of correlation with cLogP and inhibition of the growth of M. tuberculosis. Finally, members of two classes of adamantyl ureas that contained polar components to increase their bioavailability, but lacked efficacy against growing M. tuberculosis, were found to taken up by the bacterium as effectively as a highly active apolar urea suggesting that these modifications to increase bioavailability affected the interaction of the urea against its target rather than making them unable to enter the bacterium.


Subject(s)
Adamantane/chemistry , Antitubercular Agents/pharmacology , Antitubercular Agents/pharmacokinetics , Drug Evaluation, Preclinical , Mycobacterium tuberculosis/drug effects , Urea/pharmacology , Urea/pharmacokinetics , Adamantane/pharmacokinetics , Adamantane/pharmacology , Animals , Antitubercular Agents/chemistry , Biological Availability , Humans , Mice , Microbial Sensitivity Tests , Molecular Structure , Urea/chemistry
13.
Nat Chem Biol ; 8(4): 334-41, 2012 Feb 19.
Article in English | MEDLINE | ID: mdl-22344175

ABSTRACT

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.


Subject(s)
Adamantane/analogs & derivatives , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cell Membrane/metabolism , Mycobacterium tuberculosis/drug effects , Mycolic Acids/metabolism , Phenylurea Compounds/pharmacology , Adamantane/chemistry , Adamantane/pharmacology , Anti-Bacterial Agents/pharmacokinetics , Bacterial Proteins/metabolism , Biological Transport/drug effects , Cell Membrane/drug effects , Cord Factors , Drug Evaluation, Preclinical/methods , Drug Resistance, Bacterial , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Phenylurea Compounds/chemistry , Small Molecule Libraries , Trehalose/metabolism
14.
Microbiology (Reading) ; 156(Pt 5): 1497-1504, 2010 May.
Article in English | MEDLINE | ID: mdl-20093291

ABSTRACT

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6'-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.


Subject(s)
Cell Wall/metabolism , Mycobacteriophages/enzymology , Mycobacterium smegmatis/metabolism , Viral Proteins/metabolism , Cell Wall/chemistry , Esters/metabolism , Galactans/metabolism , Hydrolysis , Membrane Lipids/metabolism , Mycobacterium bovis/metabolism , Mycobacterium tuberculosis/metabolism , Mycolic Acids/metabolism , Polysaccharides, Bacterial/metabolism , Substrate Specificity , Trehalose/metabolism
15.
Bioorg Med Chem ; 18(2): 896-908, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-19969466

ABSTRACT

High-throughput screening of 201,368 compounds revealed that 1-(3-(5-ethyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one (SID 7975595) inhibited RmlC a TB cell wall biosynthetic enzyme. SID 7975595 acts as a competitive inhibitor of the enzyme's substrate and inhibits RmlC as a fast-on rate, fully reversible inhibitor. An analog of SID 7975595 had a K(i) of 62nM. Computer modeling showed that the binding of the tethered two-ringed system into the active site occurred at the thymidine binding region for one ring system and the sugar region for the other ring system.


Subject(s)
Benzimidazoles/pharmacology , Carbohydrate Epimerases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Mycobacterium tuberculosis/enzymology , Aorta/cytology , Aorta/drug effects , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Catalytic Domain , Cell Survival/drug effects , Computer Simulation , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , High-Throughput Screening Assays , Humans , Indoles/chemical synthesis , Indoles/chemistry , Models, Chemical , Structure-Activity Relationship
16.
Microbiology (Reading) ; 154(Pt 12): 3724-3730, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19047740

ABSTRACT

In Mycobacterium tuberculosis a rhamnosyltransferase (WbbL) catalyses the transfer of an alpha-L-Rhap residue from dTDP-L-rhamnose (dTDP-Rha) to decaprenyldiphosphoryl-alpha-D-N-acetylglucosamine (GlcNAc-P-P-DP) to form alpha-L-Rhap-(1-->3)-alpha-D-GlcNAc-P-P-DP, which is then further elongated with Galf and Araf units, and finally mycolylated and attached to the peptidoglycan. This enzyme is essential for M. tuberculosis viability and at the same time absent in eukaryotic cells, and is therefore a good target for the development of new antituberculosis therapeutics. Here, we report a microtitre plate-based method for the assay of this enzyme using a crude membrane preparation from an Escherichia coli strain overexpressing wbbL as an enzyme source and the natural acceptor substrate GlcNAc-P-P-DP. Initial characterization of the enzyme included unequivocal identification of the product Rha-GlcNAc-P-P-DP by liquid chromatography (LC)-MS, and the facts that WbbL shows an absolute requirement for divalent cations and that its activity is stimulated by beta-mercaptoethanol. Its pH optimum and basic kinetic parameters were also determined, and the kinetic analysis showed that WbbL uses a ternary complex mechanism. The microtitre plate-based assay for this enzyme was developed by taking advantage of the lipophilic nature of the product. This assay should be readily transferable to other glycosyltransferases which use lipid-based acceptors and aid greatly in obtaining inhibitors of such glycosyltransferases for new drug development.


Subject(s)
Bacterial Proteins/metabolism , Cell Wall/enzymology , Glycosyltransferases/metabolism , Hexosyltransferases/metabolism , Lipids/chemistry , Mycobacterium tuberculosis/enzymology , Acetylglucosamine/chemistry , Acetylglucosamine/metabolism , Bacteriological Techniques/instrumentation , Bacteriological Techniques/methods , Chromatography, Liquid , Escherichia coli/enzymology , Escherichia coli/genetics , Hydrogen-Ion Concentration , Kinetics , Mass Spectrometry , Mycobacterium tuberculosis/genetics , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...