RESUMO
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.
Assuntos
Mycobacterium tuberculosis , Proteínas de Bactérias/genética , Ácido Graxo Sintase Tipo II , Ácidos MicólicosRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Parede Celular/metabolismo , Galactanos/metabolismo , Mycobacterium tuberculosis/metabolismo , Peptidoglicano/metabolismo , Ácidos Teicoicos/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Galactanos/genética , Mycobacterium tuberculosis/genética , Peptidoglicano/genética , Ácidos Teicoicos/genéticaRESUMO
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.
Assuntos
Adamantano/análogos & derivados , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Etilenodiaminas/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Adamantano/farmacologia , Antibacterianos/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Membrana Celular , Fatores Corda/metabolismo , Farmacorresistência Bacteriana Múltipla , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Ácidos Micólicos/metabolismo , Compostos de Fenilureia/farmacologia , Piperazinas/farmacologia , Ionóforos de Próton/farmacologia , Pirróis/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Valinomicina/farmacologia , Vitamina K 2/metabolismoRESUMO
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.
Assuntos
Adamantano/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/metabolismo , Compostos de Fenilureia/farmacologia , Adamantano/química , Adamantano/farmacologia , Antibacterianos/farmacocinética , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Fatores Corda , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Bacteriana , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Compostos de Fenilureia/química , Bibliotecas de Moléculas Pequenas , Trealose/metabolismoRESUMO
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.
Assuntos
Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/antagonistas & inibidores , Feniltioureia/análogos & derivados , Tioacetazona/farmacologia , Alelos , Antituberculosos/farmacologia , Parede Celular/metabolismo , Cromatografia Líquida/métodos , Ácido Graxo Sintases/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Genoma Bacteriano , Lipídeos/química , Espectrometria de Massas/métodos , Modelos Químicos , Feniltioureia/farmacologia , Proteínas Recombinantes/química , Análise de Sequência de DNA , Fatores de TempoRESUMO
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.
Assuntos
Adamantano/química , Antituberculosos/farmacologia , Antituberculosos/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Ureia/farmacologia , Ureia/farmacocinética , Adamantano/farmacocinética , Adamantano/farmacologia , Animais , Antituberculosos/química , Disponibilidade Biológica , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ureia/químicaRESUMO
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.
Assuntos
Mycobacterium tuberculosis , Tioacetazona , Proteínas de Bactérias/metabolismo , Ácidos Micólicos/química , Tioacetazona/metabolismo , Tioacetazona/farmacologia , Hidroliases/química , Hidroliases/metabolismo , Hidroliases/farmacologiaRESUMO
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.
Assuntos
Úlcera de Buruli , Macrolídeos/metabolismo , Infecções por Mycobacterium , Mycobacterium ulcerans , Adaptação Biológica , Animais , Úlcera de Buruli/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Infecções por Mycobacterium/microbiologia , Mycobacterium ulcerans/genéticaRESUMO
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.
Assuntos
Parede Celular/metabolismo , Micobacteriófagos/enzimologia , Mycobacterium smegmatis/metabolismo , Proteínas Virais/metabolismo , Parede Celular/química , Ésteres/metabolismo , Galactanos/metabolismo , Hidrólise , Lipídeos de Membrana/metabolismo , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Especificidade por Substrato , Trealose/metabolismoRESUMO
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.
Assuntos
Benzimidazóis/farmacologia , Carboidratos Epimerases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Mycobacterium tuberculosis/enzimologia , Aorta/citologia , Aorta/efeitos dos fármacos , Benzimidazóis/síntese química , Benzimidazóis/química , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Humanos , Indóis/síntese química , Indóis/química , Modelos Químicos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas de Bactérias/genética , Desidratação , Simulação de Dinâmica Molecular , Mutação , Mycobacterium tuberculosis/enzimologia , Ácidos Micólicos/análiseRESUMO
The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5Î-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.
Assuntos
Expressão Gênica , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Pentosiltransferases/genética , Tuberculose/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Pentosiltransferases/metabolismo , Uracila/metabolismo , Uracila/farmacologia , VirulênciaRESUMO
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.
RESUMO
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.
Assuntos
Parede Celular/metabolismo , Lipopolissacarídeos/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Lipopolissacarídeos/metabolismo , Dados de Sequência MolecularRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antígenos de Bactérias/metabolismo , Azóis/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Compostos Organosselênicos/farmacologia , Anti-Inflamatórios não Esteroides/química , Antígenos de Bactérias/genética , Azóis/química , Isoindóis , Proteínas de Membrana , Modelos Moleculares , Estrutura Molecular , Mutação , Mycobacterium tuberculosis/genética , Compostos Organosselênicos/química , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiaeRESUMO
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.