RESUMO
BACKGROUND AND AIMS: Chronic liver disease leads to ~2 million deaths annually. Cyclic AMP (cAMP) signaling has long been studied in liver injury, particularly in the regulation of fatty acid (FA) ß-oxidation and pro-inflammatory polarization of tissue-resident lymphocytes. Phosphodiesterase 4B inhibition has been explored as a therapeutic modality, but these drugs have had limited success and are known to cause significant adverse effects. The PDE4 inhibitor 2-(4-([2-(5-Chlorothiophen-2-yl)-5-ethyl-6-methylpyrimidin-4-yl]amino)phenyl)acetic acid) (known as A-33) has yet to be explored for the treatment of metabolic diseases. APPROACH AND RESULTS: Herein, we evaluated the efficacy of A-33 in the treatment of animal models of alcohol-associated liver disease and steatotic liver disease. We demonstrated that A-33 effectively ameliorated the signs and symptoms of chronic liver disease, resulting in significant decreases in serum alanine aminotransferase and aspartate aminotransferase levels, decreased overall fat and collagen deposition in the liver, decreased intrahepatic triglyceride concentrations, and normalized expression of genes related to ß-oxidation of fatty acids, inflammation, and extracellular matrix deposition. We also designed and synthesized a novel analog of A-33, termed MDL3, which inhibited both phosphodiesterase 4B and PDE5A and was more effective in ameliorating pathophysiological signs and symptoms of liver injury and inflammation. In addition, MDL3 re-sensitized obese mice to glucose and significantly inhibited the pathological remodeling of adipose tissue, which was not observed with A-33 administration. CONCLUSIONS: In conclusion, we synthesized and demonstrated that MDL3, a novel phosphodiesterase 4B and PDE5A inhibitor, presents a promising avenue of exploration for treating chronic liver disease.
RESUMO
The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.
Assuntos
Elementos de DNA Transponíveis/genética , Transposases/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Catálise , Cristalização , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Dimerização , Ativação Enzimática , Helicobacter pylori/enzimologia , Ligação de Hidrogênio , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transposases/química , Transposases/genética , Tirosina/genética , Tirosina/metabolismoRESUMO
The Mycobacterium tuberculosis rv3802c gene encodes an essential enzyme with thioesterase and phospholipase A activity. Overexpression of Rv3802 orthologs in Mycobacterium smegmatis and Corynebacterium glutamicum increases mycolate content and decreases glycerophospholipids. Although a role in modulating the lipid composition of the unique mycomembrane has been proposed, the true biological function of Rv3802 remains uncertain. In this study, we present the first M. tuberculosis Rv3802 X-ray crystal structure, solved to 1.7 Å resolution. On the basis of the binding of PEG molecules to Rv3802, we identified its lipid-binding site and the structural basis for phosphatidyl-based substrate binding and phospholipase A activity. We found that movement of the α8-helix affords lipid binding and is required for catalytic turnover through covalent tethering. We gained insights into the mechanism of acyl hydrolysis by observing differing arrangements of PEG and water molecules within the active site. This study provides structural insights into biological function and facilitates future structure-based drug design toward Rv3802.
Assuntos
Proteínas de Bactérias/química , Lipídeos/química , Mycobacterium tuberculosis/enzimologia , Fosfolipases/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Fosfolipases/metabolismo , Ligação ProteicaRESUMO
Mycobacterium tuberculosis antigen 85 (Ag85) enzymes catalyze the transfer of mycolic acid (MA) from trehalose monomycolate to produce the mycolyl arabinogalactan (mAG) or trehalose dimycolate (TDM). These lipids define the protective mycomembrane of mycobacteria. The current model of substrate binding within the active sites of Ag85s for the production of TDM is not sterically and geometrically feasible; additionally, this model does not account for the production of mAG. Furthermore, this model does not address how Ag85s limit the hydrolysis of the acyl-enzyme intermediate while catalyzing acyl transfer. To inform an updated model, we obtained an Ag85 acyl-enzyme intermediate structure that resembles the mycolated form. Here, we present a 1.45-Å X-ray crystal structure of M. tuberculosis Ag85C covalently modified by tetrahydrolipstatin (THL), an esterase inhibitor that suppresses M. tuberculosis growth and mimics structural attributes of MAs. The mode of covalent inhibition differs from that observed in the reversible inhibition of the human fatty-acid synthase by THL. Similarities between the Ag85-THL structure and previously determined Ag85C structures suggest that the enzyme undergoes structural changes upon acylation, and positioning of the peptidyl arm of THL limits hydrolysis of the acyl-enzyme adduct. Molecular dynamics simulations of the modeled mycolated-enzyme form corroborate the structural analysis. From these findings, we propose an alternative arrangement of substrates that rectifies issues with the previous model and suggest a direct role for the ß-hydroxy of MA in the second half-reaction of Ag85 catalysis. This information affords the visualization of a complete mycolyltransferase catalytic cycle.
Assuntos
Aciltransferases/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Orlistate/metabolismo , Processamento de Proteína Pós-Traducional , Acilação , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Aciltransferases/genética , Substituição de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Biocatálise , Configuração de Carboidratos , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Orlistate/química , Conformação Proteica , Proteólise , Proteínas Recombinantes , Trealose/química , Trealose/metabolismoRESUMO
α,α'-Trehalose plays roles in the synthesis of several cell wall components involved in pathogenic mycobacteria virulence. Its absence in mammalian biochemistry makes trehalose-related biochemical processes potential targets for chemotherapy. The trehalose 6-phosphate synthase (TPS)/trehalose 6-phosphate phosphatase (TPP) pathway, also known as the OtsA/OtsB2 pathway, is the major pathway involved in the production of trehalose in Mycobacterium tuberculosis (Mtb). In addition, TPP is essential for Mtb survival. We describe the synthesis of α,α'-trehalose derivatives in the forms of the 6-phosphonic acid 4 (TMP), the 6-methylenephosphonic acid 5 (TEP), and the 6-N-phosphonamide 6 (TNP). These non-hydrolyzable substrate analogues of TPP were examined as inhibitors of Mtb, Mycobacterium lentiflavum (Mlt), and Mycobacterium triplex (Mtx) TPP. In all cases the compounds were most effective in inhibiting Mtx TPP, with TMP [IC50 =(288±32)â µm] acting most strongly, followed by TNP [IC50 =(421±24)â µm] and TEP [IC50 =(1959±261)â µm]. The results also indicate significant differences in the analogue binding profile when comparing Mtb TPP, Mlt TPP, and Mtx TPP homologues.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Trealose/farmacologia , Configuração de Carboidratos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosiltransferases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade , Trealose/síntese química , Trealose/químicaRESUMO
MTAN (5'-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.
Assuntos
Formicinas/química , Helicobacter pylori/enzimologia , Purina-Núcleosídeo Fosforilase/química , S-Adenosil-Homocisteína/química , Adenina/análogos & derivados , Adenina/química , Domínio Catalítico/genética , Cristalografia por Raios X , Desoxiadenosinas/química , Helicobacter pylori/química , Ligação de Hidrogênio , Modelos Moleculares , Nêutrons , Ligação Proteica , Prótons , Purina-Núcleosídeo Fosforilase/genética , Pirrolidinas/química , Especificidade por Substrato , Tionucleosídeos/químicaRESUMO
Tetrahydrolipstatin (THL) is a covalent inhibitor of many serine esterases. In mycobacteria, THL has been found to covalently react with 261 lipid esterases upon treatment of Mycobacterium bovis cell lysate. However, the covalent adduct is considered unstable in some cases because of the hydrolysis of the enzyme-linked THL adduct resulting in catalytic turnover. In this study, a library of THL stereoderivatives was tested against three essential Mycobacterium tuberculosis lipid esterases of interest for drug development to assess how the stereochemistry of THL affects respective enzyme inhibition and allows for cross enzyme inhibition. The mycolyltransferase Antigen 85C (Ag85C) was found to be stereospecific with regard to THL; covalent inhibition occurs within minutes and was previously shown to be irreversible. Conversely, the Rv3802 phospholipase A/thioesterase was more accepting of a variety of THL configurations and uses these compounds as alternative substrates. The reaction of the THL stereoderivatives with the thioesterase domain of polyketide synthase 13 (Pks13-TE) also leads to hydrolytic turnover and is nonstereospecific but occurs on a slower, multihour time scale. Our findings suggest the stereochemistry of the ß-lactone ring of THL is important for cross enzyme reactivity, while the two stereocenters of the peptidyl arm can affect enzyme specificity and the catalytic hydrolysis of the ß-lactone ring. The observed kinetic data for all three target enzymes are supported by recently published X-ray crystal structures of Ag85C, Rv3802, and Pks13-TE. Insights from this study provide a molecular basis for the kinetic modulation of three essential M. tuberculosis lipid esterases by THL and can be applied to increase potency and enzyme residence times and enhance the specificity of the THL scaffold.
Assuntos
Esterases/antagonistas & inibidores , Lactonas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Esterases/química , Humanos , Hidrólise , Cinética , Lactonas/química , Lipase/química , Lipídeos/antagonistas & inibidores , Lipídeos/química , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Orlistate , Tuberculose/enzimologia , Tuberculose/microbiologiaRESUMO
S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5'-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a Kd value of 11.3 ± 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of l-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5'-methylthioadenosine, AMP, and ADP.
Assuntos
Polarização de Fluorescência/métodos , S-Adenosil-Homocisteína/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Helicobacter pylori/enzimologia , Cinética , Limite de Detecção , Metiltransferases/química , Metiltransferases/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/isolamento & purificação , Rodaminas/síntese química , Rodaminas/químicaRESUMO
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
2-Alkyl-1,2-benzisoselenazol-3(2H)-ones, represented by ebselen (1a), are being studied intensively for a range of medicinal applications. We describe both a new thermal and photoinduced copper-mediated cross-coupling between potassium selenocyanate (KSeCN) and N-substituted ortho-halobenzamides to form 2-alkyl-1,2-benzisoselenazol-3(2H)-ones containing a C-Se-N bond. The copper ligand (1,10-phenanthroline) facilitates C-Se bond formation during heating via a mechanism that likely involves atom transfer (AT), whereas, in the absence of ligand, photoinduced activation likely proceeds through a single electron transfer (SET) mechanism. A library of 15 2-alkyl-1,2-benzisoselenazol-3(2H)-ones was prepared. One member of the library was azide-containing derivative 1j that was competent to undergo a strain-promoted azide-alkyne cycloaddition. The library was evaluated for inhibition of Mycobacterium tuberculosis (Mtb) growth and Mtb Antigen 85C (Mtb Ag85C) activity. Compound 1f was most potent with a minimal inhibitory concentration (MIC) of 12.5 µg/mL and an Mtb Ag85C apparent IC50 of 8.8 µM.
Assuntos
Antituberculosos/farmacologia , Cobre/química , Mycobacterium tuberculosis/efeitos dos fármacos , Compostos de Selênio/farmacologia , Antituberculosos/química , Carbono/química , Testes de Sensibilidade Microbiana , Processos Fotoquímicos , Compostos de Selênio/químicaRESUMO
Correction for 'Zwitterionic pyrrolidene-phosphonates: inhibitors of the glycoside hydrolase-like phosphorylase Streptomyces coelicolor GlgEI-V279S' by Sri Kumar Veleti et al., Org. Biomol. Chem., 2017, 15, 3884-3891.
RESUMO
We synthesized and evaluated new zwitterionic inhibitors against glycoside hydrolase-like phosphorylase Streptomyces coelicolor (Sco) GlgEI-V279S which plays a role in α-glucan biosynthesis. Sco GlgEI-V279S serves as a model enzyme for validated anti-tuberculosis (TB) target Mycobacterium tuberculosis (Mtb) GlgE. Pyrrolidine inhibitors 5 and 6 were designed based on transition state considerations and incorporate a phosphonate on the pyrrolidine moiety to expand the interaction network between the inhibitor and the enzyme active site. Compounds 5 and 6 inhibited Sco GlgEI-V279S with Ki = 45 ± 4 µM and 95 ± 16 µM, respectively, and crystal structures of Sco GlgE-V279S-5 and Sco GlgE-V279S-6 were obtained at a 3.2 Å and 2.5 Å resolution, respectively.
Assuntos
Glicosídeo Hidrolases/antagonistas & inibidores , Organofosfonatos/química , Fosforilases/antagonistas & inibidores , Pirróis/química , Pirróis/farmacologia , Streptomyces coelicolor/enzimologia , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Fosforilases/química , Conformação ProteicaRESUMO
Tuberculosis (TB) and its drug resistant forms kills more people than any other infectious disease. This fact emphasizes the need to identify new drugs to treat TB. 2-Aminothiophenes (2AT) have been reported to inhibit Pks13, a validated anti-TB drug target. We synthesized a library of 42 2AT compounds. Among these, compound 33 showed remarkable potency against Mycobacterium tuberculosis (Mtb) H37RV (MIC = 0.23 µM) and showed an impressive potency (MIC = 0.20-0.44 µM) against Mtb strains resistant to isoniazid, rifampicin and fluoroquinolones. The site of action for the compound 33 is presumed to be Pks13 or an earlier enzyme in the mycolic acid biosynthetic pathway. This inference is based on structural similarity of the compound 33 with known Pks13 inhibitors, which is corroborated by mycolic acid biosynthesis studies showing that the compound strongly inhibits the biosynthesis of all forms of mycolic acid in Mtb. In summary, these studies suggest 33 represents a promising anti-TB lead that exhibits activity well below toxicity to human monocytic cells.
Assuntos
Antituberculosos/síntese química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tiofenos/síntese química , Tiofenos/farmacologia , Antituberculosos/química , Técnicas de Química Sintética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Tiofenos/químicaRESUMO
The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.
Assuntos
Aciltransferases/química , Antígenos de Bactérias/química , Cisteína/química , Inibidores Enzimáticos/química , Aciltransferases/genética , Aciltransferases/metabolismo , Regulação Alostérica , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Azóis/química , Azóis/farmacologia , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Cloromercurobenzoatos/química , Cloromercurobenzoatos/farmacologia , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ligação de Hidrogênio/efeitos dos fármacos , Iodoacetamida/química , Iodoacetamida/farmacologia , Isoindóis , Modelos Moleculares , Estrutura Molecular , Mutação , Compostos Organosselênicos/química , Compostos Organosselênicos/farmacologia , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
DNA extraction represents a significant bottleneck in nucleic acid analysis. In this study, hydrophobic magnetic ionic liquids (MILs) were synthesized and employed as solvents for the rapid and efficient extraction of DNA from aqueous solution. The DNA-enriched microdroplets were manipulated by application of a magnetic field. The three MILs examined in this study exhibited unique DNA extraction capabilities when applied toward a variety of DNA samples and matrices. High extraction efficiencies were obtained for smaller single-stranded and double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III) ([(C8)3BnN(+)][FeCl3Br(-)]) MIL, while the dicationic 1,12-di(3-hexadecylbenzimidazolium)dodecane bis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III) ([(C16BnIM)2C12(2+)][NTf2(-), FeCl3Br(-)]) MIL produced higher extraction efficiencies for larger DNA molecules. The MIL-based method was also employed for the extraction of DNA from a complex matrix containing albumin, revealing a competitive extraction behavior for the trihexyl(tetradecyl)phosphonium tetrachloroferrate(III) ([P6,6,6,14(+)][FeCl4(-)]) MIL in contrast to the [(C8)3BnN(+)][FeCl3Br(-)] MIL, which resulted in significantly less coextraction of albumin. The MIL-DNA method was employed for the extraction of plasmid DNA from bacterial cell lysate. DNA of sufficient quality and quantity for polymerase chain reaction (PCR) amplification was recovered from the MIL extraction phase, demonstrating the feasibility of MIL-based DNA sample preparation prior to downstream analysis.
Assuntos
DNA/isolamento & purificação , Líquidos Iônicos/química , Imãs/química , DNA/análise , Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Fenômenos Magnéticos , Plasmídeos/análise , Plasmídeos/isolamento & purificação , SolventesRESUMO
Correction for 'Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S' by Sandeep Thanna et al., Org. Biomol. Chem., 2015, DOI: 10.1039/c5ob00867k.
RESUMO
Streptomyces coelicolor (Sco) GlgEI is a glycoside hydrolase involved in α-glucan biosynthesis and can be used as a model enzyme for structure-based inhibitor design targeting Mycobacterium tuberculosis (Mtb) GlgE. The latter is a genetically validated drug target for the development of anti-Tuberculosis (TB) treatments. Inhibition of Mtb GlgE results in a lethal buildup of the GlgE substrate maltose-1-phosphate (M1P). However, Mtb GlgE is difficult to crystallize and affords lower resolution X-ray structures. Sco GlgEI-V279S on the other hand crystallizes readily, produces high resolution X-ray data, and has active site topology identical to Mtb GlgE. We report the X-ray structure of Sco GlgEI-V279S in complex with 2-deoxy-2,2-difluoro-α-maltosyl fluoride (α-MTF, 5) at 2.3 Å resolution. α-MTF was designed as a non-hydrolysable mimic of M1P to probe the active site of GlgE1 prior to covalent bond formation without disruption of catalytic residues. The α-MTF complex revealed hydrogen bonding between Glu423 and the C1F which provides evidence that Glu423 functions as proton donor during catalysis. Further, hydrogen bonding between Arg392 and the axial C2 difluoromethylene moiety of α-MTF was observed suggesting that the C2 position tolerates substitution with hydrogen bond acceptors. The key step in the synthesis of α-MDF was transformation of peracetylated 2-fluoro-maltal 1 into peracetylated 2,2-difluoro-α-maltosyl fluoride 2 in a single step via the use of Selectfluor®.
Assuntos
Glicosídeo Hidrolases/química , Maltose/análogos & derivados , Maltose/química , Maltose/síntese química , Streptomyces coelicolor/enzimologia , Biocatálise/efeitos dos fármacos , Bioensaio , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Maltose/farmacologia , Modelos Moleculares , Especificidade por Substrato/efeitos dos fármacosRESUMO
Long treatment times, poor drug compliance, and natural selection during treatment of Mycobacterium tuberculosis (Mtb) have given rise to extensively drug-resistant tuberculosis (XDR-TB). As a result, there is a need to identify new antituberculosis drug targets. Mtb GlgE is a maltosyl transferase involved in α-glucan biosynthesis. Mutation of GlgE in Mtb increases the concentration of maltose-1-phosphate (M1P), one substrate for GlgE, causing rapid cell death. We have designed 2,5-dideoxy-3-O-α-d-glucopyranosyl-2,5-imino-d-mannitol (9) to act as an inhibitor of GlgE. Compound 9 was synthesized using a convergent synthesis by coupling thioglycosyl donor 14 and 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-ß-d-fructopyranose (23) to form disaccharide 24. A reduction and intramolecular reductive amination transformed the intermediate disaccharide 24 to the desired pyrolidine 9. Compound 9 inhibited both Mtb GlgE and a variant of Streptomyces coelicolor (Sco) GlgEI with Ki = 237 ± 27 µM and Ki = 102 ± 7.52 µM, respectively. The results confirm that a Sco GlgE-V279S variant can be used as a model for Mtb GlgE. In conclusion, we designed a lead transition state inhibitor of GlgE, which will be instrumental in further elucidation of the enzymatic mechanism of Mtb GlgE.
Assuntos
Antituberculosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Dissacarídeos/síntese química , Farmacorresistência Bacteriana/efeitos dos fármacos , Glucanos/biossíntese , Glucanos/química , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/química , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efeitos dos fármacos , Fosfatos Açúcares/química , Antituberculosos/química , Antituberculosos/farmacologia , Dissacarídeos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/metabolismo , Estrutura Molecular , Mycobacterium tuberculosis/metabolismoRESUMO
The emergence of extensively drug-resistant tuberculosis (XDR-TB) necessitates the need to identify new anti-tuberculosis drug targets as well as to better understand essential biosynthetic pathways. GlgE is a Mycobacterium tuberculosis (Mtb) encoded maltosyltransferase involved in α-glucan biosynthesis. Deletion of GlgE in Mtb results in the accumulation of M1P within cells leading to rapid death of the organism. To inhibit GlgE a maltose-C-phosphonate (MCP) 13 was designed to act as an isosteric non-hydrolysable mimic of M1P. MCP 13, the only known inhibitor of Mtb GlgE, was successfully synthesized using a Wittig olefination as a key step in transforming maltose to the desired product. MCP 13 inhibited Mtb GlgE with an IC50=230 ± 24 µM determined using a coupled enzyme assay which measures orthophosphate release. The requirement of M1P for the assay necessitated the development of an expedited synthetic route to M1P from an intermediate used in the MCP 13 synthesis. In conclusion, we designed a substrate analogue of M1P that is the first to exhibit Mtb GlgE inhibition.
Assuntos
Antituberculosos/síntese química , Inibidores Enzimáticos/síntese química , Maltose/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Organofosfonatos/síntese química , Ácidos Fosforosos/química , Fosfatos Açúcares/química , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glucanos/biossíntese , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Maltose/síntese química , Maltose/química , Maltose/farmacologia , Mycobacterium tuberculosis/metabolismo , Organofosfonatos/química , Organofosfonatos/farmacologia , Fosfatos Açúcares/síntese química , Fosfatos Açúcares/farmacologiaRESUMO
The DNA replication stress (DRS) response is a crucial homeostatic mechanism for maintaining genome integrity in the face of intrinsic and extrinsic barriers to DNA replication. Importantly, DRS is often significantly increased in tumor cells, making tumors dependent on the cellular DRS response for growth and survival. Rad9-Hus1-Rad1 Interacting Nuclear Orphan 1 (RHNO1), a protein involved in the DRS response, has recently emerged as a potential therapeutic target in cancer. RHNO1 interacts with the 9-1-1 checkpoint clamp and TopBP1 to activate the ATR/Chk1 signaling pathway, the crucial mediator of the DRS response. Moreover, RHNO1 was also recently identified as a key facilitator of theta-mediated end joining (TMEJ), a DNA repair mechanism implicated in cancer progression and chemoresistance. In this literature review, we provide an overview of our current understanding of RHNO1, including its structure, function in the DRS response, and role in DNA repair, and discuss its potential as a cancer therapeutic target. Therapeutic targeting of RHNO1 holds promise for tumors with elevated DRS as well as tumors with DNA repair deficiencies, including homologous recombination DNA repair deficient (HRD) tumors. Further investigation into RHNO1 function in cancer, and development of approaches to target RHNO1, are expected to yield novel strategies for cancer treatment.