RESUMO
Nucleic acid chemistry is a huge research area that has received new impetus due to the recent explosive success of oligonucleotide therapy. In order for an oligonucleotide to become clinically effective, its monomeric parts are subjected to modifications. Although a large number of redesigned natural nucleic acids have been proposed in recent years, the vast majority of them are combinations of simple modifications proposed over the past 50 years. This review is devoted to the main modifications of the sugar phosphate backbone of natural nucleic acids known to date. Here, we propose a systematization of existing knowledge about modifications of nucleic acid monomers and an acceptable classification from the point of view of chemical logic. The visual representation is intended to inspire researchers to create a new type of modification or an original combination of known modifications that will produce unique oligonucleotides with valuable characteristics.
Assuntos
Ácidos Nucleicos , Fosfatos Açúcares , Ácidos Nucleicos/química , Fosfatos Açúcares/química , Fosfatos Açúcares/metabolismo , Oligonucleotídeos/química , Conformação de Ácido NucleicoRESUMO
Sedoheptulose 7-phosphate (SH7P) cyclases are a subset of sugar phosphate cyclases that are known to catalyze the first committed step in many biosynthetic pathways in primary and secondary metabolism. Among them are 2-epi-5-epi-valiolone synthase (EEVS) and 2-epi-valiolone synthase (EVS), two closely related SH7P cyclases that catalyze the conversion of SH7P to 2-epi-5-epi-valiolone and 2-epi-valiolone, respectively. However, how these two homologous enzymes use a common substrate to produce stereochemically different products is unknown. Two competing hypotheses have been proposed for the stereospecificity of EEVS and EVS: (1) variation in aldol acceptor geometry during enzyme catalysis, and (2) preselection of the α-pyranose or ß-pyranose forms of the substrate by the enzymes. Yet, there is no direct evidence to support or rule out either of these hypotheses. Here we report the synthesis of the carba-analogs of the α-pyranose and ß-pyranose forms of SH7P and their use in probing the stereospecificity of ValA (EEVS from Streptomyces hygroscopicus subsp. jinggangensis) and Amir_2000 (EVS from Actinosynnema mirum DSM 43827). Kinetic studies of the enzymes in the presence of the synthetic compounds as well as docking studies of the enzymes with the α- and ß-pyranose forms of SH7P suggest that the inverted configuration of the products of EEVS and EVS is not due to the preselection of the different forms of the substrate by the enzymes.
Assuntos
Heptoses , Fosfatos Açúcares , Fosfatos Açúcares/metabolismo , Fosfatos Açúcares/química , Heptoses/química , Heptoses/metabolismo , Estereoisomerismo , Especificidade por Substrato , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
Trifluoromethyl analogues of methylerythritol phosphate (MEP) and 2-C-methyl-erythritol 2,4-cyclodiphosphate (MEcPP), natural substrates of key enzymes from the MEP pathway, were prepared starting from d-glucose as the chiral template to secure absolute configurations. The obligate trifluoromethyl group was inserted with complete diastereoselectivity using the Ruppert-Prakash nucleophile. Target compounds were assayed against the corresponding enzymes showing that trifluoro-MEP did not disrupt IspD activity, whereas trifluoro-MEcPP induced 40% inhibition of IspG at 1 mM.
Assuntos
Fosfatos , Fosfatos Açúcares , Carboidratos , Eritritol , Fosfatos Açúcares/químicaRESUMO
The enzymes of the 2-C-methylerythritol-d-erythritol 4-phosphate (MEP) pathway (MEP pathway or non-mevalonate pathway) are responsible for the synthesis of universal precursors of the large and structurally diverse family of isoprenoids. This pathway is absent in humans, but present in many pathogenic organisms and plants, making it an attractive source of drug targets. Here, we present a high-throughput screening approach that led to the discovery of a novel fragment hit active against the third enzyme of the MEP pathway, PfIspD. A systematic SAR investigation afforded a novel chemical structure with a balanced activity-stability profile (16). Using a homology model of PfIspD, we proposed a putative binding mode for our newly identified inhibitors that sets the stage for structure-guided optimization.
Assuntos
Eritritol , Fosfatos Açúcares , Eritritol/análogos & derivados , Eritritol/química , Eritritol/metabolismo , Eritritol/farmacologia , Humanos , Fosfatos Açúcares/químicaRESUMO
The synthesis of analogues of natural enzyme substrates can be used to help deduce enzymatic mechanisms. N-Acetylmannosamine-6-phosphate 2-epimerase is an enzyme in the bacterial sialic acid catabolic pathway. To investigate whether the mechanism of this enzyme involves a re-protonation mechanism by the same neighbouring lysine that performed the deprotonation or a unique substrate-assisted proton displacement mechanism involving the substrate C5 hydroxyl, the syntheses of two analogues of the natural substrate, N-acetylmannosamine-6-phosphate, are described. In these novel analogues, the C5 hydroxyl has been replaced with a proton and a methyl ether respectively. As recently reported, Staphylococcus aureus N-acetylmannosamine-6-phosphate 2-epimerase was co-crystallized with these two compounds. The 5-deoxy variant bound to the enzyme active site in a different orientation to the natural substrate, while the 5-methoxy variant did not bind, adding to the evidence that this enzyme uses a substrate-assisted proton displacement mechanism. This mechanistic information may help in the design of potential antibacterial drug candidates.
Assuntos
Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/metabolismo , Hexosaminas/biossíntese , Fosfatos Açúcares/biossíntese , Proteínas de Bactérias/química , Configuração de Carboidratos , Carboidratos Epimerases/química , Hexosaminas/química , Staphylococcus aureus/enzimologia , Fosfatos Açúcares/químicaRESUMO
There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the ß-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.
Assuntos
Proteínas de Bactérias/química , Carboidratos Epimerases/química , Hexosaminas/química , Staphylococcus aureus/enzimologia , Fosfatos Açúcares/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Carboidratos Epimerases/genética , Catálise , Hexosaminas/genética , Hexosaminas/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica em Folha beta , Domínios Proteicos , Staphylococcus aureus/genética , Fosfatos Açúcares/genética , Fosfatos Açúcares/metabolismoRESUMO
The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.
Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Estresse Fisiológico , Fosfatos Açúcares/metabolismo , Regulação Alostérica , Sítios de Ligação , Sequência Conservada , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/ultraestrutura , Evolução Molecular , Guanosina Difosfato/metabolismo , Células HEK293 , Humanos , Leucoencefalopatias/patologia , Ligantes , Metaboloma , Modelos Moleculares , Mutação/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Fosfatos Açúcares/químicaRESUMO
Mycobacterium tuberculosis HddA enzyme phosphorylates the M7P substrate and converts it to M7PP product in GDP-D-α-D-heptose biosynthetic pathway. For structural and functional studies on MtbHddA, we have purified the enzyme, which eluted as a monomer from size exclusion column. Purified MtbHddA had ATPase activity. The SAXS analysis supported globular monomeric scattering profile of MtbHddA in solution. The CD analysis showed that MtbHddA contains 45% α-helix, 18% ß-stands, and 32% random coil structures and showed unfolding temperature (TM) ~ 47.5 °C. The unfolding temperature of MtbHddA is enhanced by 1.78±0.41 °C in ATP+Mg2+ bound state, 2.12±0.41 °C in Mannose bound state and 3.07±0.41 °C in Mannose+ ATP+Mg2+ bound state. The apo and M7P +ATP + Mg2+ complexed models of MtbHddA showed that enzyme adopts a classical GHMP sugar kinase fold with conserved ATP+Mg2+ and M7P binding sites. The dynamics simulation analysis on four MtbHddA models showed that ATP+Mg2+ and M7P binding enhanced the stability of active site conformation of MtbHddA. Our study provides important insights into MtbHddA structure and activity, which can be targeted for therapeutic development against M. tuberculosis.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Magnésio/química , Mycobacterium tuberculosis/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfatos Açúcares/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , 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 , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Fosfatos Açúcares/metabolismo , TermodinâmicaRESUMO
The domestication and breeding of crops has been a major achievement for mankind enabling the development of stable societies and civilisation. Crops have become more productive per unit area of cultivated land over the course of domestication supporting a current global population of 7.8 billion. Food security crops such as wheat and maize have seen large changes compared with early progenitors. Amongst processes that have been altered in these crops, is the allocation of carbon resources to support larger grain yield (grain number and size). In wheat, reduction in stem height has enabled diversion of resources from stems to ears. This has freed up carbon to support greater grain yield. Green revolution genes responsible for reductions in stem height are known, but a unifying mechanism for the active regulation of carbon resource allocation towards and within sinks has however been lacking. The trehalose 6-phosphate (T6P) signalling system has emerged as a mechanism of resource allocation and has been implicated in several crop traits including assimilate partitioning and improvement of yield in different environments. Understanding the mode of action of T6P through the SnRK1 protein kinase regulatory system is providing a basis for a unifying mechanism controlling whole-plant resource allocation and source-sink interactions in crops. Latest results show it is likely that the T6P/SnRK1 pathway can be harnessed for further improvements such as grain number and grain filling traits and abiotic stress resilience through targeted gene editing, breeding and chemical approaches.
Assuntos
Ácido Abscísico/metabolismo , Produtos Agrícolas/genética , Fosfatos Açúcares/química , Trealose/análogos & derivados , Processamento Alternativo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Cromatina/metabolismo , Genoma de Planta , Homeostase , Fosfoproteínas Fosfatases/genética , Isoformas de Proteínas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Spliceossomos/metabolismo , Estresse Fisiológico , Trealose/químicaRESUMO
BACKGROUND: Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand. METHODS: Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions. RESULTS: We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity. CONCLUSION: G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Pseudomonas aeruginosa/enzimologia , Fosfatos Açúcares/farmacologia , Inibidores Enzimáticos/química , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/metabolismo , Ligantes , Modelos Moleculares , Conformação Proteica , Fosfatos Açúcares/químicaRESUMO
Trehalose-6-phosphate phosphatase (T6PP) catalyzes the dephosphorylation of trehalose 6-phosphate (T6P) to the disaccharide trehalose. The enzyme is not present in mammals but is essential to the viability of multiple lower organisms as trehalose is a critical metabolite, and T6P accumulation is toxic. Hence, T6PP is a target for therapeutics of human pathologies caused by bacteria, fungi, and parasitic nematodes. Here, we report the X-ray crystal structures of Salmonella typhimurium T6PP (StT6PP) in its apo form and in complex with the cofactor Mg2+ and the substrate analogue trehalose 6-sulfate (T6S), the product trehalose, or the competitive inhibitor 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (OGS). OGS replaces the substrate phosphoryl group with a sulfate group and the glucosyl ring distal to the sulfate group with an octylphenyl moiety. The structures of these substrate-analogue and product complexes with T6PP show that specificity is conferred via hydrogen bonds to the glucosyl group proximal to the phosphoryl moiety through Glu123, Lys125, and Glu167, conserved in T6PPs from multiple species. The structure of the first-generation inhibitor OGS shows that it retains the substrate-binding interactions observed for the sulfate group and the proximal glucosyl ring. The OGS octylphenyl moiety binds in a unique manner, indicating that this subsite can tolerate various chemotypes. Together, these findings show that these conserved interactions at the proximal glucosyl ring binding site could provide the basis for the development of broad-spectrum therapeutics, whereas variable interactions at the divergent distal subsite could present an opportunity for the design of potent organism-specific therapeutics.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Salmonella typhimurium/enzimologia , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Monoéster Fosfórico Hidrolases/química , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Especificidade por Substrato , Fosfatos Açúcares/química , Trealose/química , Trealose/metabolismoRESUMO
Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Simulação por Computador , Inibidores Enzimáticos/química , Glucosiltransferases/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Fosfatos Açúcares/química , Trealose/química , Trealose/metabolismoRESUMO
ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.
Assuntos
Miopatias Distais/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Hexosaminas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pró-Fármacos/farmacologia , Fosfatos Açúcares/farmacologia , Animais , Células CHO , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cricetulus , Miopatias Distais/metabolismo , Miopatias Distais/patologia , Relação Dose-Resposta a Droga , Hexosaminas/síntese química , Hexosaminas/química , Humanos , Estrutura Molecular , Ácido N-Acetilneuramínico/análise , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-Atividade , Fosfatos Açúcares/síntese química , Fosfatos Açúcares/químicaRESUMO
α-Phosphomannomutase/phosphoglucomutase (αPMM/PGM) from P. aeruginosa is involved in bacterial cell wall assembly and is implicated in P. aeruginosa virulence, yet few studies have addressed αPMM/PGM inhibition from this important Gram-negative bacterial human pathogen. Four structurally different α-d-glucopyranose 1-phosphate (αG1P) derivatives including 1-C-fluoromethylated analogues (1-3), 1,2-cyclic phosph(on)ate analogues (4-6), isosteric methylene phosphono analogues (7 and 8), and 6-fluoro-αG1P (9), were synthesized and assessed as potential time-dependent or reversible αPMM/PGM inhibitors. The resulting kinetic data were consistent with the crystallographic structures of the highly homologous Xanthomonas citri αPGM with inhibitors 3 and 7-9 binding to the enzyme active site (1.65-1.9 Å). These structural and kinetic insights will enhance the design of future αPMM/PGM inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Fosfoglucomutase/antagonistas & inibidores , Fosfotransferases (Fosfomutases)/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Fosfatos Açúcares/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cinética , Modelos Moleculares , Estrutura Molecular , Fosfoglucomutase/metabolismo , Fosfotransferases (Fosfomutases)/metabolismo , Pseudomonas aeruginosa/enzimologia , Fosfatos Açúcares/síntese química , Fosfatos Açúcares/químicaRESUMO
Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS-TLR4-MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein-protein interaction (PPI) in TLR4-MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4-MD-2) and dimerization (MD-2-TLR4*) protein-protein interaction interfaces in TLR4-MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4-MD-2 protein-protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in µM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4-MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.
Assuntos
Simulação por Computador , Antígeno 96 de Linfócito/antagonistas & inibidores , Antígeno 96 de Linfócito/química , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/química , Sequência de Aminoácidos , Sítios de Ligação , Dissacarídeos/química , Dissacarídeos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligação de Hidrogênio , Ligantes , Lipopolissacarídeos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Relação Estrutura-Atividade , Fosfatos Açúcares/química , Fosfatos Açúcares/metabolismoRESUMO
Malaria continues to impinge heavily on mankind, with five continents still under its clasp. Widespread and rapid emergence of drug resistance in the Plasmodium parasite to current therapies accentuate the quest for novel drug targets and antimalarial compounds. Plasmodium parasites, maintain a non-photosynthetic relict organelle known as Apicoplast. Among the four major pathways of Apicoplast, biosynthesis of isoprenoids via Methylerythritol phosphate (MEP) pathway is the only indispensable function of Apicoplast that occurs during different stages of the malaria parasite. Moreover, the human host lacks MEP pathway. MEP pathway is a validated repertoire of novel antimalarial and antibacterial drug targets. Fosmidomycin, an efficacious antimalarial compound against IspC enzyme of MEP pathway is already in clinical trials as a combination drugs. Exploitation of other enzymes of MEP pathway would provide a much-needed impetus to the antimalarial drug discovery programs for the elimination of malaria. We outline the cardinal features of the MEP pathway enzymes and progress made towards the characterization of new inhibitors.
Assuntos
Apicoplastos/metabolismo , Eritritol/análogos & derivados , Plasmodium falciparum/metabolismo , Fosfatos Açúcares/metabolismo , Antimaláricos/química , Antimaláricos/farmacologia , Apicoplastos/efeitos dos fármacos , Eritritol/antagonistas & inibidores , Eritritol/química , Eritritol/metabolismo , Humanos , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Fosfatos Açúcares/antagonistas & inibidores , Fosfatos Açúcares/química , Terpenos/química , Terpenos/metabolismo , Transferases/antagonistas & inibidores , Transferases/metabolismoRESUMO
Despite significant advances made in the last decade in the understanding of molecular mechanisms of sepsis and in the development of clinically relevant therapies, sepsis remains the leading cause of mortality in intensive care units with increasing incidence worldwide. Toll-like receptorâ 4 (TLR4)-a transmembrane pattern-recognition receptor responsible for propagating the immediate immune response to Gram-negative bacterial infection-plays a central role in the pathogenesis of sepsis and chronic inflammation-related disorders. TLR4 is complexed with the lipopolysaccharide (LPS)-sensing protein myeloid differentiation-2 (MD-2) which represents a preferred target for establishing new anti-inflammatory treatment strategies. Herein we report the development, facile synthesis, and biological evaluation of novel disaccharide-based TLR4â MD-2 antagonists with potent anti-endotoxic activity at micromolar concentrations. A series of synthetic anionic glycolipids entailing amide-linked ß-ketoacyl lipid residues was prepared in a straightforward manner by using a single orthogonally protected nonreducing diglucosamine scaffold. Suppression of the LPS-induced release of interleukin-6 and tumor necrosis factor was monitored and confirmed in human immune cells (MNC and THP1) and mouse macrophages. Structure-activity relationship studies and molecular dynamics simulations revealed the structural basis for the high-affinity interaction between anionic glycolipids and MD-2, and highlighted two compounds as leads for the development of potential anti-inflammatory therapeutics.
Assuntos
Anti-Inflamatórios/farmacologia , Dissacarídeos/farmacologia , Fosfatos Açúcares/farmacologia , Tensoativos/farmacologia , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Dissacarídeos/síntese química , Dissacarídeos/química , Escherichia coli/química , Humanos , Inflamação/induzido quimicamente , Interleucina-6/metabolismo , Lipopolissacarídeos , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Fosfatos Açúcares/síntese química , Fosfatos Açúcares/química , Tensoativos/síntese química , Tensoativos/química , Células THP-1 , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfatos Açúcares/metabolismo , Deleção de Genes , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Hidrólise , Monoéster Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Fosfatos Açúcares/químicaRESUMO
The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (C16H26) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C16 alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with 13C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis. 13C{1H} NMR spectroscopy facilitated the localization of 13C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphate to sodorifen.
Assuntos
Compostos Bicíclicos com Pontes/metabolismo , Eritritol/análogos & derivados , Metiltransferases/metabolismo , Octanos/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , S-Adenosilmetionina/metabolismo , Serratia/metabolismo , Sesquiterpenos/metabolismo , Fosfatos Açúcares/metabolismo , Compostos Bicíclicos com Pontes/química , Ciclização , Eritritol/química , Eritritol/metabolismo , Metilação , Estrutura Molecular , Octanos/química , Fosfatos de Poli-Isoprenil/química , S-Adenosilmetionina/química , Serratia/enzimologia , Sesquiterpenos/química , Fosfatos Açúcares/químicaRESUMO
Biosynthesis of isoprenoids (MEP Pathway) in apicoplast has an important role during the erythrocytic stages of Plasmodium, as it is the sole pathway to provide the major isoprene units required as metabolic precursor for various housekeeping activities. With the intensifying need to identify a novel therapeutic drug target against Plasmodium, the MEP pathway and its components are considered as potential therapeutic targets, due to the difference in the isoprenoid synthesis route (MVA) functional in the host cells. While few major components have already been studied from this pathway for their potential as a drug target, IspD (2-C-methyl-D-erythritol-4-phosphate cytidyltransferase) enzyme, the enzyme catalyzing the third step of the pathway has only been tested against a synthetic compound from Malaria box called MMV008138, which also has not shown adequate inhibitory activity against P. vivax IspD. In the present study, to validate the potential of PvIspD as a drug target, various antimicrobial agents were screened for their inhibition possibilities, using in-vitro High Throughput Screening (HTS) technique. Shortlisted antimicrobial drug molecules like Cefepime, Tunicamycin and Rifampicin were further validated by in-vitro biochemical enzyme inhibition assays where they showed activity at nanomolar concentrations suggesting them or their derivatives as prospective future antimalarials. This study also confirmed the in-vivo expression of PvIspD protein during asexual stages by sub-cellular localization in apicoplast and explores the importance of the IspD enzyme in the development of new therapeutics.