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1.
Eur J Med Chem ; 216: 113308, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33713976

RESUMO

Unique structure, characteristic reactivity, and facile synthesis of metal complexes have made them efficient ligands in drug development research. Among them, rhodium complexes have a limited history and there are a few discussions about their biological activities documented in the literature. However, investigation of kinetically inert rhodium complexes has recently attracted lots of attention and especially there are various evidences on their anti-cancer activity. It seems that they can be investigated as a versatile surrogates or candidates for the existing drugs which do not affect selectively or suffer from various side effects. In recent years, there has been an increasing interest in the use of mononuclear rhodium (III) organometallo drugs due to its versatile structurally important aspects to inhibit various enzymes. It has been demonstrated that organometallic Rh complexes profiting from both organic and inorganic aspects have shown more potent biological activities than classical inorganic compartments. In this respect, smart design, use of the appropriate organic ligands, and efficient and user-friendly synthesis of organometallic Rh complexes have played crucial roles in the inducing desirable biological activities. In this review, we focused on the recent advances published on the bioactivity of Rh (III/II/I) complexes especially inhibitory activity, from 2013 till now. Accordingly, considering the structure-activity relationship (SAR), the effect of oxidation state (+1, +2, and +3) and geometry (dimer or monomer complexes with coordination number of 4 and 6) of Rh complexes as well as various ligands on in vitro and in vivo studies was comprehensively discussed.


Assuntos
Complexos de Coordenação/química , Descoberta de Drogas , Ródio/química , Complexos de Coordenação/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Isomerases/antagonistas & inibidores , Isomerases/metabolismo , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade
2.
J Mol Model ; 26(8): 221, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32748070

RESUMO

The pathogen Legionella longbeachae is a causative agent of legionellosis. The antibiotic resistance is the major problem of this modern world. Thus, selective pressure warrants the need for identification of newer drug target. In current study, subtractive proteomics approach screen out SIS (sugar isomerase) domain protein as an attractive receptor molecule for rational drug design. This protein is involved in lipopolysaccharide biosynthesis and catalyzes the isomerization of sedoheptulose 7-phosphate in D-glycero-D-manno-heptose 7-phosphate. Molecular docking revealed compound 1 (2-(6-(N,N-dimethyl sulfamoyl)pipridin-4-yl)pyrazin-2-yl)imidazol-3-ium-1-ide) as the potent inhibitor having GOLD fitness score of 69. The complex is affirmed by half-site effect via simulation analysis. Complex stability was investigated via several approaches that follows dynamic simulation and binding energies. Trajectory analysis revealed slight change in ring positioning of inhibitor inside the active pocket during 130 ns (nanosecond). Interestingly, it was affirmed via binding interactions' density distribution. Hence, radial distribution function (RDF) inferred that SER55 and SER83 are the major residues that take part in hydrogen bonding and complex stability. Furthermore, an indigenously developed method axial frequency distribution (AFD) has revealed that ligand moved closer to the active site with both the residues SER55 and SER83 binding to the ligand. The phenomena was observed via rotating motion with respect to receptor center cavity. Thus, inhibitor movement towards allosteric site was observed at the end of simulations. Finally, binding free energy calculations by MMPB/GBSA predicts high compound affinity for the complex. Hence, findings from the current study will aid in the novel drug discovery and future experimental studies. Graphical abstract.


Assuntos
Inibidores Enzimáticos/química , Isomerases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos , Açúcares/química , Sítio Alostérico , Domínio Catalítico , Fenômenos Químicos , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Ligação de Hidrogênio , Isomerases/antagonistas & inibidores , Estrutura Molecular , Ligação Proteica , Proteômica , Relação Quantitativa Estrutura-Atividade
3.
Biosci Biotechnol Biochem ; 84(4): 789-796, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31889476

RESUMO

We previously demonstrated the efficient production of trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) in Lactococcus lactis by ectopically expressing a Propionibacterium acnes isomerase (pai) gene and also mentioned that a recombinant strain was unable to accumulate t10c12-CLA product, despite the normal transcription. Here, the molecular analysis indicated that this mutated strain harbors a pai gene with a single-nucleotide mutation converting GC50A to GTA, leading to a corresponding change of Alanine residue into Valine. The expression of the reverse mutation resulted in the recovery for enzyme activity. Site-directed mutagenesis indicated that the codon usage of Val17 was not responsible for the enzyme inactivation in the Ala17Val mutation. Western blot analysis revealed that the recombinant PAI protein was not detectable in the His tag-marked Ala17Val mutant. It is, therefore, reasonable to assume that Ala17 residue is critical for PAI functionality.Abbreviations: pai: propionibacterium acnes isomerase; CLA: conjugated linoleic acid; t10c12-CLA: trans 10, cis 12-CLA; LA: linoleic acid (18:2n-6); FAD: flavin adenine dinucleotide.


Assuntos
Substituição de Aminoácidos , Flavina-Adenina Dinucleotídeo/metabolismo , Isomerases/antagonistas & inibidores , Propionibacterium acnes/enzimologia , Western Blotting , Códon , Cristalografia por Raios X , Isomerases/química , Isomerases/genética , Isomerases/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Mutação com Perda de Função , Propionibacterium acnes/genética , Conformação Proteica
4.
Br J Pharmacol ; 176 Suppl 1: S297-S396, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31710714

RESUMO

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14752. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.


Assuntos
Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Isomerases/antagonistas & inibidores , Ligases/antagonistas & inibidores , Liases/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Transferases/antagonistas & inibidores , Animais , Bases de Dados de Produtos Farmacêuticos , Inibidores Enzimáticos/química , Humanos , Hidrolases/química , Hidrolases/metabolismo , Isomerases/química , Isomerases/metabolismo , Ligantes , Ligases/química , Ligases/metabolismo , Liases/química , Liases/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Transferases/química , Transferases/metabolismo
5.
J Recept Signal Transduct Res ; 39(1): 28-38, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31241401

RESUMO

Vanishing white matter (VWM) is a hereditary human disease, mostly prevalent in childhood caused by the defects in the eukaryotic initiation factor beta subunits. It is the first disease involved in the translation initiation factor, eIF2B. There is no specific treatment for VWM which mainly affect the brain and ovaries. The gray matter remains normal in all characteristics while the white matter changes texture, coming to the pathophysiology, many initiation factors are involved in the initiation of translation of mRNAs into polypeptides. In this study, the three-dimensional structure of PhMTNA protein was modeled and the stability ascertained through Molecular dynamic simulation (MDS) for 100 ns. The active site residues are conserved with the reported BsMTNA structure which is also confirmed through sitemap prediction. Through virtual screening and induced fit docking, top five leads against PhMTNA protein was identified based on their binding mode and affinity. ADME properties and DFT (Density Functional Theory) studies of these compounds were studied. In addition to that, computational mutagenesis studies were performed to identify the hotspot residues involved in the protein-ligand interactions. Overall analysis showed that the compound NCI_941 has a highest binding energy of -46.256 kcal mol-1 in the Arg57Ala mutant. Thus, the results suggest that NCI_941 would act as a potent inhibitor against PhMTNA protein.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Isomerases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Doenças do Sistema Nervoso/tratamento farmacológico , Domínio Catalítico , Humanos , Isomerases/metabolismo , Ligantes , Ligação Proteica
6.
Org Biomol Chem ; 17(5): 1206-1214, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30652178

RESUMO

δ-Cadinene synthase (DCS) is a high-fidelity sesquiterpene synthase that generates δ-cadinene as the sole detectable organic product from its natural substrate (E,E)-FDP. Previous work with this enzyme using substrate analogues revealed the ability of DCS to catalyse both 1,10- and 1,6-cyclisations of substrate analogues. To test whether this apparent promiscuity was an artefact of alternate substrate use or an inherent property of the enzyme, aza analogues of the proposed α-bisabolyl cation intermediate were prepared since this cation would be formed after an initial 1,6-cyclisation of FDP. In the presence of 250 µM inorganic disphosphate both (R)- and (S)-aza-bisaboyl cations were potent competitive inhibitors of DCS (Ki = 2.5 ± 0.5 mM and 3.44 ± 1.43 µM, respectively). These compounds were also shown to be potent inhibitors of the 1,6-cyclase amorpha-4,11-diene synthase but not of the 1,10-cyclase aristolochene synthase from Penicillium roquefortii, demonstrating that the 1,6-cyclase activity of DCS is most likely an inherent property of the enzyme even when the natural substrate is used and not an artefact of the use of substrate analogues.


Assuntos
Isomerases/metabolismo , Terpenos/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Catálise , Cátions , Ciclização , Inibidores Enzimáticos/farmacologia , Isomerases/antagonistas & inibidores , Penicillium/enzimologia , Fosfatos/química , Estereoisomerismo , Especificidade por Substrato
7.
J Med Chem ; 60(12): 4904-4922, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28513176

RESUMO

A series of Hoechst 33258 based mono- and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC50 values <5.0 µM. Several bisbenzimidazoles (3, 6, 7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I. Bisbenzimidazoles inhibit bacterial growth much better than monobenzimidazoles for Gram-positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram positive bacteria (Enterococcus spp. and Staphylococcus spp., including two MRSA strains 0.3-8 µg/mL) than for the majority of Gram negative bacteria (Pseudomonas aeruginosa, 16-32 µg/mL, Klebsiella pneumoniae > 32 µg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2-23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. Modeling studies suggest critical interactions between the inhibitor side chain and amino acids of the active site of DNA topoisomerase I.


Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Bisbenzimidazol/química , Escherichia coli/efeitos dos fármacos , Inibidores da Topoisomerase I/farmacologia , Antibacterianos/química , Benzimidazóis/química , Linhagem Celular Tumoral , Técnicas de Química Sintética , DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Isomerases/antagonistas & inibidores , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Inibidores da Topoisomerase I/química
8.
Carbohydr Res ; 419: 1-7, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26595659

RESUMO

Uridine diphosphate-galactopyranose mutase (UGM), an enzyme found in many eukaryotic and prokaryotic human pathogens, catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf), the latter being used as the biosynthetic precursor of the galactofuranose polymer portion of the mycobacterium cell wall. We report here the synthesis of a sulfonium and selenonium ion with an appended polyhydroxylated side chain. These compounds were designed as transition state mimics of the UGM-catalyzed reaction, where the head groups carrying a permanent positive charge were designed to mimic both the shape and positive charge of the proposed galactopyranosyl cation-like transition state. An HPLC-based UGM inhibition assay indicated that the compounds inhibited about 25% of UGM activity at 500 µM concentration.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Galactose/análogos & derivados , Isomerases/antagonistas & inibidores , Difosfato de Uridina/análogos & derivados , Biocatálise , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Galactose/metabolismo , Hidroxilação , Isomerases/metabolismo , Mycobacterium tuberculosis/enzimologia , Compostos de Selênio/síntese química , Compostos de Selênio/química , Compostos de Selênio/farmacologia , Compostos de Sulfônio/síntese química , Compostos de Sulfônio/química , Compostos de Sulfônio/farmacologia , Difosfato de Uridina/metabolismo
9.
Chem Biol ; 21(1): 156-64, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24374163

RESUMO

Rhamnolipids are multipurpose surface-active molecules produced by the bacterium Pseudomonas aeruginosa from L-rhamnose and R-3-hydroxyalkanoate (C10±2) precursors. R-3-hydroxyalkanoate precursor is believed to be synthesized de novo. We demonstrate, however, that ß-oxidation is the predominant source of this precursor. Inhibition of ß-oxidation sharply decreases rhamnolipids production, even when using a nonfatty acid carbon source (glycerol). Isotope tracing shows that ß-oxidation intermediates are direct precursors of rhamnolipids. A mutant-based survey revealed an operon coding for enoyl-CoA hydratases/isomerases (ECH/I), named RhlYZ, implicated in rhamnolipids production via an axial role in 3-hydroxyalkanoate synthesis. In vitro, RhlZ is an R-ECH/I transforming 2-decenoyl-CoA, a ß-oxidation intermediate, into R-3-hydroxydecanoyl-CoA, the potential rhamnolipids precursor. Interestingly, polyhydroxyalkanoates share with rhamnolipids the RhlYZ-generated R-3-hydroxyalkanoates pool, as demonstrated by the decrease of polyhydroxyalkanoates upon mutation of rhlYZ and the increase of rhamnolipids in a polyhydroxyalkanoates-defective mutant.


Assuntos
Enoil-CoA Hidratase/metabolismo , Glicolipídeos/biossíntese , Isomerases/metabolismo , Pseudomonas aeruginosa/enzimologia , Tensoativos/metabolismo , Enoil-CoA Hidratase/antagonistas & inibidores , Enoil-CoA Hidratase/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicolipídeos/química , Isomerases/antagonistas & inibidores , Isomerases/genética , Estrutura Molecular , Oxirredução , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/química , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Tensoativos/química
10.
Anal Chim Acta ; 773: 24-36, 2013 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-23561903

RESUMO

High quality assays are needed in drug discovery to reduce the high attrition rate of lead compounds during primary screening. Capillary electrophoresis (CE) represents a versatile micro-separation technique for resolution of enzyme-catalyzed reactions, including substrate(s), product(s), cofactor(s) and their stereoisomers, which is needed for reliable characterization of biomolecular interactions in free solution. This review article provides a critical overview of new advances in CE for drug screening over the past five years involving biologically relevant enzymes of therapeutic interest, including transferases, hydrolases, oxidoreductases, and isomerases. The basic principles and major configurations in CE, as well as data processing methods needed for rigorous characterization of enzyme inhibition are described. New developments in functional screening of small molecules that modulate the activity of disease-related enzymes are also discussed. Although inhibition is a widely measured response in most enzyme assays, other important outcomes of ligand interactions on protein structure/function that impact the therapeutic potential of a drug will also be highlighted, such as enzyme stabilization, activation and/or catalytic uncoupling. CE offers a selective platform for drug screening that reduces false-positives while also enabling the analysis of low amounts of complex sample mixtures with minimal sample handling.


Assuntos
Eletroforese Capilar , Preparações Farmacêuticas/química , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Hidroliases/antagonistas & inibidores , Hidroliases/metabolismo , Isomerases/antagonistas & inibidores , Isomerases/metabolismo , Espectrometria de Massas , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Estereoisomerismo , Transferases/antagonistas & inibidores , Transferases/metabolismo
11.
J Med Chem ; 55(22): 10148-59, 2012 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-23067344

RESUMO

The Plasmodium falciparum orthologue of the human cytokine, macrophage migratory inhibitory factor (PfMIF), is produced by the parasite during malaria infection and modulates the host's immune response. As for other MIF orthologues, PfMIF has tautomerase activity, whose inhibition may influence the cytokine activity. To identify small-molecule inhibitors of the tautomerase activity of PfMIF, virtual screening has been performed by docking 2.1 million compounds into the enzymatic site. Assaying of 17 compounds identified four as active. Substructure search for the most potent of these compounds, a 4-phenoxypyridine analogue, identified four additional compounds that were purchased and also shown to be active. Thirty-one additional analogues were then designed, synthesized, and assayed. Three were found to be potent PfMIF tautomerase inhibitors with K(i) of ∼40 nM; they are also highly selective with K(i) > 100 µM for human MIF.


Assuntos
Antimaláricos/farmacologia , Ensaios de Triagem em Larga Escala , Isomerases/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Antimaláricos/química , Humanos , Ligação de Hidrogênio , Malária/metabolismo , Malária/parasitologia , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade
12.
Endocrinology ; 153(4): 1925-35, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22374975

RESUMO

Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD2: PGD2 receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct.


Assuntos
Células Epiteliais/enzimologia , Receptor alfa de Estrogênio/metabolismo , Inflamação/metabolismo , Isomerases/metabolismo , Oviductos/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Receptor alfa de Estrogênio/deficiência , Receptor alfa de Estrogênio/genética , Ciclo Estral/metabolismo , Feminino , Técnicas In Vitro , Inflamação/fisiopatologia , Oxirredutases Intramoleculares , Isomerases/antagonistas & inibidores , Isomerases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Oviductos/citologia , Piperidinas/farmacologia , RNA Mensageiro/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo
13.
J Phys Chem B ; 116(23): 6889-97, 2012 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-22417185

RESUMO

4-Oxalocrotonate tautomerase (4-OT), a member of tautomerase superfamily, is an essential enzyme in the degradative metabolism pathway occurring in the Krebs cycle. The proton transfer process catalyzed by 4-OT has been explored previously using both experimental and theoretical methods; however, the elaborate catalytic mechanism of 4-OT still remains unsettled. By combining classical molecular mechanics with quantum mechanics, our results demonstrate that the native hexametric 4-OT enzyme, including six protein monomers, must be employed to simulate the proton transfer process in 4-OT due to protein-protein steric and electrostatic interactions. As a consequence, only three out of the six active sites in the 4-OT hexamer are observed to be occupied by three 2-oxo-4-hexenedioates (2o4hex), i.e., half-of-the-sites occupation. This agrees with experimental observations on negative cooperative effect between two adjacent substrates. Two sequential proton transfers occur: one proton from the C3 position of 2o4hex is initially transferred to the nitrogen atom of the general base, Pro1. Subsequently, the same proton is shuttled back to the position C5 of 2o4hex to complete the proton transfer process in 4-OT. During the catalytic reaction, conformational changes (i.e., 1-carboxyl group rotation) of 2o4hex may occur in the 4-OT dimer model but cannot proceed in the hexametric structure. We further explained that the docking process of 2o4hex can influence the specific reactant conformations and an alternative substrate (2-hydroxymuconate) may serve as reactant under a different reaction mechanism than 2o4hex.


Assuntos
Isomerases/metabolismo , Prótons , Biocatálise , Isomerases/antagonistas & inibidores , Isomerases/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Ácido Sórbico/análogos & derivados , Ácido Sórbico/química , Ácido Sórbico/metabolismo , Eletricidade Estática
14.
J Pharmacol Exp Ther ; 339(2): 716-25, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21865441

RESUMO

Prostaglandins (PGs) are a family of cellular messengers exerting diverse homeostatic and pathophysiologic effects. Recently, several studies reported significant increases of PGI(2) and PGF(2α) after the inhibition of microsomal PGE synthase-1 (mPGES-1) expression, which indicated that PGH(2) metabolism might be redistributed when the PGE(2) pathway is blocked. To address the determinants that govern the relative amounts of PGs, we developed an in vitro cell-free method, based on liquid chromatography-tandem mass spectrometry, to measure the exact amounts of these PGs formed in response to the addition of recombinant isomerases and their selective inhibitors. Our in vitro cell-free assay results were confirmed in cells using bone marrow-derived macrophage. Initially, we determined the in vitro stability of PGH(2) and noted that there was spontaneous nonenzymatic conversion to PGD(2) and PGE(2). mPGES-1 markedly increased the conversion to PGE(2) and decreased conversion to PGD(2). Reciprocally, the addition of hematopoietic or lipocalin PGD synthase resulted in a relative increase of PGD(2) and decrease of PGE(2). A detailed titration study showed that the ratio of PGE(2)/PGD(2) was closely correlated with the ratio of PGE synthase/PGD synthase. Our redistribution results also provide the foundation for understanding how PGH(2) metabolism is redistributed by the presence of distal isomerases or by blocking the major metabolic outlet, which could determine the relative benefits and risks resulting from interdiction in nonrated-limiting components of PG synthesis pathways.


Assuntos
Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Macrófagos/fisiologia , Prostaglandina D2/metabolismo , Prostaglandina H2/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Animais , Células Cultivadas , Dibenzocicloeptenos/farmacologia , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Humanos , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/metabolismo , Isomerases/antagonistas & inibidores , Isomerases/metabolismo , Isomerases/fisiologia , Lipocalinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/farmacologia , Prostaglandina-E Sintases , Ovinos , Tiofenos/farmacologia
15.
Proc Natl Acad Sci U S A ; 108(9): 3554-9, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21321225

RESUMO

In histidine and tryptophan biosynthesis, two related isomerization reactions are generally catalyzed by two specific single-substrate enzymes (HisA and TrpF), sharing a similar (ß/α)(8)-barrel scaffold. However, in some actinobacteria, one of the two encoding genes (trpF) is missing and the two reactions are instead catalyzed by one bisubstrate enzyme (PriA). To unravel the unknown mechanism of bisubstrate specificity, we used the Mycobacterium tuberculosis PriA enzyme as a model. Comparative structural analysis of the active site of the enzyme showed that PriA undergoes a reaction-specific and substrate-induced metamorphosis of the active site architecture, demonstrating its unique ability to essentially form two different substrate-specific actives sites. Furthermore, we found that one of the two catalytic residues in PriA, which are identical in both isomerization reactions, is recruited by a substrate-dependent mechanism into the active site to allow its involvement in catalysis. Comparison of the structural data from PriA with one of the two single-substrate enzymes (TrpF) revealed substantial differences in the active site architecture, suggesting independent evolution. To support these observations, we identified six small molecule compounds that inhibited both PriA-catalyzed isomerization reactions but had no effect on TrpF activity. Our data demonstrate an opportunity for organism-specific inhibition of enzymatic catalysis by taking advantage of the distinct ability for bisubstrate catalysis in the M. tuberculosis enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Domínio Catalítico , Histidina/biossíntese , Isomerases/química , Isomerases/metabolismo , Mycobacterium tuberculosis/enzimologia , Triptofano/biossíntese , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Biocatálise/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Isomerases/antagonistas & inibidores , Isomerismo , Ligantes , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Estrutura Secundária de Proteína , Especificidade por Substrato/efeitos dos fármacos
16.
Org Lett ; 13(5): 1202-5, 2011 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-21271717

RESUMO

Trigonal iminium halides of (4aS,7S)-1,4a-dimethyl- and (4aS,7S)-4a-methyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium ions, aimed to mimic transition states associated with the aristolochene synthase-catalyzed cyclization of (-)-germacrene A to eudesmane cation, were evaluated under standard kinetic steady-state conditions. In the presence of inorganic diphosphate, these analogues were shown to competitively inhibit the enzyme, suggesting a stabilizing role for the diphosphate leaving group in this apparently endothermic transformation.


Assuntos
Iminas/farmacologia , Isomerases/antagonistas & inibidores , Sesquiterpenos de Eudesmano/química , Sesquiterpenos de Germacrano/química , Sesquiterpenos/síntese química , Aspergillus/enzimologia , Catálise , Ciclização , Iminas/síntese química , Iminas/química , Isomerases/metabolismo , Modelos Moleculares , Estrutura Molecular , Penicillium/enzimologia , Sais , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Estereoisomerismo , Nicotiana/enzimologia
17.
Biochemistry ; 48(26): 6175-83, 2009 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-19489610

RESUMO

(+)-Delta-cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 A resolution and the structure of its complex with three putative Mg(2+) ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 A resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich D(307)DTYD(311) motif on helix D that interacts with Mg(2+)(A) and Mg(2+)(C). However, DCS appears to be unique among terpenoid cyclases in that it does not contain the "NSE/DTE" motif on helix H that specifically chelates Mg(2+)(B), which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg(2+)(B) ligands). Instead, DCS contains a second aspartate-rich motif, D(451)DVAE(455), that interacts with Mg(2+)(B). In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg(2+)(B) binding motif. Analyses of DCS mutants with alanine substitutions in the D(307)DTYD(311) and D(451)DVAE(455) segments reveal the contributions of these segments to catalysis.


Assuntos
Evolução Molecular , Gossypium/enzimologia , Isomerases/química , Isomerases/metabolismo , Metais/metabolismo , Substituição de Aminoácidos , Bário/química , Sítios de Ligação/genética , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Concentração Inibidora 50 , Isomerases/antagonistas & inibidores , Isomerases/genética , Cinética , Magnésio/química , Modelos Moleculares , Fosfatos de Poli-Isoprenil/química , Conformação Proteica , Proteínas Recombinantes/química
18.
Neuroscience ; 163(1): 296-307, 2009 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-19531375

RESUMO

Cerebral ischemia/reperfusion injury is characterized by the development of inflammatory response, in which vascular macrophages and endogenous microglia are involved. Recent studies showed marked induction of hematopoietic prostaglandin D synthase (HPGDS) after ischemic/reperfusion injury and its localization in microglia, but the molecular mechanism(s) of HPGDS actions in cerebral ischemia is not clear. To clarify the role of HPGDS in cerebral ischemia, C57BL/6 mice and bone marrow chimera mice with cerebral ischemia/reperfusion injury were treated with (4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79), a specific inhibitor of HPGDS. The bone marrow chimera mice exhibit expression of enhanced green fluorescent protein (EGFP) in bone marrow/blood-derived monocytes/macrophages. Mice were subjected to ischemia/reperfusion and either treated with HQL-79 (n=44) or vehicle (n=44). Brain sections prepared at 72 h and 7 days after reperfusion were analyzed for neuronal nuclei (NeuN), HPGDS, ionized calcium-binding adapter molecule 1 (Iba1), inducible NO synthase (iNOS), nitrotyrosine, nuclear factor kappa B (NF-kB) and cyclooxygenase-2 (COX-2). The mortality rate (80%) and infarct size were larger in HQL-79- than vehicle-treated mice (58.7+/-8.5 versus 45.2+/-4.9 mm(3); mean+/-SEM, P<0.0001) at 7 days after reperfusion. HQL-79 reduced NeuN expression in the transition area and Iba1 expression (P<0.0001) in the ischemic peri- and penumbra area, but increased COX-2 (P<0.05) and NF-kB expression (P<0.05) in ischemic penumbra and increased formation of nitrotyrosine (P<0.0001) and iNOS (P<0.0001) in the ischemic core area at 72 h and 7 days after reperfusion. In EGFP chimera mice, HQL-79 increased the migration of Iba1/EGFP-positive bone marrow-derived monocytes/macrophages, and simultaneously upregulated iNOS expression in the ischemic core area (P<0.0001), but increased intrinsic microglia/macrophages in ischemic peri-area and penumbra (P<0.0001) at 72 h and 7 days after reperfusion, suggesting involvement of monocytes/macrophages in HQL-79-induced expansion of ischemic injury. Our results demonstrated that the neuroprotective effects of HPGDS in our model are mediated by suppression of activation and infiltration of inflammatory cells.


Assuntos
Encéfalo/efeitos dos fármacos , Encefalite/tratamento farmacológico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Oxirredutases Intramoleculares/metabolismo , Ataque Isquêmico Transitório/tratamento farmacológico , Isomerases/metabolismo , Lipocalinas/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Biomarcadores/metabolismo , Transplante de Medula Óssea/métodos , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Modelos Animais de Doenças , Encefalite/fisiopatologia , Encefalite/prevenção & controle , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteínas de Fluorescência Verde/genética , Hipóxia-Isquemia Encefálica/fisiopatologia , Hipóxia-Isquemia Encefálica/prevenção & controle , Oxirredutases Intramoleculares/antagonistas & inibidores , Ataque Isquêmico Transitório/fisiopatologia , Ataque Isquêmico Transitório/prevenção & controle , Isomerases/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico Sintase Tipo II/metabolismo , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/prevenção & controle , Quimeras de Transplante
19.
Org Biomol Chem ; 5(20): 3287-98, 2007 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-17912381

RESUMO

Analogues of farnesyl diphosphate (FPP, ) containing phenyl substituents in place of methyl groups have been prepared in syntheses that feature use of a Suzuki-Miyaura reaction as a key step. These analogues were found not to act as substrates of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti (AS). However, they were potent competitive inhibitors of AS with K(I)-values ranging from 0.8 to 1.2 microM. These results indicate that the diphosphate group contributes the largest part to the binding of the substrate to AS and that the active sites of terpene synthases are sufficiently flexible to accommodate even substrate analogues with large substituents suggesting a potential way for the generation of non-natural terpenoids. Molecular mechanics simulations of the enzyme bound inhibitors suggested that small changes in orientations of active site residues and subtle alterations of the conformation of the backbones of the inhibitors are sufficient to accommodate the phenyl-farnesyl-diphosphates.


Assuntos
Difosfatos/química , Inibidores Enzimáticos/química , Isomerases/antagonistas & inibidores , Penicillium/enzimologia , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/química , Terpenos/química , Sítios de Ligação , Difosfatos/farmacologia , Inibidores Enzimáticos/farmacologia , Isomerases/química , Conformação Proteica , Terpenos/farmacologia
20.
Biochemistry ; 44(21): 7725-37, 2005 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-15909987

RESUMO

4-Oxalocrotonate tautomerase (4-OT), a homohexameric enzyme, converts the unconjugated enone, 2-oxo-4-hexenedioate (1), to the conjugated enone, 2-oxo-3-hexenedioate (3), via a dienolic intermediate, 2-hydroxymuconate (2). Pro-1 serves as the general base, and both Arg-11 and Arg-39 function in substrate binding and catalysis in an otherwise hydrophobic active site. Although 4-OT exhibits hyperbolic kinetics and no structural asymmetry either by X-ray or by NMR, inactivation by two affinity labels showed half-site stoichiometry [Stivers, J. T., et al. (1996) Biochemistry 35, 803-813; Johnson, W. H., Jr., et al. (1997) Biochemistry 36, 15724-15732], and titration of the R39Q mutant with cis,cis-muconate showed negative cooperativity [Harris, T. K., et al. (1999) Biochemistry 38, 12343-12357]. To test for anticooperativity during catalysis, 4-OT was titrated with equilibrium mixtures (> or = 81% product) of the reactive dicarboxylate or monocarboxylate intermediates, 2 or 2-hydroxy-2,4-pentadienoate (4), respectively, in three types of NMR experiments: two-dimensional 1H-15N HSQC titrations of backbone NH and of Arg N epsilonH resonances and one-dimensional 15N NMR titrations of Arg N epsilon resonances. All titrations showed substoichiometric binding of the equilibrium mixtures to 3 +/- 1 sites per hexamer with apparent dissociation constants comparable to the Km values of the intermediates. Compound 4 also bound 1 order of magnitude less tightly at another site, suggesting negative cooperativity. Consistent with negative cooperativity, asymmetry of the resulting complexes at saturating levels of 2 and 4 is indicated by splitting of the backbone NH resonances of 11 residues and 10 residues of 4-OT, respectively. The dicarboxylate competitive inhibitor, (2E)-fluoromuconate (5), with a KI of 45 +/- 7 microM, also exhibited substoichiometric binding to 3 +/- 1 sites per hexamer, with a KD of 25 +/- 18 microM, and splitting of the backbone NH resonance of L8. The monocarboxylate inhibitors (2E)- (6) and (2Z)-2-fluoro-2,4-pentadienoate (7) showed much weaker binding (KD = 3.1 +/- 1.3 mM), as well as splitting of two and five backbone NH resonances, respectively, indicating asymmetry of the complexes. The N epsilon resonances of both Arg-11 and Arg-39 were shifted downfield, and that of Pro-1N was broadened by all ligands, consistent with the major catalytic roles of these residues. Structural pathways for the site-site interactions which result in negative cooperativity are proposed on the basis of the X-ray structures of free and affinity-labeled 4-OT. Selective resonance broadenings induced by the binding of inactive analogues and active intermediates indicate residues which may be mobilized during reversible ligand binding and during catalysis, respectively.


Assuntos
Isomerases/química , Isomerases/metabolismo , Ácido Sórbico/análogos & derivados , Adipatos , Arginina/química , Sítios de Ligação , Ligação Competitiva , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Ácidos Dicarboxílicos/química , Ácidos Dicarboxílicos/metabolismo , Indução Enzimática , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Hidrogênio , Isomerases/antagonistas & inibidores , Isomerases/biossíntese , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Isótopos de Nitrogênio/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Ácido Sórbico/química , Especificidade por Substrato , Titulometria
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