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1.
J Enzyme Inhib Med Chem ; 35(1): 1292-1299, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32515610

RESUMO

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (ß-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world's leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, ß, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only ß- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The ß-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of ß-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Trichomonas vaginalis/enzimologia , Cinética , Conformação Proteica
2.
J Enzyme Inhib Med Chem ; 32(1): 5-12, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27766895

RESUMO

Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO2 hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.


Assuntos
Anidrases Carbônicas/metabolismo , Estresse Oxidativo , Compostos de Sulfidrila/química , Sequência de Aminoácidos , Anidrases Carbônicas/química , Cristalografia por Raios X , Homologia de Sequência de Aminoácidos
4.
J Enzyme Inhib Med Chem ; 31(sup4): 1-6, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27541737

RESUMO

The multi-component fingerprint and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products. We investigated the quantitative content of biologically active compounds (anthocyanins and chlorogenic acid) of microwave-assisted blueberry extracts from 14 different Italian cultivars, using validated high-performance liquid chromatography-photodiode array detector (HPLC-PDA) method and routinely instrument configuration. The carbonic anhydrase (CA, EC 4.2.1.1) inhibition profiles against several pharmacologically relevant CA isoforms of blueberry extracts and some bioactive compounds were also investigated. The various cultivars showed a highly variable content in anthocyanins and chlorogenic acid, and their CA inhibitory effects were also highly variable. Overall these data prove that antioxidant natural products found in blueberries may be useful for designing pharmacological agents in which various CAs are involved, e.g., antiobesity, antitumor, or anticonvulsants agents.


Assuntos
Mirtilos Azuis (Planta)/química , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica I/antagonistas & inibidores , Inibidores da Anidrase Carbônica/isolamento & purificação , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Micro-Ondas , Extratos Vegetais/farmacologia , Anidrase Carbônica I/metabolismo , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/química , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Relação Estrutura-Atividade
5.
Int J Mol Sci ; 16(7): 15456-80, 2015 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-26184158

RESUMO

Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.


Assuntos
Anidrases Carbônicas/metabolismo , Bactérias/enzimologia , Carbonato de Cálcio/metabolismo , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Cinética , Engenharia de Proteínas , Estrutura Terciária de Proteína
6.
Biopolymers ; 101(7): 769-78, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24374484

RESUMO

Carbonic anhydrase isoform XIV (CA XIV) is the last member of the human (h) CA family discovered so far, being localized in brain, kidneys, colon, small intestine, urinary bladder, liver, and spinal cord. It has recently been described as a possible drug target for treatment of epilepsy, some retinopathies as well as some skin tumors. Human carbonic anhydrase (hCA) XIV is a membrane-associated protein consisting of an N-terminal extracellular domain, a putative transmembrane region, and a small cytoplasmic tail. In this article, we report the expression, purification, and the crystallographic structure of the entire extracellular domain of this enzyme. The analysis of the structure revealed the typical α-CA fold, in which a 10-stranded ß-sheet forms the core of the molecule, while the comparison with all the other membrane associated isoforms (hCAs IV, IX, and XII) allowed to identify the diverse oligomeric arrangement and the sequence and structural differences observed in the region 127-136 as the main factors to consider in the design of selective inhibitors for each one of the membrane associated α-CAs.


Assuntos
Anidrases Carbônicas/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Cromatografia em Gel , Cromatografia Líquida , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Cinética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Células Sf9 , Espectrometria de Massas por Ionização por Electrospray
7.
Amino Acids ; 46(2): 279-88, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23604465

RESUMO

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (ß-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the ß-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.


Assuntos
Aminoácido Oxirredutases/química , Proteínas Fúngicas/química , Glucose/química , Produtos Finais de Glicação Avançada/química , Lactoglobulinas/química , Leite/química , Sequência de Aminoácidos , Animais , Armazenamento de Alimentos , Frutosamina/química , Concentração de Íons de Hidrogênio , Lactose/química , Dados de Sequência Molecular , Pasteurização
8.
J Enzyme Inhib Med Chem ; 29(4): 500-4, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23895630

RESUMO

C3 and C4 plant carbonic anhydrases (CAs) are zinc-enzymes that catalyze the reversible hydration of CO2. They are sub-divided in three classes: α, ß and γ, being distributed between both photosynthetic subtypes. The C4 dicotyledon species Flaveria bidentis (L.) "Kuntze" contains a small gene family encoding three distinct ß-CAs, named FbiCA1, FbiCA2 and FbiCA3. We have expressed and purified recombinant FbiCA1, which is localized in the chloroplast where it is thought to play a role in lipid biosynthesis and antioxidant activity, and biochemically characterized it by spectroscopic and inhibition experiments. FbiCA1 is a compact octameric protein that is moderately inhibited by carboxylate molecules. Surprisingly, pyruvate, but not lactate, did not inhibit FbiCA1 at concentrations up to 10 mM, suggesting that its capacity to tolerate high pyruvate concentration reflects the high concentration of pyruvate in the chloroplasts of bundle-sheath and mesophyll cells involved in C4 photosynthesis.


Assuntos
Antioxidantes/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Ácidos Carboxílicos/farmacologia , Flaveria/enzimologia , Sequência de Aminoácidos , Antioxidantes/isolamento & purificação , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/isolamento & purificação , Ácidos Carboxílicos/química , Relação Dose-Resposta a Droga , Lipídeos/biossíntese , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
9.
Biol Chem ; 394(10): 1343-8, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23851572

RESUMO

Human carbonic anhydrase (hCA) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Recently, S-glutathionylation of two cysteine residues from the enzyme was revealed, suggesting a new role as oxygen radical scavenger. We analyzed the effect of native and tetramutated hCA VII (all cysteines mutated into serines) in a eukaryotic system by stressing cells with an oxidant agent. Results clearly show that native hCA VII can protect cells from oxidative damage by preventing the apoptosis cascade and that cysteines play a leading role in this process. Our findings definitively confirm hCA VII protective role toward oxidative insult.


Assuntos
Anidrases Carbônicas/metabolismo , Estresse Oxidativo/fisiologia , Western Blotting , Imunofluorescência , Células HeLa , Humanos
10.
Bioorg Med Chem Lett ; 22(4): 1560-4, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22277279

RESUMO

Human carbonic anhydrase (CA, EC 4.2.1.1) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Here we report an unexpected S-glutathionylation of hCA VII which has also been observed earlier in vivo for hCA III, another cytosolic isoform. Cys183 and Cys217 were found to be the residues involved in reaction with glutathione for hCA VII. The two reactive cysteines were then mutated and the corresponding variant (C183S/C217S) expressed. The native enzyme, the variant and the S-glutathionylated adduct (sgCA VII) as well as hCA III were fully characterized for their CO(2) hydration, esterase/phosphatase activities, and inhibition with sulfonamides. Our findings suggest that hCA VII could use the in vivo S-glutathionylation to function as an oxygen radical scavenger for protecting cells from oxidative damage, as the activity and affinity for inhibitors of the modified enzyme are similar to those of the wild type.


Assuntos
Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/metabolismo , Sulfonamidas/química , Sequência de Aminoácidos , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/genética , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Isoformas de Proteínas/genética , S-Nitrosoglutationa/química , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Sulfonamidas/farmacologia
11.
J Pept Sci ; 17(9): 604-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21538708

RESUMO

We have recently reported on a class of IgE-binding peptides designed based on the crystallographic structure of the high affinity FcεRI. Peptides contain receptor key residues located within the two distinct binding sites for IgE and selectively bind IgE with an affinity ranging between 6 and 60 µM. We have here designed and characterized a new molecule containing the receptor loops C'-E and B-C and an optimized linker for joining them made of a Lys side chain and a ß-Ala. This new peptide shows an increased affinity (around 30 times) compared to the parent loop C'-E + B-C previously described, while retaining the same two-site mechanism of binding and the same selectivity. It also blocks the binding of IgE to the cell-anchored receptor and efficiently prevents histamine release from mast cells. These properties make the peptide a useful scaffold for the development of new anti-allergic drugs.


Assuntos
Liberação de Histamina , Peptídeos/metabolismo , Receptores de IgE/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/genética , Receptores de IgE/química
12.
Chem Commun (Camb) ; 56(85): 13033-13036, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33000794

RESUMO

To date, catechols have been only poorly investigated as carbonic anhydrase (CA) inhibitors. Here we report the first structural information on the CA inhibition mechanism of these molecules, showing that they adopt a peculiar binding mode to the enzyme active site which involves the zinc-bound water molecule and the "deep water".


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Catecóis/farmacologia , Inibidores da Anidrase Carbônica/química , Catecóis/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
13.
ACS Med Chem Lett ; 11(5): 1000-1005, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32435417

RESUMO

We report the synthesis and biochemical evaluation of a series of substituted 4-(4-aroylpiperazine-1-carbonyl)benzenesulfonamides (5a-s) developed as inhibitors of druggable carbonic anhydrase (CA) isoforms, as tools for the identification of new therapeutics. X-ray crystallography confirmed that this class of benzenesulfonamides binds CAs through the canonical anchoring of the benzenesulfonamide moiety to the metal ion and a tail-mediated recognition of the middle/top area of the active site cavity. Compound 5e (R = 2-Cl) demonstrated relevant selectivity toward brain-expressed hCA VII. The best balancing in binding affinity and selectivity toward tumor-expressed hCA IX/hCA XII over ubiquitous hCA I/hCA II was found for inhibitor 5o (R = 3-NO2). Notably 5b (R = 2-F) proved to be the most efficacious inhibitor of hCA XII for which computational studies elucidated the CA recognition process.

14.
Protein Expr Purif ; 59(2): 302-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18420420

RESUMO

PLD's (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx(4)Dx(6)GSxN motifs. PLD1 interacts with the small phosphoprotein PED-PEA15 by an unknown mechanism that, by enhancing PLD1 stability, apparently increases its enzymatic activity; the minimum interacting region of PLD1 was previously identified as spanning residues 712-1074 (D4 region). Since the D4/PED-PEA15 interaction has been claimed to be one of the multiple molecular events that can trigger type 2 diabetes, we purified the two recombinant proteins to study in vitro this binding by both ELISA and SPR techniques. Whilst PED-PEA15 was easily expressed and purified, expression of recombinant D4 was more problematic and only the fusion protein with Thioredoxin A and a six Histidine Tag (Trx-His(6)-D4) demonstrated sufficient stability for further characterization. We have found that Trx-His(6)-D4 is present as two different oligomeric forms, though only the monomeric variant is able to interact with PED-PEA15. All these findings may have important implications for both the mechanisms of phospholipase activity and PED-PEA15 regulative functions.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Fosfolipase D/química , Fosfolipase D/isolamento & purificação , Fosfoproteínas/química , Proteínas Reguladoras de Apoptose , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Fosfolipase D/biossíntese , Estrutura Terciária de Proteína/genética , Ressonância de Plasmônio de Superfície
15.
Oxid Med Cell Longev ; 2018: 2018306, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30154947

RESUMO

Under oxidative stress conditions, several constitutive cellular defense systems are activated, which involve both enzymatic systems and molecules with antioxidant properties such as glutathione and vitamins. In addition, proteins containing reactive sulfhydryl groups may eventually undergo reversible redox modifications whose products act as protective shields able to avoid further permanent molecular oxidative damage either in stressful conditions or under pathological circumstances. After the recovery of normal redox conditions, the reduced state of protein sulfhydryl groups is restored. In this context, carbonic anhydrases (CAs) III and VII, which are human metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate and proton, have been identified to play an antioxidant role in cells where oxidative damage occurs. Both proteins are mainly localized in tissues characterized by a high rate of oxygen consumption, and contain on their molecular surface two reactive cysteine residues eventually undergoing S-glutathionylation. Here, we will provide an overview on the molecular and functional features of these proteins highlighting their implications into molecular processes occurring during oxidative stress conditions.


Assuntos
Antioxidantes/química , Anidrase Carbônica III/metabolismo , Anidrases Carbônicas/metabolismo , Estresse Oxidativo/fisiologia , Animais , Humanos , Isoenzimas/metabolismo , Oxirredução
16.
Curr Top Med Chem ; 16(21): 2369-78, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27072690

RESUMO

Bacterial infections constitute an always growing health problem worldwide. The resistance to antibiotics of an increasing number of bacterial pathogens necessitates a permanent search for new molecules with different mechanisms of action. Histidine biosynthesis is an ancient pathway found in bacteria, archaebacteria, fungi and plants but absent in mammals. This feature makes it very interesting for the study of new strategies aimed to develop novel classes of antibacterial agents. In particular, one of the enzymes involved in the histidine biosynthesis, i.e. L-histidinol dehydrogenase (HDH), has been demonstrated to be essential for the survival of bacteria associated to several infections, such as brucellosis and tubercolosis. HDH is a Zn2+ enzyme which catalyzes the last two steps in the biosynthesis of Lhistidine: sequential NAD-dependent oxidations of L-histidinol to L-histidinaldehyde and then to L-histidine. This review will be focused on the biochemical and structural studies performed on HDH so far with the purpose to provide a structural background for the rational drug design of potent HDH inhibitors.


Assuntos
Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/química , Sequência de Aminoácidos , Humanos
17.
Chem Commun (Camb) ; 51(2): 302-5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25407638

RESUMO

A structural study of the adduct which 2-benzylsulfinylbenzoic acid forms with human carbonic anhydrase II is reported, showing a binding mode completely different from any other class of carbonic anhydrase inhibitors investigated so far; this carboxylate binds in a pocket situated out of the enzyme active site.


Assuntos
Ácido Benzoico/química , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/química , Ácido Benzoico/farmacologia , Sítios de Ligação , Anidrase Carbônica II/química , Inibidores da Anidrase Carbônica/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular
18.
Chem Commun (Camb) ; 51(57): 11519-22, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26094945

RESUMO

Hydroxylamine-O-sulfonamide, a molecule incorporating two zinc-binding groups (ZBGs), has been investigated as a carbonic anhydrase inhibitor (CAI) by means of kinetic, crystallographic and Raman spectroscopy studies, highlighting interesting results on its mechanism of action. These data can be exploited to design new, effective and selective CAIs.


Assuntos
Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/metabolismo , Hidroxilamina/química , Sulfonamidas/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Domínio Catalítico , Desenho de Fármacos , Humanos , Hidroxilamina/farmacologia , Simulação de Acoplamento Molecular , Sulfonamidas/farmacologia
20.
PLoS One ; 9(12): e113988, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25438149

RESUMO

The biological control of insect pests is based on the use of natural enemies. However, the growing information on the molecular mechanisms underpinning the interactions between insects and their natural antagonists can be exploited to develop "bio-inspired" pest control strategies, mimicking suppression mechanisms shaped by long co-evolutionary processes. Here we focus on a virulence factor encoded by the polydnavirus associated with the braconid wasp Toxoneuron nigriceps (TnBV), an endophagous parasitoid of noctuid moth larvae. This virulence factor (TnBVANK1) is a member of the viral ankyrin (ANK) protein family, and appears to be involved both in immunosuppression and endocrine alterations of the host. Transgenic tobacco plants expressing TnBVANK1 showed insecticide activity and caused developmental delay in Spodoptera littoralis larvae feeding on them. This effect was more evident in a transgenic line showing a higher number of transcripts of the viral gene. However, this effect was not associated with evidence of translocation into the haemocoel of the entire protein, where the receptors of TnBVANK1 are putatively located. Indeed, immunolocalization experiments evidenced the accumulation of this viral protein in the midgut, where it formed a thick layer coating the brush border of epithelial cells. In vitro transport experiments demonstrated that the presence of recombinant TnBVANK1 exerted a dose-dependent negative impact on amino acid transport. These results open new perspectives for insect control and stimulate additional research efforts to pursue the development of novel bioinsecticides, encoded by parasitoid-derived genes. However, future work will have to carefully evaluate any effect that these molecules may have on beneficial insects and on non-target organisms.


Assuntos
Anquirinas/farmacologia , Inseticidas/farmacologia , Lepidópteros/fisiologia , Nicotiana/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Fatores de Virulência/farmacologia , Animais , Anquirinas/genética , Arginina/metabolismo , Dicroísmo Circular , Vetores Genéticos/administração & dosagem , Larva/fisiologia , Lepidópteros/embriologia , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/parasitologia , Polydnaviridae/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/parasitologia , Proteínas Virais/genética , Proteínas Virais/farmacologia , Fatores de Virulência/genética
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