RESUMO
We investigated the cloning, catalytic activity and anion inhibition of the ß-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Legionella pneumophila. Two such enzymes, lpCA1 and lpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4-8.3)×10(5) s(-1) and kcat/KM values of (4.7-8.5)×10(7) M(-1) s(-1). A set of inorganic anions and small molecules was investigated to identify inhibitors of these enzymes. Perchlorate and tetrafluoroborate were not acting as inhibitors (KI >200 mM), whereas sulfate was a very weak inhibitor for both lpCA1 and lpCA2 (KI values of 77.9-96.5 mM). The most potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KI values ranging from 6 to 94 µM. The most potent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with KI values ranging from 2 to 13 µM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial agents with a novel mechanism of action.
Assuntos
Anidrases Carbônicas/metabolismo , Inibidores Enzimáticos/farmacologia , Legionella pneumophila/enzimologia , Ânions/síntese química , Ânions/química , Ânions/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Two ß-carbonic anhydrases (CAs, EC 4.2.1.1) were identified, cloned and purified in the pathogenic bacterium Legionella pneumophila, denominated LpCA1 and LpCA2. They efficiently catalyze CO2 hydration to bicarbonate and protons, with kcat in the range of (3.4-8.3) × 10(5)s(-1) and kcat/Km of (4.7-8.5) × 10(7)M(-1)s(-1), and are inhibited by sulfonamides and sulfamates. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide(KIs in the range of 40.3-90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide and dichlorophenamide (KIs in the range of 25.2-88.5 nM). As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Legionella pneumophila/enzimologia , Sulfonamidas/farmacologia , Sequência de Aminoácidos , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Dados de Sequência Molecular , Estrutura Molecular , Alinhamento de Sequência , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the gene product, ataxin-3. Microarray analysis revealed a dramatic differential expression of carbonic anhydrase-related protein XI (CA-RPXI/CA11) in the presence or absence of mutant ataxin-3. Therefore, we examined the expression and distribution of all three CA-RPs (CA8, 10, and 11) in human neuronal cells that stably express mutant ataxin-3. Compared with the cells containing normal ataxin-3, protein expression of CA8 and CA11 is significantly increased in human neuroblastoma cells harboring mutant ataxin-3. Semi-quantitative RT-PCR demonstrated that all three CA-RPs exhibited significantly higher transcript levels in neuronal cells expressing mutant ataxin-3. Interestingly, CA11 is distributed not only in the cytoplasm but also within the nuclei of the stably transfected mutant cells when compared with the sole cytoplasmic distribution in cells containing normal ataxin-3. In addition, results from transient transfection assays in SK-N-SH and Neuro2a (N2a) cells also confirmed the nuclear localization of CA11 in the presence of truncated ataxin-3. Most importantly, immunohistochemical staining of the MJD transgenic mouse and post-mortem MJD human brain also revealed that CA11 is highly expressed in both cytoplasm and nuclei of the brain cells. Recruitment of CA11 into nuclear inclusions containing mutant ataxin-3 revealed a possible correlation between CA11 and disease progression. Although the exact function of CA-RPs is still undefined in the central nervous system, our findings suggest that CA-RPs, especially CA11, may play specific roles in the pathogenesis of Machado-Joseph disease.
Assuntos
Regulação da Expressão Gênica/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Animais , Ataxina-3 , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/patologia , Camundongos , Neuroblastoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Mutation of amino acid residues 94, 96 and 119 to histidine(s) in the human carbonic anhydrase (CA, EC 4.2.1.1) related proteins CARP VIII, X and XI restored the zinc binding and catalytic activity for the hydration of CO(2) to bicarbonate. CA VIII, X and XI thus obtained showed high catalytic activity (67.3-92.0% of the activity of hCA II and much higher compared to hCA I) and were inhibited in the milli-micromolar range by inorganic anions, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid. Among the three new isoforms, hCA X was the most efficient enzyme and also showed the highest affinity for anion inhibitors (K(I)s of 3.6-68 µM for phenylboronic acid, sufamic acid, sulfamide, cyanide and azide). hCA VIII was poorly inhibited by halides, cyanate, nitrate and sulfate (K(I)s of 38.4-65.4 mM), whereas CA XI had a behavior intermediate between that of hCA VIII and X, both regarding the catalytic activity and sensitivity to anion inhibitors.
Assuntos
Ânions/química , Biomarcadores Tumorais/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Biocatálise , Biomarcadores Tumorais/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Humanos , Cinética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Two new ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO(2) to bicarbonate, with k(cat) of 0.79×10(6) s(-1) and 1.0×10(6) s(-1), and k(cat)/K(m) of 5.2×10(7) M(-1) s(-1) and of 8.3×10(7) M(-1) s(-1), respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the ß-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials.
Assuntos
Ânions/química , Ânions/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Anidrases Carbônicas/farmacologia , Salmonella typhimurium/enzimologia , Sequência de Aminoácidos , Ânions/síntese química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/genética , Anidrases Carbônicas/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The two ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were investigated for their inhibition with a large panel of sulfonamides and sulfamates. Unlike inorganic anions, which are weak, millimolar inhibitors of the two enzymes [Vullo et al., Bioorg. Med. Chem. Lett.2011, 21, 3591], sulfonamides and sulfamates are effective micro-to nanomolar inhibitors of the two enzymes. Various types of inhibitors have been detected among the 38 investigated sulfonamides/sulfamates, with K(I)s in the range of 31 nM-5.87 µM. The best stCA 1 inhibitors were acetazolamide and benzolamide-based compounds, whereas the best stCA 2 inhibitors were sulfonylated benzenesulfonamides and amino-benzolamide derivatives (K(I)s in the range of 31-90 nM). 3-Fluoro-5-chloro-4-aminobenzolamide showed an inhibition constant of 51 nM against stCA 1 and of 38 nM against stCA 2, being the best inhibitor detected so far for these enzymes. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the ß-CAs investigated here may be useful for developing novel antibacterials, targeting ß-CAs which may be involved in pathogenicity and invasion of some bacteria.
Assuntos
Antibacterianos/síntese química , Inibidores da Anidrase Carbônica/síntese química , Anidrases Carbônicas/efeitos dos fármacos , Infecções por Salmonella/tratamento farmacológico , Salmonella typhimurium/efeitos dos fármacos , Sulfonamidas/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/uso terapêutico , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Infecções por Salmonella/enzimologia , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Ácidos Sulfônicos/químicaRESUMO
A ß-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA II, has been cloned, purified, and characterized kinetically. bsCA II showed high catalytic activity for the hydration of CO(2) to bicarbonate, with a k(cat) of 1.1×10(6), and k(cat)/K(m) of 8.9×10(7)M(-1)s(-1). A panel of sulfonamides and sulfamates have been investigated for inhibition of this enzyme. All types of activities, from the low nanomolar to the micromolar, have been detected for these derivatives, which showed inhibition constants in the range of 7.3nM-8.56µM. The best bsCA II inhibitors were some glycosylated sulfanilamides, aliphatic sulfamates, and halogenated sulfanilamides, with inhibition constants of 7.3-87nM. Some of these dual inhibitors of bsCA I and II, also inhibited bacterial growth in vitro, in liquid cultures. These promising data on live bacteria allow us to propose bacterial ß-CA inhibition as an approach for obtaining anti-infective agents with a new mechanism of action compared to classical antibiotics.
Assuntos
Antibacterianos/farmacologia , Brucella suis/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Sulfonamidas/farmacologia , Ácidos Sulfônicos/farmacologia , Antibacterianos/química , Brucella suis/enzimologia , Brucella suis/crescimento & desenvolvimento , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Anidrases Carbônicas/isolamento & purificação , Clonagem Molecular , Desenho de Fármacos , Descoberta de Drogas , Concentração Inibidora 50 , Cinética , Sulfonamidas/química , Ácidos Sulfônicos/químicaRESUMO
The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(- 1) and k(cat)/K(M) of 2.5.10(5) M(- 1) s(- 1). The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (K(I)s of 21-78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K(I)s around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K(I)s in the range of 10-90 microM. It is difficult to explain the inhibitory activity of carbonate (K(I) of 10 microM) against hCA III, also considering the fact that this ion has an affinity of 15-73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (K(I) of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).
Assuntos
Ânions/farmacologia , Anidrase Carbônica III/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacologia , Carbonatos , Clonagem Molecular , Citosol , Humanos , CinéticaRESUMO
BACKGROUND: Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2) to bicarbonate and a proton, and alpha-class CA has been reported to facilitate the acid acclimation of Helicobacter pylori (hpalphaCA). The purpose of this study was to characterize the beta-class CA of H. pylori (hpbetaCA) and elucidate the role of this enzyme as a possible drug target for eradication therapy. METHODS: We isolated DNA clones of independent H. pylori strains obtained from patients with gastritis (n = 15), gastric ulcer (n = 6), or gastric cancer (n = 16), and then studied genetic polymorphisms. In addition, the susceptibility of H. pylori to sulpiride, an antiulcer drug and efficient inhibitor of both hpalphaCA and hpbetaCA, was studied with an in vitro killing assay. RESULTS: DNA sequences of all 37 hpbetaCA clones encoded a 221 amino acid polypeptide with a variety of polymorphisms (57 types of amino acid substitution at 48 residue positions). There was no polymorphism functionally relevant to the gastric lesion type. One strain included unique residues that were not seen in the other 36 clones from Japanese patients but which were found in a strain obtained from the United Kingdom. Sulpiride had killing effects at concentrations greater than 200 microg/ml for H. pylori, including strains resistant to clarithromycin, metronidazole, or ampicillin. CONCLUSIONS: Helicobacter pylori might have evolved independently in the Caucasian and Japanese populations. Dual inhibition of alpha-and beta-class CAs could be applied as alternative therapy for eradication of H. pylori.
Assuntos
Anidrase Carbônica II/genética , Inibidores da Anidrase Carbônica/farmacologia , Clonagem Molecular , DNA Bacteriano/genética , Helicobacter pylori/enzimologia , Polimorfismo Genético , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/biossíntese , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Sulpirida/farmacologiaRESUMO
The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO2 hydration reaction proved hCA VI to possess a kcat of 3.4 x 10(5) s-1 and kcat/KM of 4.9 x 10(7) M-1 s-1 (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction on the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of sulfonamides and one sulfamate have been tested for their interaction with this isozyme. Simple benzenesulfonamides were rather ineffective hCA VI inhibitors, with inhibition constants in the range of 1090-6680 nM. Better inhibitors were detected among such derivatives bearing 2- or 4-amino-, 4-aminomethyl-, or 4-hydroxymethyl moieties or among halogenated sulfanilamides (KI values of 608-955 nM). Some clinically used compounds, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, sulpiride, and indisulam, or the orphan drug benzolamide, showed effective hCA VI inhibitory activity, with inhibition constants of 0.8-79 nM. The best inhibitors were brinzolamide and sulpiride (KI values of 0.8-0.9 nM), the latter compound being also a CA VI-selective inhibitor. The metallic taste reported as a side effect after the treatment with systemic sulfonamides may be due to the inhibition of the salivary CA VI. Some of the compounds investigated in this study might be used as additives in toothpastes for reducing the acidification produced by the relevant CO2 hydrase activity of enamel CA VI, which leads to the formation of protons and bicarbonate and may have a role in cariogenesis.
Assuntos
Inibidores da Anidrase Carbônica/síntese química , DNA/biossíntese , Subunidades Proteicas/antagonistas & inibidores , Sulfonamidas/síntese química , Ácidos Sulfônicos/síntese química , Sequência de Aminoácidos , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Catálise , Clonagem Molecular , Humanos , Isoenzimas/síntese química , Isoenzimas/química , Isoenzimas/farmacologia , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologiaRESUMO
We have cloned and sequenced Helicobacter pylori alpha-class carbonic anhydrase (hpCA) from patients with different gastric mucosal lesions, including gastritis (n=15), ulcer (n=6), and cancer (n=16). Although several polymorphisms were newly identified such as 12Ala, 13Thr, 16Ile, and 168Phe, there was no significant relevance of any polymorphism with gastric mucosal lesion types. A library of sulfonamides/sulfamates has been investigated for the inhibition of hpCA, whereas new derivatives have been obtained by attaching 4-tert-butyl-phenylcarboxamido/sulfonamido tails to benzenesulfonamide/1,3,4-thiadiazole-2-sulfonamide scaffolds. All types of activity for inhibition of hpCA have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak inhibitors (KI 873-4360 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (KI 413-640 nM), whereas most of the clinically used inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium-potency inhibitors (KI 105-378 nM). Some potent hpCA inhibitors were detected too (KI 12-84 nM) among acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide and some newly designed compounds incorporating lipophilic tails. Some of the newly prepared derivatives had selectivity ratios for inhibiting hpCA over hCA II in the range of 1.25-3.48, showing thus some selectivity for inhibiting the bacterial enzyme. Since hpCA is essential for the survival of the pathogen in acid, it might be used as a new pharmacologic tool in the management of drug-resistant H. pylori.
Assuntos
Inibidores da Anidrase Carbônica/síntese química , Anidrases Carbônicas/genética , DNA Bacteriano/genética , Mucosa Gástrica/microbiologia , Helicobacter pylori/enzimologia , Sulfonamidas/síntese química , Ácidos Sulfônicos/síntese química , Sequência de Aminoácidos , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Clonagem Molecular , Gastrite/microbiologia , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Neoplasias Gástricas/microbiologia , Úlcera Gástrica/microbiologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologiaRESUMO
Carbonic anhydrase-related protein VIII (CA-RP VIII) is believed to be an oncofetal antigen and is overexpressed in colorectal and non-small cell lung cancer. However, the pathobiological properties of CA-RP VIII in lung cancer remain unclear. In the present study, we examined ultrastructural changes caused by exogenous CA-RP VIII expression in a well-differentiated lung adenocarcinoma cell line, PC-9. Many vacuoles lined by cilia, sometimes large vacuoles pushing the nuclei to one side, were found in the cytoplasm of CA-RP VIII-expressing PC-9 cells, but not in control PC-9 cells. Moreover, signet-ring cells containing abundant intracytoplasmic mucin were often found among CA-RP VIII-expressing PC-9 cells, but rarely among control PC-9 cells. We subsequently examined CA-RP VIII expression in atypical adenomatous hyperplasia and early-stage lung adenocarcinoma (Stage Ia). Significant expression of CA-RP VIII was observed in invasive lung adenocarcinoma but not in noninvasive adenocarcinoma. Interestingly, CA-RP VIII was strongly expressed in signet-ring cell cancer and invasive mucinous adenocarcinoma components. CA-RP VIII also appeared to enhance the invasiveness of PC-9 cells in Matrigel invasion assay. The present findings suggest that CA-RP VIII expression in lung adenocarcinoma is related to cancer cell invasion.
Assuntos
Anidrases Carbônicas/genética , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/ultraestrutura , Proteínas do Tecido Nervoso/genética , Adenoma/genética , Adenoma/ultraestrutura , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular , Expressão Gênica , Humanos , Hiperplasia , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Microscopia Eletrônica de Transmissão , Mucinas/metabolismo , Invasividade Neoplásica , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacúolos/ultraestruturaRESUMO
Three ß-carbonic anhydrases (CAs, EC 4.2.1.1), encoded by the gene Rv1284 (mtCA 1) Rv3588c (mtCA 2) and Rv3273 (mtCA 3) are present in the human pathogen Mycobacterium tuberculosis. These enzymes were cloned and they showed appreciable catalytic activity for CO(2) hydration, with k(cat) of 3.9 x 10(5) s(-1), and k(cat)/K(m) of 3.7 x 10(7) M(-1).s1 for mtCA 1, of 9.8 x 10(5) s(-1), and k(cat)/K(m) of 9.3 x 10(7) M(-1).s(-1) for mtCA 2 and k(cat) of 4.3 x 10(5) s(-1), and a k(cat)/K(m) of 4.0 x 10(7) M(-1).s(-1) for mtCA 3, respectively. The Rv3273 gene product is predicted to be a 764 amino acid residues polypeptide, consisting of a sulfate transporter domain (amino acids 121-414) in addition to the ß-CA mentioned above (which is encoded by residues 571-741). All these enzymes were inhibited appreciably by many sulfonamides and sulfamates, in the nanomolar - micromolar range, whereas some subnanomolar inhibitors were also reported for two of them (mtCA 1 and mtCA 3). As sulfonamides also efficiently inhibit dehydropteroate synthetase (DHPS), the contribution of mtCAs and DHPS inhibition to a possible antimycobacterial action of these drugs must be better understood. It has been, however, proven that mtCAs are druggable targets, with a real potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug resistance and extensive multi-drug resistance, although for the moment no in vivo inhibition of the bacteria could be evidenced with the presently available drugs due to lack of penetrability through the mycolic acid cell wall of M. tuberculosis.
Assuntos
Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/efeitos dos fármacos , Isoenzimas/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Isoenzimas/metabolismo , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Ácidos Sulfônicos/farmacologiaRESUMO
A beta-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified, and characterized kinetically. bsCA 1 has appreciable activity as catalyst for the hydration of CO(2) to bicarbonate, with a k(cat) of 6.4 x 10(5) s(-1) and k(cat)/K(m) of 3.9 x 10(7) M(-1).s(-1). A panel of 38 sulfonamides and one sulfamate have been investigated for inhibition of this new beta-CA. All types of activities have been detected, with K(I)s in the range of 17 nM to 5.87 microM. The best bsCA 1 inhibitors were ethoxzolamide (17 nM), celecoxib (18 nM), dorzolamide (21 nM), valdecoxib, and sulpiride (19 nM). Whether bsCA 1 inhibitors may have application in the fight against brucellosis, an endemic disease and the major bacterial zoonosis, producing debilitating infection in humans and animals, warrants further studies.
Assuntos
Brucella suis/enzimologia , Brucelose/tratamento farmacológico , Anidrases Carbônicas/genética , Filogenia , Sulfonamidas/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Brucella suis/genética , Anidrases Carbônicas/efeitos dos fármacos , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Cinética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Relação Estrutura-AtividadeRESUMO
The beta-carbonic anhydrase (CA, EC 4.2.1.1) encoded by the gene Rv1284 (mtCA 1) of Mycobacterium tuberculosis shows appreciable catalytic activity for CO(2) hydration, with a k(cat) of 3.9 x 10(5) s(-1) and a k(cat)/K(m) of 3.7 x 10(7) M(-1) s(-1). A panel of 36 sulfonamides and one sulfamate, some of which are used clinically, were assayed for their effect on mtCA 1 catalytic activity. Most sulfonamides exhibited K(I) values in the range of 1-10 microM, but several derivatives, including sulfanilyl-sulfonamides acetazolamide, methazolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and the sulfamate topiramate, exhibited submicromolar inhibition (K(I) values of 0.481-0.905 microM). The best inhibitors were 3-bromosulfanilamide and indisulam (K(I) values of 97-186 nM). This study demonstrates that mtCA 1 can be inhibited by sulfonamides and sulfamates and thus has potential for developing antimycobacterial agents with an alternate mechanism of action. This is an important finding to explore further, as many strains exhibit multidrug resistance and extensive multidrug resistance to existing therapeutics.
Assuntos
Antibacterianos/química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/genética , Mycobacterium tuberculosis/enzimologia , Sulfonamidas/química , Ácidos Sulfônicos/química , Sequência de Aminoácidos , Sequência de Bases , Anidrases Carbônicas/química , Anidrases Carbônicas/isolamento & purificação , Catálise , Clonagem Molecular , Farmacorresistência Bacteriana Múltipla , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
The Rv3273 gene product of Mycobacterium tuberculosis, a beta-carbonic anhydrase (CA, EC 4.2.1.1), mtCA 3, shows appreciable catalytic activity for CO(2) hydration (k(cat) of 4.3 x 10(5) s(-1), and k(cat)/K(m) of 4.0 x 10(7) M(-1) x s(-1)). A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 3. Sulfanilyl-sulfonamides, acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and zonisamide, showed effective, submicromolar inhibition (K(I)s of 104-611 nM), the best inhibitor being 2-amino-pyrimidin-4-yl-sulfanilamide (K(I) of 91 nM).
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Biocatálise , Anidrases Carbônicas/química , Clonagem Molecular , Dados de Sequência Molecular , Sulfonamidas/farmacologia , Ácidos Sulfônicos/farmacologiaRESUMO
DNA clones for the beta-class carbonic anhydrase (CA, EC 4.2.1.1) of Helicobactor pylori (hpbetaCA) were obtained. A recombinant hpbetaCA protein lacking the N-terminal 15-amino acid residues was produced and purified, representing a catalytically efficient CA. hpbetaCA was strongly inhibited (K(I)s in the range of 24-45 nM) by many sulfonamides/sulfamates, among which acetazolamide, ethoxzolamide, topiramate, and sulpiride, all clinically used drugs. The dual inhibition of alpha- and/or beta-class CAs of H. pylori might represent a useful alternative for the management of gastritis/gastric ulcers, as well as gastric cancer. This is also the first study showing that a bacterial beta-CA can be a drug target.
Assuntos
Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Química Farmacêutica/métodos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Helicobacter pylori/enzimologia , Sulfonamidas/antagonistas & inibidores , Sulfonamidas/química , Sequência de Aminoácidos , Clonagem Molecular , Desenho de Fármacos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Neoplasias Gástricas/tratamento farmacológico , Úlcera Gástrica/tratamento farmacológicoRESUMO
The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(-1) and k(cat)/K(M) of 2.5 x 10(5) M(-1) s(-1), being a slower catalyst for the physiological reaction as compared to the genetically related cytosolic isoforms hCA I and II. An inhibition study with a library of sulfonamides and one sulfamate, some which are clinically used compounds, is reported. hCA III is less prone to be inhibited by these compounds as compared to hCA I and II for which many low nanomolar inhibitors were detected earlier. The best hCA III inhibitors were prontosil, sulpiride, indisulam, benzolamide, aminobenzolamide, and 4-amino-6-chloro-benzene-1,3-disulfonamide which showed K(I)s in the range of 2.3-18.1 microM. Clinically used compounds such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, topiramate, zonisamide, celecoxib, and valdecoxib were less effective hCA III inhibitors, with affinities in the range of 154-2200 microM. This is the first study in which low micromolar hCA III inhibitors are reported.
Assuntos
Anidrase Carbônica III/antagonistas & inibidores , Anidrase Carbônica III/genética , Inibidores da Anidrase Carbônica/farmacologia , Citosol/enzimologia , Sulfonamidas/farmacologia , Sequência de Aminoácidos , Anidrase Carbônica III/isolamento & purificação , Inibidores da Anidrase Carbônica/química , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , Sulfonamidas/química , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologiaRESUMO
Increased expression of carbonic anhydrase-related protein (CA-RP) VIII has previously been shown in colorectal carcinoma. Since CA-RP has no catalytic carbonic anhydrase (CA) activity, the present study attempted to elucidate its biological significance in colon cancer cells. From a colon cancer cell line (LoVo), we established clones that overexpressed CA-RP VIII (LoVo-CA8) and a control transfectant with a vector alone (LoVo-pCIneo) and studied alterations in the biological behaviors of the tumor cells both in vitro and in vivo. LoVo-CA8 cells showed significantly increased mRNA and protein expressions of CA-RP VIII as compared to LoVo-pCIneo cells. Cell proliferation, colony formation, and cell invasion assays showed that LoVo-CA8 cells had significantly higher cell proliferative and invasive abilities as compared to parental LoVo and LoVo-pCIneo cells in vitro. In an in vivo xenograft assay, LoVo-CA8 cells showed a higher tumor growth rate than parental LoVo cells. Further, small interfering RNA (siRNA)-mediated knockdown of CA-RP VIII revealed significant inhibition in cell proliferation and colony formation of a colon cancer cell line HCT116, which showed high endogenous expression of CA-RP VIII. These findings indicated that CA-RP VIII plays a role in the growth of colon cancer cells.
Assuntos
Biomarcadores Tumorais/fisiologia , Neoplasias do Colo/patologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Ensaio de Unidades Formadoras de Colônias , Regulação Neoplásica da Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/patologia , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A library of sulfonamides/sulfamates has been investigated for the inhibition of the carboxyterminal truncated form of the alpha-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the gastric pathogen Helicobacter pylori (hpCA). This enzyme, incorporating 202 amino acid residues, showed a catalytic activity similar to that of the full length hpCA, with k(cat) of 2.35 x 10(5)s(-1) and k(cat)/K(M) of 1.56 x 10(7)M(-1)s(-1) at 25 degrees C and pH of 8.9, for the CO(2) hydration reaction. All types of activity for inhibition of the bacterial enzyme have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak hpCA inhibitors (inhibition constants, K(I)s, in the range of 830-4310 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (K(I)s in the range of 310-562 nM), whereas most of the clinically used CA inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium potency hpCA inhibitors (K(I)s in the range of 124-287 nM). Some potent hpCA inhibitors were detected too (K(I)s in the range of 20-96 nM) such as acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide, 4-sulfanilyl-aminoethyl-benzenesulfonamide, and 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. Most of the investigated derivatives acted as better inhibitors of the human isoform hCA II than as hpCA inhibitors. Since hpCA is essential for the survival of the pathogen in acid, its inhibition by compounds such as those investigated here might be used as a new pharmacologic tool in the management of drug resistant H. pylori.