RESUMEN
The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, ß- and γ-classes. Here we report and anion inhibition study of the ß-CA, VchCAß with anions and other small molecules which inhibit metalloenzymes. The best VchCAß anion inhibitors were sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed KIs in the range of 54-86µM. Diethyldithiocarbonate was also an effective VchCAß inhibitor, with an inhibition constant of 0.73mM. The halides, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrate, nitrite, stannate, selenate, tellurate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanide, trithiocarbonate, and fluorosulfonate showed affinity in the low millimolar range, with KIs of 2.3-9.5mM. Identification of selective inhibitors of VchCAß (over the human CA isoforms) may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzyme.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Vibrio cholerae/enzimología , Aniones/química , Aniones/farmacología , Arsenicales/química , Arsenicales/farmacología , Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Ácidos Sulfónicos/química , Ácidos Sulfónicos/farmacologíaRESUMEN
Three series of sulfonamides incorporating long, bulky tails were obtained by applying synthetic strategies in which substituted anthranilic acids, quinazolines and aromatic sulfonamides have been used as starting materials. They incorporate long, bulky diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl moieties in their molecules, and were investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides showed excellent inhibitory effects against the four isoforms, with KIs of 7.6-322nM against hCA I, of 0.06-85.4nM against hCA II; of 6.7-152nM against hCA IX and of 0.49-237nM against hCA XII; respectively. However no relevant isoform-selective behavior has been observed for any of them, although hCA II and XII, isoforms involved in glaucoma-genesis were the most inhibited ones. The structure-activity relationship for inhibiting the four CAs with these derivatives is discussed in detail.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Anhidrasas Carbónicas/metabolismo , Humanos , Isoformas de Proteínas/metabolismo , Relación Estructura-Actividad , BencenosulfonamidasRESUMEN
By using SLC-0111 (4-fluorophenylureido-benzenesulfonamide), a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in Phase I clinical trials as an antitumor agent as lead molecule, a series of benzenesulfonamide derivatives incorporating ureido moieties was synthesized. The new compounds contain a 4-N-substituted piperazine fragment in which the ureido linker has been included, and were tested as inhibitors of the cytosolic human (h) hCA I and II isoforms, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. Depending on the substitution pattern at the piperazine ring, low nanomolar inhibitors were detected against all four isoforms, making the new class of sulfonamides of interest for various pharmacologic applications.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Piperazinas/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Estructura Molecular , Piperazina , Piperazinas/química , Relación Estructura-Actividad , Sulfonamidas/síntesis químicaRESUMEN
A series of potent inhibitors of human carbonic anhydrase (CA) isoforms I and II has been prepared via a direct, chemoselective sulfochlorination of a range of 1,3-oxazolyl benzenes and thiophenes, followed by primary sulfonamide synthesis. The latter functionality is a known zinc-binding group (ZBG) responsible for anchoring the inhibitors to the CA's zinc metal ion. The compound's periphery as well as the overall scaffold geometry was designed to enable optimal interactions with the two distinct sides of the enzyme's active site, one of which is lined with hydrophobic residues and while the other is predominantly hydrophilic. As a result, several compounds inhibiting the therapeutically important cytosolic CA I and CA II in picomolar range have been identified. These compounds are one of the most potent CA inhibitors identified to-date. Not only the remarkable (>10 000-fold), cytosolic CA I and CA II selectivity vs. the membrane-bound CA IX and CA XII isoforms, but also the pronounced CA II/I selectivity observed in some cases, allow considering this series as a set of isoform-selective chemical biology tools and promising starting points for drug candidate development.
Asunto(s)
Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/química , Anhidrasa Carbónica I/antagonistas & inhibidores , Anhidrasa Carbónica I/química , Inhibidores de Anhidrasa Carbónica/farmacología , Oxazoles/farmacología , Sulfonamidas/farmacología , Anhidrasa Carbónica I/metabolismo , Anhidrasa Carbónica II/metabolismo , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Dominio Catalítico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Estructura Molecular , Oxazoles/química , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
The oral pathogenic bacterium involved in human dental caries formation Streptococcus mutans, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the α- and the other one to the ß-class. This last enzyme (SmuCA) has been cloned, characterized and investigated for its inhibition profile with a major class of CA inhibitors, the inorganic anions. Here we show that SmuCA has a good catalytic activity for the CO2 hydration reaction, with kcat 4.2×10(5)s(-1) and kcat/Km of 5.8×10(7)M(-1)×s(-1), being inhibited by cyanate, carbonate, stannate, divannadate and diethyldithiocarbamate in the submillimolar range (KIs of 0.30-0.64mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KIs of 15-46µM). The anion inhibition profile of the S. mutans enzyme is very different from other α- and ß-CAs investigated earlier. Identification of effective inhibitors of this new enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.
Asunto(s)
Arsenicales/química , Proteínas Bacterianas/antagonistas & inhibidores , Ácidos Borónicos/química , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Streptococcus mutans/enzimología , Ácidos Sulfónicos/química , Secuencia de Aminoácidos , Aniones , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/genética , Clonación Molecular , Caries Dental/microbiología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cinética , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Especificidad de la Especie , Streptococcus mutans/clasificación , Streptococcus mutans/genética , Streptococcus mutans/aislamiento & purificaciónRESUMEN
Streptococcus mutans, the oral pathogenic bacterium provoking dental caries formation, encodes for a ß-class carbonic anhydrase (CA, EC 4.2.1.1), SmuCA. This enzyme was cloned, characterized and investigated for its inhibition profile with the major class of CA inhibitors, the primary sulfonamides. SmuCA has a good catalytic activity for the CO2 hydration reaction, with a kcat of 4.2×10(5) s(-1) and kcat/Km of 5.8×10(7) M(-1)×s(-1), and is efficiently inhibited by most sulfonamides (KIs of 246 nM-13.5 µM). The best SmuCA inhibitors were bromosulfanilamide, deacetylated acetazolamide, 4-hydroxymethylbenzenesulfonamide, a pyrimidine-substituted sulfanilamide derivative, aminobenzolamide and compounds structurally similar to it, as well as acetazolamide, methazolamide, indisulam and valdecoxib. These compounds showed inhibition constants ranging between 246 and 468 nM. Identification of effective inhibitors of this enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.
Asunto(s)
Antibacterianos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Streptococcus mutans/enzimología , Sulfonamidas/farmacología , Secuencia de Aminoácidos , Antibacterianos/química , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Estructura Molecular , Filogenia , Sulfonamidas/químicaRESUMEN
In this study, paraoxonase 1 (PON1; EC 3.1.8.1) was purified from bull semen, and some characteristics of the enzyme were investigated. In vitro inhibition effect of some heavy metals, including Cu(2+), Mn(2+), Cd(2+), Zn(2+), Ni(2+), and Pb(2+), on the activity of the purified enzyme was also investigated. The purification of bull semen PON1 procedure was composed of two steps: ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The enzyme, having a specific activity of 288 EU/mg proteins, was purified 22.67-fold with a yield of 89 %. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme showed the presence of a single band with an apparent MW of 66 kDa. The V max and K M values for the paraoxon substrate were determined as 100 EU and 8.0 × 10(-5) M, respectively. The inhibitory effects of different heavy metals on PON1 activity were determined by using the paraoxon as a substrate. The results showed that all the metals, except for Cd(2+), inhibited the PON1 enzyme activity in a concentration-dependent fashion. IC50 values of Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) were found as 2.59 × 10(-3), 1.17 × 10(-3), 42.74 × 10(-3), 99.10 × 10(-3), 48.80 × 10(-3) mM, respectively. Conversely, Cd(2+) increased the bull semen PON1 enzyme activity. The present study has demonstrated that Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) are serious toxic metals, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.
Asunto(s)
Arildialquilfosfatasa/aislamiento & purificación , Cromatografía en Agarosa/métodos , Metales Pesados/farmacología , Semen/enzimología , Sulfato de Amonio/química , Animales , Arildialquilfosfatasa/antagonistas & inhibidores , Arildialquilfosfatasa/metabolismo , Bovinos , Precipitación Química , Cobre/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Plomo/farmacología , Masculino , Manganeso/farmacología , Peso Molecular , Níquel/farmacología , Paraoxon/metabolismo , Sefarosa/química , Especificidad por Sustrato , Zinc/farmacologíaRESUMEN
The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K(M) and V(Max) values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 x 10(- 3) M & 0.723 EU/mL and 9.465 x 10(- 3) M & 0.722 EU/mL, respectively. Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5-11.0 and 5-50 degrees C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC(50) values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, beta-mercaptoethanol and syringic acid were determined as 9.1 x 10(- 4), 2.3 x 10(- 4) M, 1.5 x 10(- 4) M, 3.8 x 10(- 7) M, 1.2 x 10(- 4) M, 4.9 x 10(- 4) M, and 4 x 10(- 4) M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.