RESUMEN
PURPOSE: Adrenocortical carcinoma (ACC), a rare malignancy of the adrenocortex, is characterized by a crosstalk between the adipose microenvironment and tumor. Here, we assessed the involvement of carbonic anhydrase (CA) enzymes III and IX (CAIII and CAIX), in the metabolic alterations of the adipose tissue characterizing obesity and in the local crosstalk between the tumor adipose microenvironment and ACC. RESULTS/METHODS: CAIII and CAIX expression is altered in visceral adipose tissue (VAT) in obesity and in ACC. A significant CAIX upregulation was present in ACC at advanced stages (n = 14) (fold increase FI = 7.4 ± 0.1, P < 0.05) associated with lower CAIII levels (FI = 0.25 ± 0.06, P < 0.001), compared with lower stages (n = 9). In vitro coculture between visceral adipose stem cells (ASCs) and ACC cell lines, H295R and MUC-1, mimicking the interaction occurring between VAT and advanced ACC, showed a significant CAIX upregulation in H295R but not in MUC-1 cells, and a decreased expression of CAIII. The effect on adipose cells was different when cocultured with H295R or MUC-1 cells. Coculture did not modulate CAIII expression in ASCs, which, however, was significantly downregulated with H295R (FI = 0.34 ± 0.11, P < 0.05) and upregulated by MUC-1 when cocultured ASCs were induced to differentiate toward adipocytes, with an expression profile similar to what found in VAT of obese subjects. CAIX expression was markedly increased in ASCs cocultured with H295R and to a less extent following adipogenesis induction (FI = 150.9 ± 46.5 and FI = 4.6 ± 1.1, P < 0.01, respectively). CONCLUSION: Our findings highlight a modulation of CAIII and CAIX in the metabolic crosstalk between ACC and its local adipose microenvironment, suggesting that CAs might represent a potential target for novel anticancer therapies.
Asunto(s)
Neoplasias de la Corteza Suprarrenal , Carcinoma Corticosuprarrenal , Anhidrasa Carbónica III , Anhidrasas Carbónicas , Humanos , Anhidrasa Carbónica IX , Antígenos de Neoplasias/metabolismo , Anhidrasas Carbónicas/metabolismo , Obesidad , Microambiente TumoralRESUMEN
A successful spawning migration in salmon depends on their athletic ability, and thus on efficient cardiovascular oxygen (O2) transport. Most teleost fishes have highly pH-sensitive haemoglobins (Hb) that can release large amounts of O2 when the blood is acidified at the tissues. We hypothesized that plasma-accessible carbonic anhydrase (paCA; the enzyme that catalyses proton production from CO2) is required to acidify the blood at the tissues and promote tissue O2 extraction. Previous studies have reported an elevated tissue O2 extraction in hypoxia-acclimated teleosts that may also be facilitated by paCA. Thus, to create experimental contrasts in tissue O2 extraction, Atlantic salmon were acclimated to normoxia or hypoxia (40% air saturation for more than six weeks), and the role of paCA in enhancing tissue O2 extraction was tested by inhibiting paCA at rest and during submaximal exercise. Our results show that: (i) in both acclimation groups, the inhibition of paCA increased cardiac output by one-third, indicating a role of paCA in promoting tissue O2 extraction during exercise, recovery and at rest; (ii) the recruitment of paCA was plastic and increased following hypoxic acclimation; and (iii) maximal exercise performance in salmon, and thus a successful spawning migration, may not be possible without paCA.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Consumo de Oxígeno , Oxígeno/sangre , Salmo salar/metabolismo , Aclimatación , Anaerobiosis , Animales , Transporte BiológicoRESUMEN
Synthetic sulfonamide derivatives are a class of potent matrix metalloproteinase inhibitors (MMPI) that have potential for the treatment of diseases related to uncontrolled expression of these enzymes. The lack of selectivity of the large majority of such inhibitors, leading to the inhibition of MMPs in tissues other than the targeted one, has dramatically reduced the therapeutic interest in MMPIs. The recent development of efficient drug delivery systems that allow the transportation of a selected drug to its site of action has opened the way to new perspectives in the use of MMPIs. Here, a PAMAM-based divalent dendron with two sulfonamidic residues was synthesized. This nanomolar inhibitor binds to the catalytic domain of two MMPs as well as to the transmembrane human carbonic anhydrases (hCAs) XII, which is present in the eye and considered an antiglaucoma target. In the animal model of an experimental dry eye, no occurrence of dotted staining in eyes treated with our inhibitor was observed, indicating no symptoms of corneal desiccation.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Síndromes de Ojo Seco/tratamiento farmacológico , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloproteinasas de la Matriz/química , Animales , Sistemas de Liberación de Medicamentos , Humanos , Metaloproteinasas de la Matriz/metabolismoRESUMEN
A mild, efficient and one pot procedure to access benzoxazoles using easily accessible acylselenoureas as starting materials has been discovered. Mechanistic studies revealed a pH dependent intramolecular oxidative deselenization, with ring closure due to an intramolecular nucleophilic attack of a phenoxide ion. All the benzoxazoles herein reported possessed a primary sulfonamide zinc binding group and showed effective inhibitory action on the enzymes, carbonic anhydrases.
Asunto(s)
Amidas/farmacología , Benzoxazoles/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Compuestos de Organoselenio/química , Urea/análogos & derivados , Amidas/síntesis química , Amidas/química , Benzoxazoles/síntesis química , Benzoxazoles/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Oxidación-Reducción , Relación Estructura-Actividad , Urea/químicaRESUMEN
Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm.
Asunto(s)
Acrosoma/enzimología , Anhidrasas Carbónicas/metabolismo , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Animales , Células Cultivadas , Activación Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
6-Sulfamoyl-saccharin was investigated as an inhibitor of 11 α-carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, hCA I-XIV, and X-ray crystallographic data were obtained for its adduct with hCA II, the physiologically dominant isoform. This compound possesses two potential zinc-binding groups, the primary sulfamoyl one and the secondary, acylatedsulfonamide. Saccharin itself binds to the Zn(II) ion from the CA active site coordinating with this last group, in deprotonated (SO2N(-)CO) form. Here we explain why 6-sulfamoyl-saccharin, unlike saccharin, binds to the metal ion from the hCA II active site by its primary sulfonamide moiety and not the secondary one as saccharin itself. Our study is useful for shedding new light to the structure-based drug design of isoform-selective CA inhibitors of the sulfonamide type.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Sacarina/análogos & derivados , Sacarina/farmacología , Sulfonamidas/farmacología , Inhibidores de Anhidrasa Carbónica/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Sacarina/química , Sulfonamidas/químicaRESUMEN
4-Sulfamoyl-N-(3-morpholinopropyl)benzamide (I-1), N-(3-morpholinopropyl)benzene-1,4-disulfonamide (I-2) and N-(4-diethylaminoethoxybenzyl)benzene-1,4-bis(sulfonamide (I-3), were prepared and assayed as inhibitors of four carbonic anhydrase (CA) isoenzymes hCA I, hCA II, hCA IV and hCA XII. These compounds exhibited nanomolar half maximal inhibitory concentration (IC(50)) ranging from 58 to 740 nmol/L. All three aromatic sulfonamides show different activities for the isoenzymes studied with lowest affinity against isoenzyme hCA XII.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Sulfonamidas/farmacología , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Three different honey extracts from the endemic plant in the Black Sea region Rhododendron ponticum, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), more precisely the human (h) isoforms hCA I and hCA II. Hexane, methanol, ethanol, and water solid-phase extractions (SPEs) showed inhibitory activity towards the two CA isozymes which were related to the total phenolic content. The highest inhibitory effects (0.036-0.039 mg/mL) were those of propolis methanolic extract. Among the three different samples investigated here, the aqueous extracts showed lower inhibitory effects compared to the organic solvent SPE extracts (in the range of 1.150- 5.144 mg/mL). The studied honey extracts constitute an interesting source of phenolic derivatives that might serve to identify lead compounds, targeting the physiologically relevant enzymes CA I and CA II.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Miel , Extractos Vegetales/farmacología , Polen/química , Própolis/química , Zinc/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/aislamiento & purificación , Activación Enzimática/efectos de los fármacos , Humanos , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extracción en Fase Sólida , Relación Estructura-ActividadRESUMEN
We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).
Asunto(s)
Acuaporina 1/metabolismo , Dióxido de Carbono/metabolismo , Membrana Eritrocítica/metabolismo , Animales , Bicarbonatos/metabolismo , Transporte Biológico , Permeabilidad de la Membrana Celular/fisiología , Membrana Eritrocítica/fisiología , Humanos , Concentración de Iones de Hidrógeno , Oocitos , Isótopos de Oxígeno/metabolismo , Xenopus laevisRESUMEN
Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidence proteins identified include metabolic transporters, ß1 integrins, integrin-associated protein CD98hc and matrix metalloprotease 14 (MMP14). Biochemical studies validate the association of CAIX with α2ß1 integrin, CD98hc and MMP14, and immunofluorescence microscopy demonstrates colocalization of CAIX with α2ß1 integrin and MMP14 in F-actin/cofilin-positive lamellipodia/pseudopodia, and with MMP14 to cortactin/Tks5-positive invadopodia. Modulation of CAIX expression and activity results in significant changes in cell migration, collagen degradation and invasion. Mechanistically, we demonstrate that CAIX associates with MMP14 through potential phosphorylation residues within its intracellular domain, and that CAIX enhances MMP14-mediated collagen degradation by directly contributing hydrogen ions required for MMP14 catalytic activity. These findings establish hypoxia-induced CAIX as a novel metabolic component of cellular migration and invasion structures, and provide new mechanistic insights into its role in tumor cell biology.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/enzimología , Anhidrasa Carbónica IX/metabolismo , Movimiento Celular/fisiología , Neoplasias Mamarias Experimentales/enzimología , Metaloproteinasa 14 de la Matriz/metabolismo , Animales , Antígenos de Neoplasias/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Anhidrasa Carbónica IX/genética , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Metaloproteinasa 14 de la Matriz/genética , Ratones , Podosomas/enzimología , Podosomas/genética , Podosomas/patología , TransfecciónRESUMEN
The possibility to develop new antibacterial agents raised much interest recently. The main classes of antibiotics clinically used nowadays act towards the inhibition of four classical targets: a) cell wall biosynthesis; b) protein biosynthesis; c) DNA and RNA biosynthesis; d) folate biosynthesis. Recently, carbonic anhydrases (CAs, EC 4.2.1.1) started to be investigated in detail in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many bacteria, CAs are essential for the life cycle of the organism and that their inhibition leads to growth impairment or growth defects of the pathogen. CAs catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. Several classes of CA inhibitors (CAIs) are known to date: the metal complexing anions and the unsubstituted sulfonamides, which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere, generating trigonal- bipyramidal species are the classical, most frequently investigated ones. In many cases effective inhibitors were detected, some of which also inhibited the bacterial growth in vivo. However, very few of the detected inhibitors were also selective for the bacterial over the human, off target isoforms such as hCA II. Using structure-based drug design processes, we estimate that it will be possible to achieve the desired selectivity for inhibiting preferentially the bacterial but not the host CA isoforms.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Antibacterianos/química , Bacterias/enzimología , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The presence and degree of dysplasia are important diagnostic and prognostic criteria for oral leukoplakia, but evaluation of dysplasia is difficult and subjective. Carbonic anhydrase-IX (CA-IX) is expressed primarily in tumor cells and is considered a specific hypoxia marker. We investigated the role of CA-IX in oral leukoplakia. We investigated 30 specimens of oral leukoplakia and 35 dysplasia specimens adjacent to the tumor margin. We analyzed clinical variables including age, sex, degree of dysplasia, and smoking, clinical appearance of leukoplakia, number of lesions, location, size, clinical monitoring, malignant transformation and recurrence. For the immunohistochemical study, we used a noncommercial monoclonal antibody against human CA-IX MAb M75. We found greater CA-IX positivity in nonsmokers, erythroplakia and mottled leukoplakia, those located on the tongue, patients with multiple lesions, 2-4 cm leukoplakias and in recurrent cases, although differences were not statistically significant. All lesions in all samples without dysplasia were negative for CA-IX; however, for all other categories of dysplasia, the percentages of positivity and negativity varied. Regarding the diagnostic index values, we found a sensitivity of 32%, specificity of 100%, a positive predictive value of 100% and a negative predictive value of 13%. Leukoplakias appear mainly in females and potentially are malignant; more than 90% have some degree of dysplasia, and therefore require close clinical and histopathological monitoring. The CA-IX immunohistochemical marker may be useful for screening samples without dysplasia owing to its high specificity.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/análisis , Anhidrasas Carbónicas/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Leucoplasia Bucal/diagnóstico , Neoplasias de la Boca/diagnóstico , Adulto , Anciano , Anhidrasa Carbónica IX , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Leucoplasia Bucal/patología , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/patología , Lesiones Precancerosas/patología , PronósticoRESUMEN
The 14 different carbonic anhydrase (CA, EC 4.2.1.1) isozymes as well as the 23 different matrix metalloproteinases (MMPs) isolated up to now in higher vertebrates play important physiological functions in these organisms. Unsubstituted sulfonamides act as high-affinity inhibitors for the first type of these enzymes, whereas hydroxamates strongly inhibit the latter ones. Since the active site geometry around the zinc ion in these two types of metalloenzymes is rather similar, we tested whether sulfonylated amino acid hydroxamates of the type RSO(2)NX-AA-CONHOH (X = H, benzyl, substituted benzyl; AA = amino acid moiety, such as Gly, Ala, Val, Leu) with well-known inhibitory properties against MMPs and Clostridium histolyticum collagenase (ChC, another zinc enzyme related to the MMPs) might also act as CA inhibitors. We also investigated whether N-hydroxysulfonamides of the type RSO(2)NHOH (which are effective CA inhibitors) inhibit MMPs and ChC. Here we report several potent sulfonylated amino acid hydroxamate CA inhibitors (with inhibition constants in the range of 5-40 nM, against the human isozymes hCA I and hCA II, and 10-50 nM, against the bovine isozyme bCA IV), as well as preliminary SAR for this new class of non-sulfonamide CA inhibitors. Some N-hydroxysulfonamides also showed inhibitory properties (in the micromolar range) against MMP-1, MMP-2, MMP-8, MMP-9, and ChC. Thus, the SO(2)NHOH group is a new zinc-binding function for the design of MMP inhibitors. Both CA as well as MMPs are involved, among others, in carcinogenesis and tumor invasion processes. On the basis of these findings, we suggest that the mechanism of antitumor action with some hydroxamate inhibitors might also involve inhibition of some CA isozymes (such as CA IX, CA XII, and CA XIV) present only in tumor cell membranes, in addition to collagenases/gelatinases of the MMP type. Our data also suggest that it should be possible to develop dual enzyme inhibitors that would strongly inhibit both these metalloenzymes, CAs and MMPs, based on the nature of the R, AA, and X moieties in the above formula. Compact X (such as H) and AA (such as Gly) moieties favor CA over MMP inhibition, whereas bulkier X (benzyl, substituted benzyl, etc.) and AA (such as Val, Leu) moieties and substituted-aryl R groups are advantageous for obtaining potent MMP and ChC inhibitors, which show lower affinity for CA.
Asunto(s)
Aminoácidos/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Anhidrasas Carbónicas/metabolismo , Ácidos Hidroxámicos/síntesis química , Inhibidores de Proteasas/síntesis química , Sulfonamidas/síntesis química , Sulfonas/síntesis química , Animales , Inhibidores de Anhidrasa Carbónica/química , Bovinos , Clostridium/química , Cobalto , Humanos , Ácidos Hidroxámicos/química , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/química , Espectrofotometría , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonas/química , ZincRESUMEN
A series of compounds was prepared by reaction of alkyl/arylsulfonyl halides with N-4-nitrobenzylglycine, followed by conversion of the COOH to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-4-nitrobenzylglycine with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. Another subseries of derivatives was prepared from sulfanilyl- or metanilyl-4-nitrobenzylglycine by reaction with arylsulfonyl isocyanates, followed by conversion of the COOH to the hydroxamate moiety. The new compounds were assayed as inhibitors of four matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-8, and MMP-9, and of the Clostridium histolyticum collagenase (ChC). Some of the prepared hydroxamate derivatives proved to be very effective collagenase/gelatinase inhibitors, depending on the substitution pattern at the sulfonamido moiety. Substitutions leading to best inhibitors of MMP-1, a short pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethylphenylsulfonyl moieties at P(1') (K(I)'s of 3-5 nM). For MMP-2, MMP-8, and MMP-9 (deep-pocket enzymes), best inhibitors were especially those containing long perfluoroalkylsulfonyl and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl, 3- and 4-carboxyphenylsulfonyl, arylsulfonylureido, or arylsulfonylureidosulfanilyl/metanilyl moieties, at P(1'). Bulkier groups in this position, such as 1- and 2-naphthyl, substituted-naphthyl, or quinolin-8-yl moieties among others, led to less effective MMP/ChC inhibitors. Best ChC inhibitors were again those containing pentafluorophenylsulfonyl or 3- and 4-protected-aminophenylsulfonyl P(1') anchoring groups, suggesting that this protease is also a short-pocket wider-neck one (more similar to MMP-1). This study also proves that the 4-nitrobenzyl moiety is an efficient P(2') anchoring moiety and that sulfonylureido, ureido, or carboxythioureido substitutions at P(1') are also tolerated for obtaining potent sulfonylated amino acid hydroxamate-like MMP/ChC inhibitors.
Asunto(s)
Metaloendopeptidasas/antagonistas & inhibidores , Colagenasa Microbiana/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Humanos , Peso Molecular , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
A series of sulfonyl amino acyl hydroxamates incorporating alkyl/arylsulfonyl-N-2-nitrobenzyl-L-alanine was prepared. Related compounds were obtained by reaction of N-2-nitrobenzyl-L-Ala with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase (ChC), a bacterial protease involved in the degradation of extracellular matrix. Many of the obtained hydroxamates proved to be effective bacterial collagenase inhibitors, the main contributor to activity being the substitution pattern at the sulfonamido moiety. The best ChC inhibitors were those containing pentafluorophenylsulfonyl and 3- and 4-protected-aminophenylsulfonyl P(1)(') groups among others, with affinities in the low nanomolar range. This study also proves that the 2-nitrobenzyl- moiety, similarly to the 4-nitrobenyl one previously investigated (Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2000, 43, 1858-1865) is an efficient P(2)(') anchoring moiety for obtaining potent bacterial collagenase inhibitors.
Asunto(s)
Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Colagenasa Microbiana/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Sulfonas/síntesis química , Sulfonas/farmacología , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Inhibidores de Proteasas/farmacología , Espectrofotometría Infrarroja , Espectroscopía Infrarroja por Transformada de Fourier , Relación Estructura-ActividadRESUMEN
Aromatic/heterocyclic sulfonamides act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA; EC 4.2.1.1), but the presently available compounds do not generally discriminate between the 14 isozymes isolated in higher vertebrates. Thus, clinically used drugs from this class of pharmacological agents show many undesired side effects due to unselective inhibition of all CA isozymes present in a tissue/organ. Here we propose a new approach for the selective in vivo inhibition of membrane-bound versus cytosolic CA isozymes with a new class of positively charged, membrane-impermeant sulfonamides. This approach is based on the attachment of trisubstituted-pyridinium-methylcarboxy moieties (obtained from 2,4, 6-trisubstituted-pyrylium salts and glycine) to the molecules of classical aromatic/heterocyclic sulfonamides possessing free amino, imino, hydrazino, or hydroxyl groups in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes: i.e., hCA I, hCA II (cytosolic forms), and bCA IV (membrane-bound isozyme) (h = human isozyme; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, and ethoxzolamide), are unable to penetrate through biological membranes, as shown by ex vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors undoubtedly proved that: (i) when using the new type of positively charged sulfonamides, only the membrane-bound enzyme (CA IV) was inhibited, whereas the cytosolic isozymes (CA I and II) were not affected; (ii) in the experiments in which the classical compounds (acetazolamide, benzolamide, etc.) were used, unselective inhibition of all CA isozymes (I, II, and IV) has been evidenced.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/síntesis química , Isoenzimas/metabolismo , Sulfonamidas/síntesis química , Animales , Inhibidores de Anhidrasa Carbónica/metabolismo , Inhibidores de Anhidrasa Carbónica/farmacología , Bovinos , Permeabilidad de la Membrana Celular , Citosol/enzimología , Membrana Eritrocítica/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Ratas , Ratas Wistar , Espectrofotometría Ultravioleta , Sulfonamidas/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Using benzamidine as a lead molecule, two series of alkyl/aralkyl/arylsulfonylguanidines/sulfonyl-O-methylisoureas+ ++ have been prepared and assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that sulfaguanidine and its corresponding O-methylisourea derivative possess moderate but intrinsically selective thrombin inhibitory properties, with K(I)'s around 100 nM against thrombin and 1350-1500 nM against trypsin. Further elaboration of these two molecules afforded compounds that inhibited thrombin with K(I)'s in the range of 12-50 nM, whereas affinity for trypsin remained relatively low. Such compounds were obtained by attaching benzyloxycarbonyl- or 4-toluenesulfonylureido-protected amino acids (such as L- and D-Phe or L-Pro) or dipeptides (such as Phe-Pro, Gly-His, beta-Ala-His, or Pro-Gly) to the two leads mentioned above, sulfaguanidine and 4-aminobenzenesulfonyl-O-methylisourea. Thus, the present study proposes two novel approaches for the preparation of high-affinity, specific thrombin inhibitors: two novel S1 anchoring moieties in the already large family of arginine/amidine-based inhibitors and novel peptidomimetic scaffolds obtained by incorporating tosylureido amino acids in the hydrophobic binding site(s). The first one is important for obtaining bioavailable thrombin inhibitors, devoid of the high basicity of the commonly used arginine/amidine-based inhibitors, whereas the second one may lead to improved water solubility of such compounds due to facilitated metal (sodium) salts formation (at the relatively acidic SO(2)NHCO protons) as well as increased stability at hydrolysis (in vivo). A QSAR study also explained the activity in terms of global properties of the molecules, electronic properties of the sulfonylguanidine/sulfonylisourea moiety, and novel descriptors, the frontier orbital phase angles (FOPA), that account for the directions of the nodes in the pi orbitals in the aromatic portion of those of the drugs in which the sulfonyl group was bound to a benzene ring. For thrombin inhibition, the size of the molecule was the dominant influence, while for trypsin inhibition the FOPA was the principal determinant of activity. The dependence of activity on the FOPA variables is perhaps the clearest example of a quantum effect in pharmacology and suggests a promising new tool for drug design.
Asunto(s)
Guanidina/análogos & derivados , Inhibidores de Serina Proteinasa/síntesis química , Trombina/antagonistas & inhibidores , Urea/análogos & derivados , Simulación por Computador , Diseño de Fármacos , Guanidina/química , Humanos , Análisis de Regresión , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-Actividad , Inhibidores de Tripsina/síntesis química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacología , Urea/químicaRESUMEN
Reaction of perfluoroalkyl/arylsulfonyl chlorides or perfluoroalkyl/arylcarbonyl chlorides with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded compounds with the general formula C(x)()F(y)()Z-A-SO(2)NH(2), where Z = SO(2)NH, SO(3), CONH, or CO(2) and A = aromatic/heterocyclic moiety. The sulfonyl chlorides used in synthesis included: CF(3)SO(2)Cl, n-C(4)F(9)SO(2)Cl, n-C(8)F(17)SO(2)Cl, and C(6)F(5)SO(2)Cl, whereas the acyl chlorides were C(8)F(17)COCl and C(6)F(5)COCl. A total of 25 different sulfonamides have been derivatized by means of the above-mentioned perfluorosulfonyl/acyl halides. These new series of sulfonamides showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA). For a given sulfonamide derivatized by the above procedures, inhibitory power was greater for the alkyl/arylsulfonylated compounds, as compared to the corresponding perfluoroalkyl/arylcarbonylated ones. In vitro inhibitory activity generally increased with the number of carbon atoms in the molecule of the acylating/sulfonylating agent, with a maximum for the perfluorophenylsulfonylated and perfluorobenzoylated derivatives. Some of the prepared CA inhibitors displayed very good water solubility (in the range of 2%) and strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. The good water solubility of these new classes of CA inhibitors, correlated with the neutral pH of their solutions used in the ophthalmologic applications, makes them attractive candidates for developing novel types of antiglaucoma drugs devoid of unpleasant ocular side effects.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/síntesis química , Anhidrasas Carbónicas/metabolismo , Presión Intraocular/efectos de los fármacos , Sulfonamidas/síntesis química , Administración Tópica , Animales , Humor Acuoso/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/química , Bovinos , Glaucoma/fisiopatología , Humanos , Isoenzimas/antagonistas & inhibidores , Pulmón/química , Conejos , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Distribución Tisular , Úvea/metabolismoRESUMEN
A series of sulfonamides has been obtained by reaction of 4-isothiocyanatobenzenesulfonamide with amines, amino acids, and oligopeptides. The new thiourea derivatives showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA, EC 4.2.1.1). In vitro inhibitory power was good (in the low-nanomolar range) for the derivatives of beta-phenylserine and alpha-phenylglycine, for those incorporating hydroxy and mercapto amino acids (Ser, Thr, Cys, Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr, DOPA), and dicarboxylic amino acids as well as di/tri/tetrapeptides among others. Such CA inhibitors displayed very good water solubility (in the range of 2-3%) mainly as sodium (carboxylate) salts, with pH values of the obtained solutions being 6.5-7.0. Some of these preparations (such as the derivatives of Ser, beta-Ph-Ser, Leu, Asn, etc.) strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. It is interesting to note that not all the powerful CA inhibitors designed in the present study showed topical IOP-lowering effects (such as, for instance, the Cys and Lys derivatives, devoid of such properties) whereas the Pro, Arg, and oligopeptidyl thiourea derivatives showed reduced efficacy when administered topically. This may be due to the very hydrophilic nature of some of these compounds, whereas inhibitors with balanced hydro- and liposolubility also showed optimal in vivo effects. The interesting pharmacological properties of this new type of CA inhibitors, correlated with the neutral pH of their solutions used in ophthalmologic applications, make them attractive candidates for developing novel antiglaucoma drugs devoid of major ocular side effects.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/síntesis química , Presión Intraocular/efectos de los fármacos , Sulfonamidas/síntesis química , Tiourea/análogos & derivados , Tiourea/síntesis química , Animales , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacocinética , Inhibidores de Anhidrasa Carbónica/farmacología , Córnea/metabolismo , Ojo/metabolismo , Glaucoma/tratamiento farmacológico , Concentración de Iones de Hidrógeno , Soluciones Oftálmicas , Conejos , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Tiourea/química , Tiourea/farmacocinética , Tiourea/farmacología , Distribución Tisular , AguaRESUMEN
Reaction of 26 aromatic/heterocyclic sulfonamides containing amino, imino, hydrazino, or hydroxyl groups with Boc-Gly, Boc-Sar, TrS-Crt, or Boc-Gly-Gly (Sar = sarcosine, N-Me-Gly; Crt = creatine, N-amidinosarcosine; TrS = tritylsulfenyl; Boc = tert-butoxycarbonyl) in the presence of carbodiimide derivatives afforded after removal of the protecting groups a series of water-soluble compounds (as salts of strong acids, such as hydrochloric, trifluoroacetic, or trifluoromethanesulfonic). The new derivatives were assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA) and more precisely of three of its isozymes, CA I, II (cytosolic forms), and IV (membrane-bound form), involved in important physiological processes. Efficient inhibition was observed against all three isozymes and especially against CA II and IV (in the nanomolar range), the two isozymes known to play a critical role in aqueous humor secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were applied as 2% water solutions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed with many of them. Thus, the aminoacyl/dipeptidyl tail conferring water solubility to these sulfonamide CA inhibitors coupled with strong enzyme inhibitory properties and balanced lipid solubility seem to be the key factors for obtaining compounds with effective topical antiglaucoma activity.