RESUMO
Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and ß of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares â¼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.
Assuntos
Benzobromarona/química , Inibidores do Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/química , Benzobromarona/metabolismo , Benzobromarona/farmacologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Humanos , Hidroxilação/efeitos dos fármacos , Hidroxilação/fisiologia , Concentração Inibidora 50 , Midazolam/metabolismo , Midazolam/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Rivaroxabana/metabolismo , Rivaroxabana/farmacologia , Testosterona/metabolismo , Testosterona/farmacologiaRESUMO
Although benzbromarone (BBR) is a conventional, highly potent uricosuric drug, it is not a standard medicine because it causes rare but fatal fulminant hepatitis. We transformed the bis-aryl ketone structure of BBR to generate novel monocyclic amide-linked phenol derivatives that should possess uric acid excretion activity without adverse properties associated with BBR. The derivatives were synthesized and tested for uric acid uptake inhibition (UUI) in two assays using either urate transporter 1-expressing cells or primary human renal proximal tubule epithelial cells. We also evaluated their inhibitory activity against mitochondrial respiration as a critical mitochondrial toxicity parameter. Some derivatives with UUI activity had no mitochondrial toxicity, including compound 3f, which effectively lowered the plasma uric acid level in Cebus apella. Thus, 3f is a promising candidate for further development as a uricosuric agent.
Assuntos
Amidas/química , Fenol/síntese química , Ácido Úrico/metabolismo , Uricosúricos/síntese química , Animais , Benzobromarona/química , Benzobromarona/farmacologia , Avaliação Pré-Clínica de Medicamentos , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Fenol/efeitos adversos , Fenol/farmacologia , Pirróis/química , Sapajus apella , Transdução de Sinais , Relação Estrutura-Atividade , Ácido Úrico/sangue , Uricosúricos/efeitos adversos , Uricosúricos/farmacocinéticaRESUMO
Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.
Assuntos
Benzobromarona/química , Reposicionamento de Medicamentos , Fármacos Neuroprotetores/química , Pré-Albumina/química , Tiroxina/química , Amiloide/antagonistas & inibidores , Benzobromarona/metabolismo , Benzoxazóis/química , Benzoxazóis/metabolismo , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Diflunisal/análogos & derivados , Diflunisal/química , Diflunisal/metabolismo , Expressão Gênica , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/metabolismo , Pré-Albumina/agonistas , Pré-Albumina/genética , Pré-Albumina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Tiroxina/metabolismo , Tolcapona/química , Tolcapona/metabolismoRESUMO
Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as livestock. Exposure to Microcystins (MCs), toxins produced by various cyanobacterial or blue green algae found in poorly treated drinking water or contaminated seafood such as fish or prawns are associated with hepatotoxicity, nephropathy and neurotoxicity and in extreme cases, death in humans. MC congeners, currently >240 known, differ dramatically in their uptake kinetics, i.e. their uptake via OATP1B1 and OATP1B3, in OATP overexpressing human HEK293â¯cells and primary human hepatocytes. It is thus likely that MC congeners will also differ with respect to the cellular efflux of the parent and conjugated congeners, e.g. via MRPs, MDRs, BCRP or BSEP. Consequently, the role and kinetics of different human efflux transporters - MRP, MDR, BCRP and BSEP in MC efflux was studied using insect membrane vesicles overexpressing the human transporters of interest. Of the efflux transporters investigated, MRP2 displayed MC transport. Michaelis-Menten kinetics displayed mild co-operativity and thus allosteric behavior of MRP2. MC transport by MRP2 was MC congener-specific, whereby MC-LF was transported more rapidly than MC-LR and -RR. Other human transporters (BCRP, BSEP, MRP1,3,5, MDR1) tested in this study did not exhibit interaction with MC. Although MRP2 showed specific MC transport, the MC-LR-GSH conjugate, was not transported suggesting the involvement of other transporters than MRP2 for the conjugate efflux.
Assuntos
Glutationa/química , Microcistinas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzobromarona/química , Benzobromarona/metabolismo , Cromatografia Líquida de Alta Pressão , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Toxinas Marinhas , Microcistinas/análise , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Espectrometria de Massas em TandemRESUMO
Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC-MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC-MS/MS method was developed with fine linearity (r2≥0.996), good reproducibility (RSD≤6.6%), high stability (92.37-114.67%), efficient recovery (91.23-109.82%) and acceptable matrix effect (110.54-115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (Km, Vmax, Clint). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1=85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1=21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic.
Assuntos
Benzobromarona/análise , Benzobromarona/metabolismo , Cromatografia Líquida/métodos , Citocromo P-450 CYP2C9/metabolismo , Espectrometria de Massas em Tandem/métodos , Benzobromarona/química , Citocromo P-450 CYP2C9/química , Humanos , Limite de Detecção , Modelos Lineares , Isoformas de Proteínas , Reprodutibilidade dos TestesRESUMO
Benzbromarone (BBR) is a potent uricosuric drug that can cause serious liver injury. Our recent study suggested that 1'-hydroxy BBR, one of major metabolites of BBR, is metabolized to a cytotoxic metabolite that could be detoxified by glutathione (GSH). The aim of this study was to clarify whether GSH adducts are formed from 1'-hydroxy BBR in human liver microsomes (HLM). Incubation of 1'-hydroxy BBR with GSH in HLM did not result in the formation of GSH adducts, but 1',6-dihydroxy BBR was formed. In addition, incubation of 1',6-dihydroxy BBR with GSH in HLM resulted in the formation of three novel GSH adducts (M1, M2 and M3). The structures of M1 and M2 were estimated to be GSH adducts in which the 1-hydroxyethyl group at the C-2 position and the hydroxyl group at the C-1' position of 1',6-dihydroxy BBR were substituted by GSH, respectively. We also found that the 6-hydroxylation of 1'-hydroxy BBR is mainly catalyzed by CYP2C9 and that several CYPs and/or non-enzymatic reaction are involved in the formation of GSH adducts from 1',6-dihydroxy BBR. The results indicate that 1'-hydroxy BBR is metabolized to reactive metabolites via 1',6-dihydroxy BBR formation, suggesting that these reactive metabolites are responsible for BBR-induced liver injury.
Assuntos
Benzobromarona/análogos & derivados , Benzobromarona/metabolismo , Glutationa/metabolismo , Microssomos Hepáticos/metabolismo , Benzobromarona/efeitos adversos , Benzobromarona/química , Glutationa/química , Humanos , Inativação Metabólica , Estrutura MolecularRESUMO
Benzbromarone (BBR) is a benzofuran derivative that has been quite useful for the treatment of gout; however, it was withdrawn from European markets in 2003 because of reported serious incidents of drug-induced liver injury. BBR-induced hepatotoxicity has been suggested to be associated with the formation of a quinone intermediate. The present study reported epoxide-derived intermediate(s) of BBR. An N-acetylcysteine (NAC) conjugate derived from epoxide metabolite(s) was detected in both microsomal incubations of BBR and urine samples of mice treated with BBR. The NAC conjugate was identified as 6-NAC BBR. Ketoconazole suppressed the bioactivation of BBR to the epoxide intermediate(s), and the CYP3A subfamily was the primary enzyme responsible for the formation of the epoxide(s). The present study provided new information on metabolic activation of BBR.
Assuntos
Benzobromarona/metabolismo , Compostos de Epóxi/metabolismo , Uricosúricos/metabolismo , Animais , Benzobromarona/química , Compostos de Epóxi/química , Humanos , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Uricosúricos/químicaRESUMO
BACKGROUND AND PURPOSE: High-throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second-generation. low MW inhibitors of the calcium-activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors. EXPERIMENTAL APPROACH: Using intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose-dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves. KEY RESULTS: The efficacy of second-generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCCinh -A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16Ainh -A01 in both preparations. CONCLUSIONS AND IMPLICATIONS: We have identified several second-generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.
Assuntos
Benzobromarona/farmacologia , Canais de Cloreto/antagonistas & inibidores , Diclorofeno/farmacologia , Trato Gastrointestinal/efeitos dos fármacos , Hexaclorofeno/farmacologia , Tiofenos/farmacologia , Animais , Anoctamina-1 , Benzobromarona/química , Canais de Cloreto/metabolismo , Diclorofeno/química , Relação Dose-Resposta a Droga , Trato Gastrointestinal/metabolismo , Hexaclorofeno/química , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Relação Estrutura-Atividade , Tiofenos/químicaRESUMO
Traditional read-across approaches typically rely on the chemical similarity principle to predict chemical toxicity; however, the accuracy of such predictions is often inadequate due to the underlying complex mechanisms of toxicity. Here, we report on the development of a hazard classification and visualization method that draws upon both chemical structural similarity and comparisons of biological responses to chemicals measured in multiple short-term assays ("biological" similarity). The Chemical-Biological Read-Across (CBRA) approach infers each compound's toxicity from both chemical and biological analogues whose similarities are determined by the Tanimoto coefficient. Classification accuracy of CBRA was compared to that of classical RA and other methods using chemical descriptors alone or in combination with biological data. Different types of adverse effects (hepatotoxicity, hepatocarcinogenicity, mutagenicity, and acute lethality) were classified using several biological data types (gene expression profiling and cytotoxicity screening). CBRA-based hazard classification exhibited consistently high external classification accuracy and applicability to diverse chemicals. Transparency of the CBRA approach is aided by the use of radial plots that show the relative contribution of analogous chemical and biological neighbors. Identification of both chemical and biological features that give rise to the high accuracy of CBRA-based toxicity prediction facilitates mechanistic interpretation of the models.
Assuntos
Substâncias Perigosas/classificação , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Anticonvulsivantes/química , Anticonvulsivantes/toxicidade , Bactérias/metabolismo , Benzobromarona/química , Benzobromarona/toxicidade , Carbamazepina/química , Carbamazepina/toxicidade , Cloranfenicol/química , Cloranfenicol/toxicidade , Bases de Dados de Compostos Químicos , Supressores da Gota/química , Supressores da Gota/toxicidade , Substâncias Perigosas/toxicidade , Fígado/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Ratos , Transcriptoma/efeitos dos fármacosRESUMO
A high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-QTOF-MS) method was employed in investigation of benzbromarone metabolites in rat plasma, urine, feces and bile samples. Meanwhile, the metabolic pathways of benzbromarone in rats were discussed. The identification was achieved on a reversed-phase C(18) column with mobile phase gradient method. The QTOF-MS was operated under full scan of MS or MS/MS in negative mode. The fragments were acquired by raising collision induced dissociation (CID) energy for speculating the structures of parent ions. According to the information from the chromatograms and mass spectra, 17 metabolites were obtained. Among them, the deoxidized phase I metabolites and an array of phase II metabolites-sulfate conjugates detected in the biological samples made the work more significant.
Assuntos
Benzobromarona/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Animais , Benzobromarona/análogos & derivados , Benzobromarona/análise , Benzobromarona/química , Ácidos e Sais Biliares/química , Fezes/química , Glucuronídeos/análise , Glucuronídeos/química , Glucuronídeos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Sulfatos/análise , Sulfatos/química , Sulfatos/metabolismoRESUMO
Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies.
Assuntos
Benzobromarona/farmacologia , Inibidores Enzimáticos/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Uricosúricos/farmacologia , Animais , Aorta/efeitos dos fármacos , Benzobromarona/análogos & derivados , Benzobromarona/química , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/química , Feminino , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Interferência de RNA , Uricosúricos/química , Peixe-ZebraRESUMO
Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system. In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (K(m) of 2.2 ± 0.3 µM and V(max) of 115 ± 26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC(50) values ≤ 1.1 µM. Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2. A highly significant correlation (r(2) = 0.959) between the in vitro IC(50) values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds. This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Sistema Biliar/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/química , Animais , Benzobromarona/química , Benzobromarona/farmacologia , Canalículos Biliares , Transporte Biológico/efeitos dos fármacos , Ciclosporina/química , Ciclosporina/farmacologia , Digoxina/química , Digoxina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Fluoresceínas/análise , Fluoresceínas/farmacocinética , Taxa de Depuração Metabólica , Propionatos/química , Propionatos/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Ratos , Ácido Taurocólico/química , Ácido Taurocólico/farmacologia , Vesículas TransportadorasRESUMO
The most common cause of cystic fibrosis is deletion of Phe508 in the first nucleotide-binding domain (NBD) of the CFTR chloride channel, which inhibits protein folding. ΔF508 CFTR can be rescued by indirect approaches such as low temperature but the protein is unstable. Here, we tested our predictions that (1) other CFTR mutants such V232D and H1085R were more stable at the cell surface than ΔF508 CFTR after low temperature rescue and (2) the advantages of rescue with specific correctors (pharmacological chaperones) are that they may stabilize ΔF508 CFTR and increase the effectiveness of the correctors by bypassing drug pumps such as P-glycoprotein (P-gp) (increased bioavailability). It was found that the stability of mutants V232D and H1085R after low-temperature (30°C) rescue was about 10-fold higher than ΔF508 CFTR. We show that the corrector, 4,5,7-trimethyl-N-phenylquinolin-2-amine (5a), could stabilize ΔF508 CFTR at the cell surface. Unlike most correctors, corrector 5a showed specificity for CFTR as it did not rescue the G268V P-gp processing mutant nor stimulate the ATPase activity of wild-type P-gp. By contrast, corrector KM11060 was a P-gp substrate as it stimulated P-gp ATPase activity and rescued the G268V mutant. Expression of wild-type P-gp reduced the effectiveness of CFTR rescue by corrector KM11060 by about 5-fold. The results underlie the importance of selecting correctors that are specific for CFTR because their efficiency can be reduced by drug pumps such as P-gp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mutação/genética , Processamento Pós-Transcricional do RNA/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Animais , Benzobromarona/química , Linhagem Celular , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células HEK293 , Humanos , Mesocricetus , Especificidade por SubstratoRESUMO
The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe(508) (DeltaF508). The DeltaF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 degrees C, we found that maturation could be restored if Val(510) was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of DeltaF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Benzobromarona/química , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/química , DNA Complementar/metabolismo , Dissulfetos/química , Humanos , Dados de Sequência Molecular , Fenilalanina/química , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
CYP2C9 substrates can exhibit both hyperbolic and atypical kinetic profiles, and their metabolism can be activated or inhibited depending on the effector studied. CYP2C9 genetic variants can also affect both substrate turnover and kinetic profile. The present study assessed whether analogs of the effector amiodarone differentially altered the atypical kinetic profile of the substrate naproxen and whether this effect was genotype-dependent. Amiodarone, desethylamiodarone, benzbromarone, and its dimethyl analog (benz(meth)arone) were incubated with naproxen and either CYP2C9.1 or CYP2C9.3. Amiodarone activated naproxen demethylation at lower concentrations, regardless of the CYP2C9 allele, and inhibited metabolism at higher concentrations without altering the kinetic profile. Desethylamiodarone was a potent inhibitor of naproxen demethylation, irrespective of the CYP2C9 allele. Benzbromarone altered naproxen demethylation kinetics from a biphasic profile to that of a hyperbolic form in CYP2C9.1 and CYP2C9.3, resulting in inhibition and activation, respectively. In contrast, benz(meth)arone activated naproxen demethylation in both CYP2C9.1 and CYP2C9.3. In addition, the kinetic profile of naproxen demethylation became more hyperbolic at lower concentrations of benz(meth)arone and then reverted back to biphasic as the benz(meth)arone was increased further. Equilibrium binding and multiple-ligand docking studies were used to propose how such similar compounds exerted very different effects on naproxen metabolism. In summary, effectors of CYP2C9 metabolism can alter not only the degree of substrate turnover (activation or inhibition) but also the kinetic profile of metabolism of CYP2C9 substrates through effects on substrate binding and orientation. In addition, these kinetics effects are concentration- and genotype-dependent.
Assuntos
Amiodarona/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Amiodarona/análogos & derivados , Amiodarona/química , Amiodarona/farmacologia , Hidrocarboneto de Aril Hidroxilases/genética , Benzobromarona/química , Benzobromarona/metabolismo , Benzobromarona/farmacologia , Citocromo P-450 CYP2C9 , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Genótipo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Metilação/efeitos dos fármacos , Estrutura Molecular , Naproxeno/química , Naproxeno/metabolismo , Naproxeno/farmacologia , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Especificidade por SubstratoRESUMO
Treatment with benzarone or benzbromarone can be associated with hepatic injury. Both drugs share structural similarities with amiodarone, a well-known mitochondrial toxin. Therefore, we investigated the hepatotoxicity of benzarone and benzbromarone as well as the analogues benzofuran and 2-butylbenzofuran. In isolated rat hepatocytes, amiodarone, benzarone, and benzbromarone (20 micromol/L) decreased mitochondrial membrane potential by 23%, 54% or 81%, respectively. Benzofuran and 2-butylbenzofuran had no effect up to 100 micromol/L. In isolated rat liver mitochondria, amiodarone, benzarone, and benzbromarone, but not benzofuran, decreased state 3 oxidation and respiratory control ratios for L-glutamate (50% decrease of respiratory control ratio at [micromol/L]: amiodarone, 12.9; benzarone, 10.8; benzbromarone, <1). Amiodarone, benzarone, and benzbromarone, but not benzofuran, also uncoupled oxidative phosphorylation. Mitochondrial beta-oxidation was decreased by 71%, 87%, and 58% with 100 micromol/L amiodarone or benzarone and 50 micromol/L benzbromarone, respectively, but was unaffected by benzofuran, whereas ketogenesis was not affected. 2-Butylbenzofuran weakly inhibited state 3 oxidation and beta-oxidation only at 100 micromol/L. In the presence of 100 micromol/L amiodarone, benzarone or benzbromarone, reactive oxygen species production was increased, mitochondrial leakage of cytochrome c was induced in HepG2 cells, and permeability transition was induced in isolated rat liver mitochondria. At the same concentrations, amiodarone, benzarone, and benzbromarone induced apoptosis and necrosis of isolated rat hepatocytes. In conclusion, hepatotoxicity associated with amiodarone, benzarone, and benzbromarone can at least in part be explained by their mitochondrial toxicity and the subsequent induction of apoptosis and necrosis. Side chains attached to the furan moiety are necessary for rendering benzofuran hepatotoxic.
Assuntos
Benzobromarona/análogos & derivados , Benzobromarona/intoxicação , Fígado/efeitos dos fármacos , Amiodarona/química , Amiodarona/intoxicação , Animais , Apoptose/efeitos dos fármacos , Benzobromarona/química , Benzofuranos/química , Benzofuranos/intoxicação , Linhagem Celular Tumoral , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/fisiologia , Corpos Cetônicos/biossíntese , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Dilatação Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Necrose , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Human cytochrome P450 (CYP) 2C19 is one of the most important CYP2C family members responsible for metabolizing commonly prescribed drugs. This research describes synthetic modifications to benzbromarone (Bzbr) to create the most potent CYP2C19 inhibitor ever reported. The most important features enabling analogues of Bzbr to bind to CYP2C19 with high affinity are low acidity (high pK(a) or nonionizability) and hydrophobic substituents adjacent to the phenol moiety. Though CYP2C19 was known to prefer neutral substrates, the extent was perhaps not realized until the anionic, parent compound Bzbr (K(i) = 3.7 microM) was compared to a less acidic dimethyl analogue (K(i) = 0.033 microM). However, differences in affinity for anionic and neutral Bzbr analogues did not appear to affect the regiospecificity of their metabolism by CYP's 2C19 and 2C9. In addition, some Bzbr analogues were metabolized both on the phenol and benzofuran rings. By using a substrate with a methyl ether in place of the Bzbr phenol, it was shown that some Bzbr analogues must be able to freely reposition after binding and oxidize the more energetically favorable position. Normally, O-demethylation of this methyl ether is favored over benzofuran hydroxylation based on ion current from LC/MS. Deuterium substitution of the methyl ether results in an inverse isotope effect on benzofuran hydroxylation (i.e. increased oxidation of this less favorable site). Likewise, Bzbr-based CoMFA models of CYP2C19 demonstrated no clear preference for any one ligand alignment. This suggests results from this modeling method must be interpreted carefully for each CYP isoform. In summary, Bzbr analogues have demonstrated they can be adapted to other CYP2C enzymes in order to probe isoform-specific properties.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Benzobromarona/metabolismo , Inibidores Enzimáticos/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Benzobromarona/química , Benzobromarona/farmacologia , Citocromo P-450 CYP2C19 , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
1. The uricosuric drug benzbromarone is extensively metabolized in man and two main metabolites are formed: the previously characterized 1'-hydroxybenzbromarone (metabolite M1) and an arylhydroxybenzbromarone (metabolite M2) of unknown structure. A dimethyl derivative was isolated from urine after methylation and was characterized by gas chromatography-mass spectrometry (g.l.c.-m.s.) and high resolution nuclear magnetic resonance spectroscopy as 4''-O-methyl-6-methoxybenzbromarone; the structure of M2 therefore is 6-hydroxybenzbromarone. 2. A minor metabolite was similarly characterized as 1'-oxobenzbromarone by comparison with authentic synthetic samples and is a product of biodegradation and not an artifact derived from the in vitro oxidation of 1'-hydroxybenzbromarone. Further minor metabolites were detected and were provisionally characterized by g.l.c.-m.s. after derivatization and include: 2'-hydroxybenzbromarone (an isomer of 1'-hydroxybenzbromarone); 1',6-dihydroxybenzbromarone; dihydroxy-aryl-benzbromarone; and two structure isomers of 6-hydroxybenzbromarone. Debrominated metabolites were not detectable. 3. Benzbromarone is hydroxylated in vivo at the prochiral centre C1' to 1'-hydroxybenzbromarone; analysis of 1'-hydroxybenzbromarone from plasma and urine extracts by h.p.l.c. using a chiral column revealed that two peaks were eluted which showed a mean enantiomeric ratio of 2.1 for plasma and 7.3 for urine; these data demonstrate that the formation and elimination of this metabolite is enantioselective; the absolute configuration of the 1'-chiral centre is presently unknown.