RESUMO
This study provides the first example of a strategy to design a practical ligand toward lysosomal acid α-glucosidase (GAA) focusing on N-alkyl derivatives of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB). The optimized N-4'-(p-trifluoromethylphenyl)butyl-DAB (5g) showed a Ki value of 0.73 µM, which was 353-fold higher affinity than N-butyl-DAB (3f) without a terminal phenyl group. Docking analysis showed that the phenyl part of 5g was accommodated in a lipophilic pocket. Furthermore, the p-trifluoromethyl group effectively suppresses the fluctuation of the phenyl group, allowing it to produce a stable bonding form with GAA. 5g increased the midpoint of the protein's protein denaturation temperature (Tm) by 6.6 °C above that in the absence of the ligand and acted as a "thermodynamic stabilizer" to improve the thermal stability of rhGAA. 5g dose-dependently increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation; its effect was comparable to that of DNJ, which is under clinical trials.
Assuntos
Doença de Depósito de Glicogênio Tipo II , alfa-Glucosidases , Humanos , alfa-Glucosidases/metabolismo , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/metabolismo , Ligantes , Lisossomos/metabolismo , FibroblastosRESUMO
L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.
Assuntos
1-Desoxinojirimicina , Inibidores de Glicosídeo Hidrolases , Imino Açúcares , alfa-Glucosidases , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/farmacologia , Aminoácidos , Domínio Catalítico , Glucose/análogos & derivados , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Humanos , Imino Açúcares/química , Imino Açúcares/farmacologia , Ligantes , Ligação Proteica , alfa-Glucosidases/químicaRESUMO
In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 µM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 µM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.
Assuntos
1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Inibidores Enzimáticos/farmacologia , alfa-Glucosidases/metabolismo , Alquilação , Inibidores Enzimáticos/síntese química , Fibroblastos/metabolismo , Doença de Depósito de Glicogênio Tipo II , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , alfa-Glucosidases/efeitos dos fármacos , alfa-Glucosidases/genéticaRESUMO
Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.
Assuntos
Antivirais/química , Desenho de Fármacos , Inibidores de Glicosídeo Hidrolases/química , Imino Açúcares/química , Simulação de Acoplamento Molecular , alfa-Glucosidases/química , 1-Desoxinojirimicina/química , Glucosamina/análogos & derivados , Glucosamina/química , HumanosRESUMO
The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.
Assuntos
Arabinose/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Imino Furanoses/farmacologia , Alcaloides de Pirrolizidina/farmacologia , Álcoois Açúcares/farmacologia , alfa-Glucosidases/metabolismo , Animais , Arabinose/química , Cães , Inibidores de Glicosídeo Hidrolases/química , Humanos , Imino Furanoses/química , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Alcaloides de Pirrolizidina/química , Álcoois Açúcares/químicaRESUMO
Proteolysis mediated by the ubiquitin-proteome system plays an important role in cancer. Recently, a deubiquitinating enzyme, ubiquitin-specific protease 7 (USP7) has attracted attention as a key regulator of the p53-human double minute 2 (HDM2) pathway in cancer cells. Although some USP7 enzyme inhibitors have been identified, issues related to activity and selectivity prevent their therapeutic application. In this study, we aimed to search for novel USP7-HDM2 protein-protein interaction (PPI) inhibitors that do not affect the USP7 enzyme activity. Using the fragment-mapping program Fsubsite and the canonical subsite-fragment database (CSFDB) developed in our laboratory, we mapped a variety of fragments onto USP7 protein and constructed 3D-pharmacophore models based on the arrangement patterns of the mapped fragments. Finally, we performed 3D pharmacophore-based virtual screening of a commercial compound database and successfully selected promising USP7-HDM2 PPI inhibitor candidates.
Assuntos
Antineoplásicos , Simulação por Computador , Descoberta de Drogas , Inibidores de Proteases , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-mdm2 , Mapeamento por Restrição/métodos , Peptidase 7 Específica de Ubiquitina , Modelos Moleculares , Inibidores de Proteases/química , Estrutura Quaternária de Proteína , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/química , Peptidase 7 Específica de Ubiquitina/químicaRESUMO
Some point mutations in ß-glucocerebrosidase cause either improper folding or instability of this protein, resulting in Gaucher disease. Pharmacological chaperones bind to the mutant enzyme and stabilize this enzyme; thus, pharmacological chaperone therapy was proposed as a potential treatment for Gaucher disease. The binding affinities of α-1-C-alkyl 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives, which act as pharmacological chaperones for ß-glucocerebrosidase, abruptly increased upon elongation of their alkyl chain. In this study, the primary causes of such an increase in binding affinity were analyzed using proteinâ»ligand docking and molecular dynamics simulations. We found that the activity cliff between α-1-C-heptyl-DAB and α-1-C-octyl-DAB was due to the shape and size of the hydrophobic binding site accommodating the alkyl chains, and that the interaction with this hydrophobic site controlled the binding affinity of the ligands well. Furthermore, based on the aromatic/hydrophobic properties of the binding site, a 7-(tetralin-2-yl)-heptyl-DAB compound was designed and synthesized. This compound had significantly enhanced activity. The design strategy in consideration of aromatic interactions in the hydrophobic pocket was useful for generating effective pharmacological chaperones for the treatment of Gaucher disease.
Assuntos
Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/antagonistas & inibidores , Imino Açúcares/química , Álcoois Açúcares/química , Sítios de Ligação , Estabilidade Enzimática/efeitos dos fármacos , Glucosilceramidase/química , Humanos , Imino Açúcares/uso terapêutico , Ligantes , Chaperonas Moleculares/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Mutação Puntual , Ligação Proteica , Álcoois Açúcares/antagonistas & inibidores , Álcoois Açúcares/uso terapêuticoRESUMO
The affinity of a series of iminosugar-based inhibitors exhibiting various ring sizes toward Hex A and their essential interactions with the enzyme active site were investigated. All the Hex A-inhibiting iminosugars tested formed hydrogen bonds with Arg178, Asp322, Tyr421 and Glu462 and had the favorable cation-π interaction with Trp460. Among them, DMDP amide (6) proved to be the most potent competitive inhibitor with a Ki value of 0.041 µM. We analyzed the dynamic properties of both DMDP amide (6) and DNJNAc (1) in aqueous solution using molecular dynamics (MD) calculations; the distance of the interaction between Asp322 and 3-OH and Glu323 and 6-OH was important for stable interactions with Hex A, reducing fluctuations in the plasticity of the active site. DMDP amide (6) dose-dependently increased intracellular Hex A activity in the G269S mutant cells and restored Hex A activity up to approximately 43% of the wild type level; this effect clearly exceeded the border line treatment for Tay-Sachs disease, which is regarded as 10-15% of the wild type level. This is a significantly greater effect than that of pyrimethamine, which is currently in Phase 2 clinical trials. DMDP amide (6), therefore, represents a new promising pharmacological chaperone candidate for the treatment of Tay-Sachs disease.
Assuntos
Domínio Catalítico , Simulação por Computador , Hexosaminidase A/metabolismo , Açúcares/metabolismo , Açúcares/farmacologia , Doença de Tay-Sachs/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Hexosaminidase A/antagonistas & inibidores , Hexosaminidase A/química , Hexosaminidase A/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Açúcares/química , Açúcares/uso terapêuticoRESUMO
Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism due to mutations in phenylalanine hydroxylase (PAH). Recently, small compounds, known as pharmacological chaperones (PhCs), have been identified that restore the enzymatic activity of mutant PAHs. Understanding the mechanism of the reduction in enzymatic activity due to a point mutation in PAH and its restoration by PhC binding is important for the design of more effective PhC drugs. Thermal fluctuations of an enzyme can alter its activity. Here, molecular dynamics simulation show the thermal fluctuation of PAH is increased by introduction of the A313T mutation. Moreover, a simulation using the A313T-PhC complex model was also performed. Thermal fluctuation of the mutant was found to be reduced upon PhC binding, which contributes to restoring its enzymatic activity.
Assuntos
Fenilalanina Hidroxilase/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/genética , Mutação Puntual , Dobramento de Proteína , Pirimidinonas/química , Pirimidinonas/metabolismo , TemperaturaRESUMO
Developing selective inhibitors for a particular kinase remains a major challenge in kinase-targeted drug discovery. Here we performed a multi-step virtual screening for dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) inhibitors by focusing on the selectivity for DYRK1A over cyclin-dependent kinase 5 (CDK5). To examine the key factors contributing to the selectivity, we constructed logistic regression models to discriminate between actives and inactives for DYRK1A and CDK5, respectively, using residue-based binding free energies. The residue-based parameters were calculated by molecular mechanics-generalized Born surface area (MM-GBSA) decomposition methods for kinase-ligand complexes modeled by computer ligand docking. Based on the findings from the logistic regression models, we built a three-dimensional (3D) pharmacophore model and chose filter criteria for the multi-step virtual screening. The virtual hit compounds obtained from the screening were assessed for their inhibitory activities against DYRK1A and CDK5 by in vitro assay. Our screening identified two novel selective DYRK1A inhibitors with IC50 values of several µM for DYRK1A and >100µM for CDK5, which can be further optimized to develop more potent selective DYRK1A inhibitors.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Bioensaio , Quinase 5 Dependente de Ciclina/metabolismo , Humanos , Ligantes , Modelos Logísticos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Máquina de Vetores de Suporte , Quinases DyrkRESUMO
We report on the synthesis and biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives as pharmacological chaperones for Gaucher disease. The parent compound, DAB, did not show inhibition of human ß-glucocerebrosidase but showed moderate intestinal α-glucosidase inhibition; in contrast, extension of α-1-C-alkyl chain length gave a series of highly potent and selective inhibitors of the ß-glucocerebrosidase. Our design of α-1-C-tridecyl-DAB (5j) produced a potent inhibitor of the ß-glucocerebrosidase, with IC50 value of 0.77 µM. A molecular docking study revealed that the α-1-C-tridecyl group has a favorable interaction with the hydrophobic pocket and the sugar analogue part (DAB) interacted with essential hydrogen bonds formed to Asp127, Glu235 and Glu340. Furthermore, α-1-C-tridecyl-DAB (5j) displayed enhancement of activity at an effective concentration 10-times lower than isofagomine. α-1-C-Tridecyl-DAB therefore provides the first example of a pyrrolidine iminosugar as a new class of promising pharmacological chaperones with the potential for treatment of Gaucher disease.
Assuntos
Doença de Gaucher/tratamento farmacológico , Imino Açúcares/química , Imino Açúcares/farmacologia , Simulação de Acoplamento Molecular , Pirrolidinas/química , Pirrolidinas/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Doença de Gaucher/metabolismo , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/metabolismo , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Humanos , Imino Açúcares/síntese química , Relação Estrutura-AtividadeRESUMO
Cordycepin, which is an analogue of a nucleoside adenosine, exhibits a wide variety of pharmacological activities including anticancer effects. In this study, ADA1- and ADA2-expressing HEK293 cells were established to determine the major ADA isoform responsible for the deamination of cordycepin. While the metabolic rate of cordycepin deamination was similar between ADA2-expressing and Mock cells, extensive metabolism of cordycepin was observed in the ADA1-expressing cells with K m and V max values of 54.9 µmol/L and 45.8 nmole/min/mg protein. Among five natural substances tested in this study (kaempferol, quercetin, myricetin, naringenin, and naringin), naringin strongly inhibited the deamination of cordycepin with K i values of 58.8 µmol/L in mouse erythrocytes and 168.3 µmol/L in human erythrocytes. A treatment of Jurkat cells with a combination of cordycepin and naringin showed significant cytotoxicity. Our in silico study suggests that not only small molecules such as adenosine derivatives but also bulky molecules like naringin can be a potent ADA1 inhibitor for the clinical usage.
RESUMO
This paper identifies the required configuration and orientation of α-glucosidase inhibitors, miglitol, α-1-C-butyl-DNJ, and α-1-C-butyl-LAB for binding to ntSI (isomaltase). Molecular dynamics (MD) calculations suggested that the flexibility around the keyhole of ntSI is lower than that of ctSI (sucrase). Furthermore, a molecular-docking study revealed that a specific binding orientation with a CH-π interaction (Trp370 and Phe648) is a requirement for achieving a strong affinity with ntSI. On the basis of these results, a new class of nortropane-type iminosugars, labystegines, hybrid iminosugars of LAB and calystegine, have been designed and synthesized efficiently from sugar-derived cyclic nitrones with intramolecular 1,3-dipolar cycloaddition or samarium iodide catalyzed reductive coupling reaction as the key step. Biological evaluation showed that our newly designed 3(S)-hydroxy labystegine (6a) inherited the selectivity against intestinal α-glucosidases from LAB, and its inhibition potency was 10 times better than that of miglitol. Labystegine, therefore, represents a promising new class of nortropane-type iminosugar for improving postprandial hyperglycemia.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Imino Açúcares/farmacologia , Nortropanos/farmacologia , Sacarase/antagonistas & inibidores , alfa-Glucosidases/metabolismo , Arabinose/química , Sítios de Ligação/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Imino Furanoses/química , Imino Açúcares/síntese química , Imino Açúcares/química , Intestinos/enzimologia , Conformação Molecular , Simulação de Dinâmica Molecular , Nortropanos/síntese química , Nortropanos/química , Sacarase/metabolismo , Álcoois Açúcares/química , Tropanos/químicaRESUMO
The pregnane X receptor [PXR (NR1I2)] induces the expression of xenobiotic metabolic genes and transporter genes. In this study, we aimed to establish a computational method for quantifying the enzyme-inducing potencies of different compounds via their ability to activate PXR, for the application in drug discovery and development. To achieve this purpose, we developed a three-dimensional quantitative structure-activity relationship (3D-QSAR) model using comparative molecular field analysis (CoMFA) for predicting enzyme-inducing potencies, based on computer-ligand docking to multiple PXR protein structures sampled from the trajectory of a molecular dynamics simulation. Molecular mechanics-generalized born/surface area scores representing the ligand-protein-binding free energies were calculated for each ligand. As a result, the predicted enzyme-inducing potencies for compounds generated by the CoMFA model were in good agreement with the experimental values. Finally, we concluded that this 3D-QSAR model has the potential to predict the enzyme-inducing potencies of novel compounds with high precision and therefore has valuable applications in the early stages of the drug discovery process.
Assuntos
Indutores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Descoberta de Drogas/métodos , Hepatócitos/efeitos dos fármacos , Modelos Moleculares , Receptores de Esteroides/agonistas , Inteligência Artificial , Citocromo P-450 CYP3A/genética , Indutores do Citocromo P-450 CYP3A/química , Indutores do Citocromo P-450 CYP3A/metabolismo , Bases de Dados de Proteínas , Transferência de Energia , Indução Enzimática/efeitos dos fármacos , Sistemas Inteligentes , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Imageamento Tridimensional , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptor de Pregnano X , Conformação Proteica , Relação Quantitativa Estrutura-Atividade , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Reprodutibilidade dos TestesRESUMO
We report on the identification of the required configuration and binding orientation of nor-tropane alkaloid calystegines against ß-glucocerebrosidase. Calystegine B2 is a potent competitive inhibitor of human lysosomal ß-glucocerebrosidase with Ki value of 3.3 µM. A molecular docking study revealed that calystegine B2 had a favorable van der Waals interactions (Phe128, Trp179, and Phe246) and the hydrogen bonding (Glu235, Glu340, Asp127, Trp179, Asn234, Trp381 and Asn396) was similar to that of isofagomine. All calystegine isomers bound into the same active site as calystegine B2 and the essential hydrogen bonds formed to Asp127, Glu235 and Glu340 were maintained. However, their binding orientations were obviously different. Calystegine A3 bound to ß-glucocerebrosidase with the same orientations as calystegine B2 (Type 1), while calystegine B3 and B4 had different binding orientations (Type 2). It is noteworthy that Type 1 orientated calystegines B2 and A3 effectively stabilized ß-glucocerebrosidase, and consequently increased intracellular ß-glucocerebrosidase activities in N370S fibroblasts, while Type 2 orientated calystegines B3 and B4 could not keep the enzyme activity. These results clearly indicate that the binding orientations of calystegines are changed by the configuration of the hydroxyl groups on the nor-tropane ring and the suitable binding orientation is a requirement for achieving a strong affinity to ß-glucocerebrosidase.
Assuntos
Tropanos/metabolismo , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Doença de Gaucher/enzimologia , Doença de Gaucher/patologia , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/metabolismo , Humanos , Ligação de Hidrogênio , Imino Piranoses/química , Imino Piranoses/metabolismo , Isomerismo , Simulação de Acoplamento Molecular , Nortropanos/química , Nortropanos/metabolismo , Alcaloides de Solanáceas/química , Alcaloides de Solanáceas/metabolismo , Eletricidade Estática , Relação Estrutura-Atividade , Tropanos/químicaRESUMO
The natural variant of the cytochrome P450 enzyme CYP2D6.1, CYP2D6.17, is most common in African populations, has three amino acid substitutions (T107I, R296C, and S486T) compared to the wild-type, and is known to have a different ligand preference from CYP2D6.1. It is becoming increasingly important to understand differences in the metabolism of medicines in different ethnic groups in order to assess the relevance of clinical data from different countries. This study investigated differences in the inhibition profiles of drugs for CYP2D6 with respect to gene polymorphisms. Firstly, we used computer docking with six drugs to several CYP2D6.1 structures, sampled from the trajectory of MD simulations, and calculated MM-GB/SA scores representing binding free energies. We then used regression analysis to predict the potency with which drugs inhibited CYP2D6.1 based on MM-GB/SA scores. The pKi-values obtained were in good agreement with experimental values measured for the six drugs (r(2) = 0.81). We carried out the same analysis for CYP2D6.17 and the pKi-values calculated were also in good agreement with experimental values (r(2) = 0.92). Finally, we were able to successfully explain the different abilities of CYP2D6.1 and CYP2D6.17 to metabolize drugs in different ethnic groups with reference to their 3D-structures.
Assuntos
Simulação por Computador , Inibidores do Citocromo P-450 CYP2D6 , Inibidores Enzimáticos/química , Polimorfismo Genético , Cocaína/química , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/genética , Fluoxetina/química , Imipramina/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Orgânicos/química , Conformação Proteica , Quinidina/química , Análise de Regressão , Estereoisomerismo , Tioridazina/químicaRESUMO
d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.
Assuntos
Aciclovir/metabolismo , Aciclovir/farmacologia , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Aciclovir/química , Algoritmos , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Benzoatos/química , Benzoatos/metabolismo , Benzoatos/farmacologia , Domínio Catalítico/genética , D-Aminoácido Oxidase/química , Relação Dose-Resposta a Droga , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , TemperaturaRESUMO
Cytochrome P450 3A4 (CYP3A4) is a member of the CYP family and is an important enzyme in drug metabolism. A compound that inhibits CYP3A4 activity could also affect the pharmacokinetics of other substrates, resulting in drug-drug interactions (DDIs) that could cause side effects. Pharmacokinetic data from drug-development studies in rats often determine the dosage used in human clinical trials. It is therefore useful to understand differences in metabolism in different species at an early stage in drug development. Human and rat CYP3A enzymes show different inhibition profiles with different drugs, although the mechanisms involved are not yet clear. Here we built three-dimensional quantitative structure-activity relationship (3D-QSAR) models using structure-based comparative molecular field analysis (CoMFA), to predict the direct inhibitory activity of ligands for human CYP3A4 and rat CYP3A1, based on computer-ligand docking. The alignment of the ligand docking poses suggested that key amino acid-ligand interactions (e.g., Thr309 in CYP3A4 and Pro310 in CYP3A1) characterized the different potencies with which the ligands inhibited CYP3A4 and CYP3A1. The 3D-QSAR models for human and rat CYP3A family inhibitors predicted the potency of inhibitors and could be useful for assessing DDIs at an early stage in drug discovery.
Assuntos
Inibidores do Citocromo P-450 CYP3A , Animais , Citocromo P-450 CYP3A , Interações Medicamentosas , Fluconazol/farmacologia , Fluvoxamina/farmacologia , Humanos , Concentração Inibidora 50 , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Ratos , Ritonavir/farmacologia , Saquinavir/farmacologiaRESUMO
We report on the synthesis and the biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-l-arabinitol (LAB) derivatives. The asymmetric synthesis of the derivatives was achieved by asymmetric allylic alkylation, ring-closing metathesis, and Negishi cross-coupling as key reactions. α-1-C-Butyl-LAB is a potent inhibitor of intestinal maltase, isomaltase, and sucrase, with IC50 values of 0.13, 4.7, and 0.032 µM, respectively. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that this compound differs from miglitol in that it does not influence oligosaccharide processing and the maturation of glycoproteins. A molecular docking study of maltase-glucoamylase suggested that the interaction modes and the orientations of α-1-C-butyl-LAB and miglitol are clearly different. Furthermore, α-1-C-butyl-LAB strongly suppressed postprandial hyperglycemia at an early phase, similar to miglitol in vivo. It is noteworthy that the effective dose was about 10-fold lower than that for miglitol. α-1-C-Butyl-LAB therefore represents a new class of promising compounds that can improve postprandial hyperglycemia.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Inibidores de Glicosídeo Hidrolases , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Imino Açúcares/uso terapêutico , Administração Oral , Animais , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/química , Imino Açúcares/administração & dosagem , Imino Açúcares/química , Imino Açúcares/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
SCOPE: Previous reports suggest that soy isoflavones have multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about whether soy isoflavones can inhibit the downregulation of adiponectin and their molecular mechanisms. In the present study, we demonstrate that genistein (Gen) or daidzein (Dai) can significantly inhibit the downregulation of adiponectin expression via unique and different molecular mechanisms. METHODS AND RESULTS: Pretreatment with Gen or Dai significantly inhibited the tumor necrosis factor-alpha (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Gen inhibited the TNF-α-induced c-Jun-NH(2) -terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on JNK1 with Gen or Dai clearly supported our experimental results. However, Dai did not significantly inhibit JNK signaling. Dai did, however, inhibit the TNF-α-induced downregulation of forkhead box-containg protein O1, which is also involved in adiponectin expression. CONCLUSION: These data demonstrate that: (i) both Gen and Dai significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes; (ii) Gen is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin; and (iii) Dai can inhibit the downregulation of adiponectin by restoring the TNF-α-mediated reduction of forkhead box-containg protein O1 protein expression.