RESUMEN
The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid ß-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.
Asunto(s)
Ácido Fólico , Defectos del Tubo Neural , Animales , Humanos , Ratones , Carbono/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Ácido Fólico/metabolismo , Formiatos/metabolismo , Glicina/metabolismo , Mitocondrias/metabolismo , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismoRESUMEN
BACKGROUND: Glycogen storage disease type Ib (GSD Ib) is a rare disorder characterized by impaired glucose homeostasis caused by mutations in the SLC37A4 gene. It is a severe inherited metabolic disease associated with hypoglycemia, hyperlipidemia, lactic acidosis, hepatomegaly, and neutropenia. Traditional treatment consists of feeding raw cornstarch which can help to adjust energy metabolism but has no positive effect on neutropenia, which is fatal for these patients. Recently, the pathophysiologic mechanism of the neutrophil dysfunction and neutropenia in GSD Ib has been found, and the treatment with the SGLT2 inhibitor empaglifozin is now well established. In 2020, SGLT2 inhibitor empagliflozin started to be used as a promising efficient remover of 1,5AG6P in neutrophil of GSD Ib patients worldwide. However, it is necessary to consider long-term utility and safety of a novel treatment. RESULTS: In this study, we retrospectively examined the clinical manifestations, biochemical examination results, genotypes, long-term outcomes and follow-up of thirty-five GSD Ib children who visited our department since 2009. Fourteen patients among them underwent empagliflozin treatment since 2020. This study is the largest cohort of pediatric GSD Ib patients in China as well as the largest cohort of pediatric GSD Ib patients treated with empagliflozin in a single center to date. The study also discussed the experience of long-term management on pediatric GSD Ib patients. CONCLUSION: Empagliflozin treatment for pediatric GSD Ib patients is efficient and safe. Increase of urine glucose is a signal for pharmaceutical effect, however attention to urinary infection and hypoglycemia is suggested.
Asunto(s)
Compuestos de Bencidrilo , Enfermedad del Almacenamiento de Glucógeno Tipo I , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Niño , Humanos , Antiportadores , Estudios de Seguimiento , Glucosa , Glucósidos , Enfermedad del Almacenamiento de Glucógeno Tipo I/tratamiento farmacológico , Hipoglucemia , Proteínas de Transporte de Monosacáridos/genética , Neutropenia , Estudios Retrospectivos , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéuticoRESUMEN
Phosphodiesterase-4D (PDE4D) has been proved to be a potential therapeutic target against strokes. In the present study, a procedure of integrating pharmacophore, molecular docking, molecular dynamics (MD) simulations, binding free energy calculations, and finally validation with bioassay was developed and described to search for novel PDE4D inhibitors from the SPECS database. Among the 29 compounds selected by our MD-augmented strategy, 15 hits were found with IC50 between 1.9 and 50 µM (a hit rate of 52%) and 6 potent hits showed IC50 less than 10 µM, which suggested that MD simulations can explore the intermolecular interactions of PDE4D-inhibitor complexes more precisely and thus significantly enhanced the hit rate of this screening. The effective and efficient integrated procedures described in this study could be readily applied to screening studies toward other drug targets.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Inhibidores de Fosfodiesterasa/química , Relación Estructura-Actividad Cuantitativa , Dominio Catalítico , Bases de Datos de Compuestos Químicos , Pruebas de Enzimas , Escherichia coli/genética , Humanos , Cinética , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Proteínas Recombinantes/química , TermodinámicaRESUMEN
In our previous kinetics studies the natural products oroxylin and wogonin were shown to have strong biological affinity for, and inhibitory effects against, human cytochrome P450 1A2, with IC(50) values of 579 and 248 nM, respectively; this might lead to the occurrence of drug-drug interactions when co-administered clinically. However, their inhibitory mechanisms against 1A2 remain elusive. In this study, molecular docking and molecular dynamics simulations were performed to better understand the molecular basis of their inhibitory mechanisms towards 1A2. Structural analysis revealed that oroxylin has a different binding pattern from wogonin and another very strongly binding inhibitor α-naphthoflavone (ANF, IC(50) = 49 nM). The O(7) atom of oroxylin forms hydrogen bonds with the OD1/OD2 atoms of Asp313, which is not observed in the 1A2-wogonin complex. Because of energetically unfavorable repulsions with the methoxy group at the 6 position of the oroxylin ring, significant conformational changes were observed for the sidechain of Thr118 in the MD simulated model. As a result, the larger and much more open binding-site architecture of the 1A2-oroxylin complex may account for its weaker inhibitory effect relative to the 1A2-ANF complex. Energy analysis indicated that oroxylin has a less negative predicted binding free energy of -19.8 kcal/mol than wogonin (-21.1 kcal/mol), which is consistent with our experimental assays. Additionally, our energy results suggest that van der Waals/hydrophobic and hydrogen-bonding interactions are important in the inhibitory mechanisms of oroxylin whereas the former is the underlying force responsible for strong inhibition by ANF and wogonin.
Asunto(s)
Inhibidores del Citocromo P-450 CYP1A2 , Medicamentos Herbarios Chinos/farmacología , Inhibidores Enzimáticos/farmacología , Flavanonas/farmacología , Flavonoides/farmacología , Benzoflavonas/metabolismo , Sitios de Unión , Fenómenos Químicos , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/metabolismo , Diseño de Fármacos , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Flavanonas/química , Flavanonas/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Reproducibilidad de los Resultados , TermodinámicaRESUMEN
Phosphodiesterase-4 (PDE4) has been identified to be a promising target for treatment of asthma. Moracin M extracted from Chinese herbal drug 'Sang-Bai-Pi' (Morus alba L.) was studied for the inhibitory affinity towards PDE4. It inhibited PDE4D2, PDE4B2, PDE5A1, and PDE9A2 with the IC(50) values of 2.9, 4.5, >40, and >100 µM, respectively. Our molecular docking and 8ns molecular dynamics (MD) simulations demonstrated that moracin M forms three hydrogen bonds with Gln369, Asn321, and Asp318 in the active site and stacks against Phe372. In addition, comparative kinetics analysis of its analog moracin C was carried out to qualitatively validate their inhibitory potency as predicted by the binding free energy calculations after MD simulations.
Asunto(s)
Benzofuranos/farmacología , Morus/química , Inhibidores de Fosfodiesterasa 4/química , Resorcinoles/farmacología , Simulación por Computador , Medicamentos Herbarios Chinos , Concentración 50 Inhibidora , Cinética , Simulación de Dinámica Molecular , Inhibidores de Fosfodiesterasa 4/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/farmacología , Unión ProteicaRESUMEN
The natural product curcumin is widely used in Asian countries for the treatment of several diseases. However, the clinical potential of curcumins remains limited due to their relatively poor bioavailability and no experimental data about their lipophilicity for bioavailability prediction. To evaluate the retention and lipophilicity of curcumin and its 31 newly synthesized analogues, they were subjected to 3D quantitative structure-retention relationship studies by RP-HPLC. Superior than the classical four-variant quantitative structure-retention relationship model (conventional r(2) =0.734), the 3D comparative molecular similarity index analysis model with combined steric, electrostatic, and H-bond donor fields, resulted in a robust structure-retention correlation (cross-validated q(2) =0.613 and r(2) =0.979). The statistical analyses indicate that the electrostatic and H-bond donor fields have a primary influence on the chromatographic retention of analytes. The predictive power and robustness of the derived comparative molecular similarity index analysis model was further confirmed by the test-set validation (q(2) =0.702, r(2) =0.905, and the slope K=1.016) and Y-randomization examination. Statistically significant and physically meaningful 3D-quantitative structure-retention relationship provided better insight into understanding the retention behaviors of curcumin and its analogues, and their separation mechanism in a given RP-HPLC system.
Asunto(s)
Curcumina/análogos & derivados , Curcumina/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Curcumina/química , Estructura Molecular , Relación Estructura-Actividad CuantitativaRESUMEN
BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is pleomorphic congenital hyperammonemia, in which the prognosis of the patient is determined both by genotype and environmental factors. This study investigated the clinical and biochemical characteristics of OTCD patients with different prognosis. METHOD: Of 35 OTCD patients, six males deceased at the first disease-onset, 17 males survived and had controllable ammonia levels after treatment, and 12 females survived through the first disease-onset but had intractable hyperammonemia and high mortality. Fasting blood samples from patients collected at three disease stages were used for the analysis of amino acid (AA) profile, acylcarnitine profile, and micronutrients. Differences in profiles between patients and healthy controls and within patient groups were studied. RESULTS: All OTCD patients had accumulation of glutamine, homocitrulline, lysine, glutamate, cystathionine, and pipecolic acid, as well as deficiency of citrulline, tryptophan, threonine, and carnitine. For male non-survivors, most other AAs and long-chain acylcarnitines were elevated at disease onset, of which the levels of creatine, N-acetylaspartic acid, and homoarginine were remarkably high. Male survivors and female patients had most other AAs at low to normal levels. Compared with male survivors, female patients had much lower protein-intolerance, as indicated by significantly lower levels of protein consumption indicators, including essential AAs, 1-methylhistidine, acylcarnitines et al., but high levels of ammonia. Female patients still had significantly higher levels of citrulline, homocitrulline, and citrulline/arginine compared to male survivors. CONCLUSION: Unique profiles were observed in each group of OTCD patients, indicating specific physiological changes that happened to them.
Asunto(s)
Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/metabolismo , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/fisiopatología , Adolescente , Adulto , Amoníaco/sangre , Arginina/sangre , Niño , Preescolar , China , Creatina/metabolismo , Femenino , Humanos , Hiperamonemia/fisiopatología , Lisina/sangre , Masculino , Ornitina/uso terapéutico , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/sangre , Urea/sangre , Adulto JovenRESUMEN
Urea cycle disorders (UCD) are inborn errors of ammonia detoxification in which early diagnosis and treatment are critical to prevent metabolic emergencies. Unfortunately, the diagnosis was often and pronounced delayed. To improve diagnosis, we developed herein a liquid chromatography-tandem mass spectrometry method to investigate the disturbance of amino acid profile caused by UCD. The method enabled absolute quantification of 48 amino acids (AAs) within 20â¯min. Only 2.5⯵L plasma was required for the analysis. The lower limits of quantification for most AAs were 0.01⯵mol/L. Method accuracies ranged from 89.9% to 113.4%. The within- and between-run coefficients of variation were 0.8-7.7% and 2.6-14.5%, respectively. With this method, age-specific reference values were established for 42 AAs by analyzing 150 samples from normal controls, and patients with different subtypes of UCD were successfully distinguished. The data of patients revealed that UCD not only disturbed the metabolism of urea cycle AAs and induced accumulation of ammonia detoxification AAs, but also interfered the metabolism of some nervous system related AAs, such as pipecolic acid and N-acetylaspartic acid. This data may provide new insight into pathogenesis for UCD.
Asunto(s)
Aminoácidos/metabolismo , Trastornos Innatos del Ciclo de la Urea/metabolismo , Aminoácidos/sangre , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Cromatografía Liquida , Femenino , Humanos , Masculino , Ácidos Pipecólicos/metabolismo , Espectrometría de Masas en Tándem , Trastornos Innatos del Ciclo de la Urea/sangreRESUMEN
Phosphodiesterase 9 (PDE9) inhibitors have been studied as potential therapeutics for treatment of diabetes and Alzheimer's disease. Here we report a potent PDE9 inhibitor 3r that has an IC50 of 0.6 nM and >150-fold selectivity over other PDEs. The HepG2 cell-based assay shows that 3r inhibits the mRNA expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase. These activities of 3r, together with the reasonable pharmacokinetic properties and no acute toxicity at 1200 mg/kg dosage, suggest its potential as a hypoglycemic agent. The crystal structure of PDE9-3r reveals significantly different conformation and hydrogen bonding pattern of 3r from those of previously published 28s. Both 3r and 28s form a hydrogen bond with Tyr424, a unique PDE9 residue (except for PDE8), but 3r shows an additional hydrogen bond with Ala452. This structure information might be useful for design of PDE9 inhibitors.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Hipoglucemiantes/farmacología , Microsomas Hepáticos/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinonas/farmacología , Animales , Western Blotting , Líquidos Corporales/química , Dominio Catalítico , Cristalografía por Rayos X , Descubrimiento de Drogas , Células Hep G2 , Humanos , Enlace de Hidrógeno , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacocinética , Pirazoles/química , Pirazoles/farmacocinética , Pirimidinonas/química , Pirimidinonas/farmacocinética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Distribución TisularRESUMEN
Phosphodiesterase-5 (PDE5) inhibitors have been approved for the treatment of erectile dysfunction and pulmonary hypertension, but enthusiasm on discovery of PDE5 inhibitors continues for their potential new applications. Reported here is discovery of a series of new PDE5 inhibitors by structure-based design, molecular docking, chemical synthesis, and enzymatic characterization. The best compound, 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one (57), has an IC50 of 17 nM against the PDE5 catalytic domain and good selectivity over other PDE families. The crystal structure of the PDE5 catalytic domain in complex with 57 was determined at 2Å resolution and showed that 57 occupies the same pocket as other PDE5 inhibitors, but has a different binding pattern in detail. On the basis of the binding pattern of 57, a novel scaffold can be proposed as a candidate of PDE inhibitors.
Asunto(s)
Inhibidores de Fosfodiesterasa 5/farmacología , Pirroles/farmacología , Cristalografía por Rayos X , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Inhibidores de Fosfodiesterasa 5/química , Pirroles/químicaRESUMEN
A new series of phosphodiesterase-9 (PDE9) inhibitors that contain a scaffold of 6-amino-pyrazolopyrimidinone have been discovered by a combination of structure-based design and computational docking. This procedure significantly saved the load of chemical synthesis and is an effective method for the discovery of inhibitors. The best compound 28 has an IC(50) of 21 nM and 3.3 µM, respectively, for PDE9 and PDE5 and about 3 orders of magnitude of selectivity against other PDE families. The crystal structure of the PDE9 catalytic domain in complex with 28 has been determined and shows a hydrogen bond between 28 and Tyr424. This hydrogen bond may account for the 860-fold selectivity of 28 against PDE1B, in comparison with about 30-fold selectivity of BAY73-6691. Thus, our studies suggest that Tyr424, a unique residue of PDE8 and PDE9, is a potential target for improvement of selectivity of PDE9 inhibitors.