RESUMO
RATIONALE: Although diabetic peripheral neuropathic pain (DPNP) and depression have been recognized for many years, their co-morbidity relationship and effective treatment choices remain uncertain. OBJECTIVES: To evaluate the antidepressant effect of carvedilol on streptozotocin-induced DPNP mice, and the relationship with gut microbiota. METHODS: The hyperalgesia and depressive behaviors of mice with comorbidity of DPNP and depression were confirmed by pain threshold of the mechanical sensitivity test (MST), immobility time of the tail suspension test (TST) and the forced swimming test (FST). The anti-depressive effect and fecal gut microbiota composition were studied in DPNP mice treated with carvedilol (10 mg/kg/day), and the relationships between them were analyzed by Spearman's correlation. RESULTS: Depression was successfully induced in DPNP mice. Carvedilol can reverse the decreased mechanical pain threshold and relieve the depressive behaviors of DPNP mice, while increasing the abundance of Prevotella, Ruminococcus, Helicobacter and Desulfovibrio, and decreasing the abundance of Akkermansia and Allobaculum. CONCLUSIONS: Carvedilol can alleviate the mechanical hyperalgesia and alter gut microbiota to ameliorate the depression-like behaviors which induced by DPNP.
Assuntos
Antidepressivos , Carvedilol , Depressão , Neuropatias Diabéticas , Microbioma Gastrointestinal , Estreptozocina , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Carvedilol/farmacologia , Carvedilol/uso terapêutico , Masculino , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Camundongos , Depressão/tratamento farmacológico , Depressão/microbiologia , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/microbiologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/psicologia , Diabetes Mellitus Experimental/microbiologia , Hiperalgesia/tratamento farmacológico , Camundongos Endogâmicos C57BLRESUMO
Pregnane X receptor (PXR) is essential in the regulation of liver homeostasis, and the gut microbiota is closely linked to liver physiologic and pathologic status. We previously found that activation of PXR significantly promotes liver enlargement through interaction with yes-associated protein (YAP). However, whether gut microbiota contributes to PXR-induced hepatomegaly and the involved mechanisms remain unclear. In this study, C57BL/6 mice were administered the mouse-specific agonist pregnenolone 16α-carbonitrile (PCN) for 5 days. Depletion of gut microbiota was achieved using broad-spectrum antibiotics (ABX) and fecal microbiota transplantation (FMT) was performed to restore the gut microbia. The composition of gut microbiota was analyzed by 16S rRNA sequencing, while the expression of PXR, YAP, and their downstream target genes and proteins were assessed. The results indicated that PCN treatment altered the composition and abundance of specific bacterial taxa. Furthermore, depletion of gut microbiota using ABX significantly attenuated PCN-induced hepatomegaly. FMT experiments further demonstrated that the fecal microbiota from PCN-treated mice could induce liver enlargement. Mechanistic studies revealed that ABX treatment impeded the PXR and YAP activation induced by PCN, as evidenced by decreased expression of PXR, YAP, and their downstream targets. Moreover, alterations in PXR and YAP activation were likely contributing to hepatomegaly in recipient mice following FMT from PCN-treated mice. Collectively, the current study demonstrated that gut microbiota is involved in PCN-induced hepatomegaly via regulating PXR and YAP activation, providing potential novel insights into the involvement of gut microbiota in PXR-mediated hepatomegaly. SIGNIFICANCE STATEMENT: This work describes that the composition of gut microbiota is altered in mouse pregnane X receptor (PXR) agonist pregnenolone 16α-carbonitrile (PCN)-induced hepatomegaly. Treatment with an antibiotic cocktail depletes the intestinal microbiota, leading to the impairment of liver enlargement caused by PCN. Additionally, fecal microbiota transplantation from PCN-treated mice induces liver enlargement. Further study revealed that gut microbiota is involved in hepatomegaly via regulating PXR and yes-associated protein activation.
Assuntos
Transplante de Microbiota Fecal , Microbioma Gastrointestinal , Hepatomegalia , Camundongos Endogâmicos C57BL , Receptor de Pregnano X , Carbonitrila de Pregnenolona , Proteínas de Sinalização YAP , Animais , Hepatomegalia/induzido quimicamente , Hepatomegalia/metabolismo , Receptor de Pregnano X/agonistas , Receptor de Pregnano X/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Camundongos , Carbonitrila de Pregnenolona/farmacologia , Proteínas de Sinalização YAP/metabolismo , Masculino , Transplante de Microbiota Fecal/métodos , Fígado/efeitos dos fármacos , Fígado/metabolismoRESUMO
The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC50 of 5.34 ± 0.65 µM, which is less potent than benzbromarone (2.01 ± 0.36 µM) but more potent than lesinurad (10.36 ± 1.23 µM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC50 of 1.04 ± 0.23 µM. Its metabolic product, gallic acid, inhibited XOD with an IC50 of 0.91 ± 0.14 µM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug.
Assuntos
Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Hiperuricemia , Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos , Hiperuricemia/tratamento farmacológico , Humanos , Animais , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Urato Oxidase/química , Descoberta de Drogas , Simulação de Acoplamento Molecular , Camundongos , Masculino , Ácido Gálico/química , Ácido Gálico/farmacologia , Ácido Gálico/análogos & derivados , Ratos Sprague-DawleyRESUMO
The NLRP3 inflammasome is a supramolecular complex that is linked to sterile and pathogen-dependent inflammation, and its excessive activation underlies many diseases. Ion flux disturbance and cell volume regulation are both reported to mediate NLRP3 inflammasome activation, but the underlying orchestrating signaling remains not fully elucidated. The volume-regulated anion channel (VRAC), formed by LRRC8 proteins, is an important constituent that controls cell volume by permeating chloride and organic osmolytes in response to cell swelling. We now demonstrate that Lrrc8a, the essential component of VRAC, plays a central and specific role in canonical NLRP3 inflammasome activation. Moreover, VRAC acts downstream of K+ efflux for NLRP3 stimuli that require K+ efflux. Mechanically, our data demonstrate that VRAC modulates itaconate efflux and damaged mitochondria production for NLRP3 inflammasome activation. Further in vivo experiments show mice with Lrrc8a deficiency in myeloid cells were protected from lipopolysaccharides (LPS)-induced endotoxic shock. Taken together, this work identifies VRAC as a key regulator of NLRP3 inflammasome and innate immunity by regulating mitochondrial adaption for macrophage activation and highlights VRAC as a prospective drug target for the treatment of NLRP3 inflammasome and itaconate related diseases.
Assuntos
Inflamassomos , Proteínas de Membrana , Camundongos , Animais , Proteínas de Membrana/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ânions/metabolismo , Mitocôndrias/metabolismoRESUMO
Herein, we present a copper-mediated C4-benzylation of 5-aminopyrazoles with 3-indoleacetic acids. Various benzylated 5-aminopyrazoles are prepared in good-to-excellent yields under basic and ligand-free conditions in the presence of copper acetate. Moreover, this benzylation method is applicable to other substrates, including naphthylamine, 2-aminochromen-4-one, and enamines. Some products exhibit antiproliferative activities against cancer cell lines. In addition, the C4-benzylated products are cyclized into 1H-pyrazolo[4',3':6,7]azepino[3,4-b]indoles with aldehydes via one-pot two-step processes; notably, the cyclized products exhibit fluorescence emissions with large Stokes shifts.
RESUMO
Previously we discovered a novel natural scaffold compound, isobavachin (4', 7-dihydroxy-8-prenylflavanone), as a potent URAT1 inhibitor by shape and structure based on a virtue screening approach. In this study, further urate-lowering mechanism, pharmacokinetics and toxicities of isobavachin were conducted. Isobavachin inhibited URAT1 with an IC50 value of 0.24 ± 0.06 µM, and residues S35, F365, I481 and R477 of URAT1 contributed to high affinity for isobavachin. Isobavachin also inhibited glucose transporter 9 (GLUT9), another pivotal urate reabsorption transporter, with an IC50 value of 1.12 ± 0.26 µM. Molecular docking and MMGBSA results indicated that isobavachin might compete residues R171, L75 and N333 with uric acid, which leads to inhibition of uric acid transport of GLUT9. Isobavachin weakly inhibited urate secretion transporters OAT1 with an IC50 value of 4.38 ± 1.27 µM, OAT3 with an IC50 of 3.64 ± 0.62 µM, and ABCG2 with an IC50 of 10.45 ± 2.17 µM. Isobavachin also inhibited xanthine oxidase (XOD) activity in vitro with an IC50 value of 14.43 ± 3.56 µM, and inhibited the hepatic XOD activities at 5-20 mg/kg in vivo. Docking and MMGBSA analysis indicated that isobavachin might bind to the Mo-Pt catalyze center of XOD, which leads to inhibition of uric acid production. In vivo, isobavachin exhibited powerful urate-lowering and uricosuric effects at 5-20 mg/kg compared with the positive drugs morin (20 mg/kg) and RDEA3170 (10 mg/kg). Safety assessments revealed that isobavachin was safe and had no obvious toxicities. Isobavachin has little cell toxicity in HK2 cells as indicated by the MTT assay. In vivo, after treatment with 50 mg/kg isobavachin for 14 days, isobavachin had little renal toxicity, as revealed by serum CR/BUN levels, and no hepatotoxicity as revealed by ALT/AST levels. Further HE examination also suggests that isobavachin has no obvious kidney/liver damage. A pharmacokinetic study in SD rats indicated isobavachin had lower bioavailability (12.84 ± 5.13 %) but long half-time (7.04 ± 2.68 h) to maintain a continuous plasma concentration. Collectively, these results indicate that isobavachin deserves further investigation as a candidate anti-hyperuricemic drug with a novel mechanism of action: selective urate reabsorption inhibitor (URAT1/GLUT9) with a moderate inhibitory effect on XOD.
Assuntos
Flavonas , Ácido Úrico , Xantina Oxidase , Animais , Ratos , Rim/efeitos dos fármacos , Rim/metabolismo , Simulação de Acoplamento Molecular , Ratos Sprague-Dawley , Ácido Úrico/metabolismo , Xantina Oxidase/antagonistas & inibidores , Flavonas/química , Flavonas/farmacologiaRESUMO
Emerging data have demonstrated the critical roles of potassium efflux in the innate immune system. However, the role of potassium efflux in TLR3/4 activation and type I interferon (IFN) responses are not well elucidated. In the present study, we found potassium efflux is essential for TLR3/4 signaling, which mediates the expression of IFN and its inducible gene Cxcl10 and proinflammatory cytokine gene TNF-α. Furthermore, pharmacological inhibition of Kv1.3 channel (PAP-1), but not Kir2.1, KCa3.1 or TWIK2, attenuated TLR3/4 receptor activation in macrophages. Mechanistically, PAP-1 suppressed LPS-induced inflammatory function through marked suppressing the activation of JNK mitogen-activated protein kinase (MAPK) and p65 subunit of nuclear factor-kB (NF-kB). Notably, PAP-1 effectively protected mice against Listeria monocytogenes induced infection. Our findings reveal that potassium efflux mediated by the Kv1.3 channel is essential for TLR3/4 activation and suggest that pharmacological inhibition of Kv1.3 may help to treat type I IFN related autoimmune diseases and bacterial infections.
Assuntos
Listeria monocytogenes , Receptor 3 Toll-Like , Animais , Listeria monocytogenes/metabolismo , Macrófagos/metabolismo , Camundongos , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dysregulation of NLRP3 inflammasome results in uncontrolled inflammation, which participates in various chronic diseases. TWIK2 potassium channel mediates potassium efflux that has been reported to be an essential upstream mechanism for ATP-induced NLRP3 inflammasome activation. Thus, TWIK2 potassium channel could be a potential drug target for NLRP3-related inflammatory diseases. In the present study we investigated the effects of known K2P channel modulators on TWIK2 channel expressed in a heterologous system. In order to increase plasma membrane expression and thus TWIK2 currents, a mutant channel with three mutations (TWIK2I289A/L290A/Y308A) in the C-terminus was expressed in COS-7 cells. TWIK2 currents were assessed using whole-cell voltage-clamp recording. Among 6 known K2P channel modulators tested (DCPIB, quinine, fluoxetine, ML365, ML335, and TKDC), ML365 was the most potent TWIK2 channel blocker with an IC50 value of 4.07 ± 1.5 µM. Furthermore, ML365 selectively inhibited TWIK2 without affecting TWIK1 or THIK1 channels. We showed that ML365 (1, 5 µM) concentration-dependently inhibited ATP-induced NLRP3 inflammasome activation in LPS-primed murine BMDMs, whereas it did not affect nigericin-induced NLRP3, or non-canonical, AIM2 and NLRC4 inflammasomes activation. Knockdown of TWIK2 significantly impaired the inhibitory effect of ML365 on ATP-induced NLRP3 inflammasome activation. Moreover, we demonstrated that pre-administration of ML365 (1, 10, 25 mg/kg, ip) dose-dependently ameliorated LPS-induced endotoxic shock in mice. In a preliminary pharmacokinetic study conducted in rats, ML365 showed good absolute oral bioavailability with F value of 22.49%. In conclusion, ML365 provides a structural reference for future design of selective TWIK2 channel inhibitors in treating related inflammatory diseases.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ligação a DNA , Inflamassomos/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RatosRESUMO
Urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) are important targets for the development of uric acid-lowering drugs. We previously showed that the flexible linkers of URAT1 inhibitors could enhance their potency. In this study we designed and synthesized CDER167, a novel RDEA3710 analogue, by introducing a linker (methylene) between the naphthalene and pyridine rings to increase flexibility, and characterized its pharmacological and pharmacokinetics properties in vitro and in vivo. We showed that CDER167 exerted dual-target inhibitory effects on both URAT1 and GLUT9: CDER167 concentration-dependently inhibited the uptake of [14C]-uric acid in URAT1-expressing HEK293 cells with an IC50 value of 2.08 ± 0.31 µM, which was similar to that of RDEA3170 (its IC50 value was 1.47 ± 0.23 µM). Using site-directed mutagenesis, we demonstrated that CDER167 might interact with URAT1 at S35 and F365. In GLUT9-expressing HEK293T cells, CDER167 concentration-dependently inhibited GLUT9 with an IC50 value of 91.55 ± 15.28 µM, whereas RDEA3170 at 100 µM had no effect on GLUT9. In potassium oxonate-induced hyperuricemic mice, oral administration of CDER167 (10 mg·kg-1 · d-1) for 7 days was more effective in lowering uric acid in blood and significantly promoted uric acid excretion in urine as compared with RDEA3170 (20 mg·kg-1 · d-1) administered. The animal experiment proved the safety of CDER167. In addition, CDER167 displayed better bioavailability than RDEA3170, better metabolic stability and no hERG toxicity at 100 µM. These results suggest that CDER167 deserves further investigation as a candidate antihyperuricemic drug targeting URAT1 and GLUT9.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose , Hiperuricemia , Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos , Humanos , Células Cultivadas , Relação Dose-Resposta a Droga , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Células HEK293 , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Estrutura Molecular , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Emphysematous pyelonephritis (EPN) is a potentially life-threatening disease caused by a gas-producing necrotizing bacterial infection that involves the renal parenchyma, collecting system, and/or perinephric tissue. EPN is often complicated by a previous diagnosis of diabetes mellitus, and venous air bubbles are an uncommon complication of it. We describe a 52-year-old woman who was admitted in coma, with a history of vomiting, and was found to have EPN with air bubbles in the uterine veins. We discuss the presentation, diagnosis, and pathogenesis of this uncommon but clinically significant event, and briefly review other case reports of venous gas or thrombosis caused by EPN. CASE PRESENTATION: We report the case of a 52-year-old woman with past history of type 2 diabetes mellitus, presenting with loss of consciousness after vomiting for half a day. Abdominal computed tomography scan revealed unilateral EPN with air bubbles in the uterine veins. The blood, pus, and urine cultures were positive for extended-spectrum beta-lactamase-producing Escherichia coli. The patient's condition improved well after conservative management comprising supportive measures, broad-spectrum antibiotics, percutaneous drainage therapy, and an open operation. CONCLUSIONS: Venous air bubbles are rare but fatal complication of EPN. Early diagnosis and treatment are critical to ensure good results.
Assuntos
Complicações do Diabetes , Diabetes Mellitus Tipo 2 , Enfisema , Pielonefrite , Antibacterianos/uso terapêutico , Complicações do Diabetes/complicações , Diabetes Mellitus Tipo 2/complicações , Enfisema/diagnóstico por imagem , Enfisema/etiologia , Enfisema/terapia , Escherichia coli , Feminino , Humanos , Pessoa de Meia-Idade , Pielonefrite/complicações , Pielonefrite/diagnóstico por imagem , Vômito/complicações , Vômito/tratamento farmacológico , beta-LactamasesRESUMO
A copper-catalyzed C-3 functionalization of imidazo[1,2-a]pyridines with 3-indoleacetic acids through an aerobic oxidative decarboxylative process has been developed. The protocol provided a series of 3-(1H-indol-3-ylmethyl)-imidazo[1,2-a]pyridines in moderate to good yields under simple reaction conditions. Importantly, some products exhibited potent antiproliferative activity in cancer cell lines.
Assuntos
Cobre , Piridinas , Catálise , Ácidos Indolacéticos , OxirreduçãoRESUMO
As a promising therapeutic target for gout, hURAT1 has attracted increasing attention. In this work, we identified a novel scaffold of hURAT1 inhibitors from a personal natural product database of verified herb-treated gout. First, we constructed more than 800 natural compounds from Chinese medicine that were verified to treat gout. Following the application of both shape-based and docking-based virtual screening (VS) methods, taking into account the shape similarity and flexibility of the target, we identified isopentenyl dihydroflavones that might inhibit hURAT1. Specifically, 9 compounds with commercial availability were tested with biochemical assays for the inhibition of 14C-uric acid uptake in high-expression hURAT1 cells (HEK293-hURAT1), and their structure-activity relationship was evaluated. As a result, 8-isopentenyl dihydroflavone was identified as a novel scaffold of hURAT1 inhibitors since isobavachin (DHF3) inhibited hURAT1 with an IC50 value of 0.39 ± 0.17 µM, which was comparable to verinurad with an IC50 value of 0.32 ± 0.23 µM. Remarkably, isobavachin also displayed an eminent effect in the decline of serum uric acid in vivo experiments. Taken together, isobavachin is a promising candidate for the treatment of hyperuricemia and gout.
Assuntos
Produtos Biológicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Flavonas/farmacologia , Hiperuricemia/tratamento farmacológico , Simulação de Acoplamento Molecular , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Animais , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Flavonas/química , Hiperuricemia/metabolismo , Masculino , Medicina Tradicional Chinesa , Camundongos , Camundongos Endogâmicos , Estrutura Molecular , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Relação Estrutura-AtividadeRESUMO
Quercetin is a natural flavonoid which has been reported to be analgesic in different animal models of pain. However, the mechanism underlying the pain-relieving effects is still unclear. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in controlling pacemaker activity in cardiac and nervous systems, making the channel a new target for therapeutic exploration. In this study, we explored a series of flavonoids for their modulation on HCN channels. Among all tested flavonoids, quercetin was the most potent inhibitor for HCN channels with an IC50 value of 27.32 ± 1.19 µM for HCN2. Furthermore, quercetin prominently left shifted the voltage-dependent activation curves of HCN channels and decelerated deactivation process. The results presented herein firstly characterize quercetin as a novel and potent inhibitor for HCN channels, which represents a novel structure for future drug design of HCN channel inhibitors.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Quercetina/farmacologia , Animais , Células COS , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Fenômenos Eletrofisiológicos , Flavonoides/química , Flavonoides/farmacologia , Flavonóis/química , Flavonóis/farmacologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio/metabolismo , Quercetina/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
hURAT1 (human urate transporter 1) is a successful target for hyperuricemia. Recently, the modification work on hURAT1 inhibitors showed that the flexible linkers would benefit biological activity. The study aimed to investigate the contribution of the linkers and give modification strategies on this kind of structures based on QSAR models (HQSAR and topomer CoMFA). The most effective HQSAR and topomer CoMFA models were generated by applying the training set containing 63 compounds, with the cross-validated q2 values of 0.869/0.818 and the non-cross-validated correlation coefficients r2 of 0.951/0.978, respectively. The Y-randomization test was applied to ensure the robustness of the models. The external predictive correlation coefficient (rpred2) grounded on the external test set (21 compounds) of two models was 0.910 and 0.907, respectively. In addition, the models were validated by Golbraikh-Tropsha and Roy methods, as well as other statistical metrics. The results showed that both models were reliable. Topomer CoMFA steric/electrostatic contours and HQSAR atomic contribution maps illustrated the structural features which governed their inhibitory potency. The dependable results could provide important insights to guide the designing of more potential hURAT1 inhibitors.
Assuntos
Descoberta de Drogas , Transportadores de Ânions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/química , Relação Quantitativa Estrutura-Atividade , Algoritmos , Descoberta de Drogas/métodos , Humanos , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidoresRESUMO
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in controlling pacemaker activity in both heart and nervous system. Developing HCN channel inhibitors has been proposed to be an important strategy for the treatment of pain, heart failure, arrhythmias, and epilepsy. One HCN channel inhibitor, ivabradine, has been clinically approved for the treatment of angina pectoris and heart failure. In this study, we designed and synthesized eight alkanol amine derivatives, and assessed their effects on HCN channels expressed in COS7 cells using a whole-cell patch clamp method. Among them, compound 4e displayed the most potent inhibitory activity with an IC50 of 2.9 ± 1.2 µM at - 120 mV on HCN2 channel expressed in COS7 cells. Further analysis revealed that application of compound 4e (10 µM) caused a slowing of activation and a hyperpolarizing shift (ΔV1/2 = - 30.2 ± 2.9 mV, n = 5) in the voltage dependence of HCN2 channel activation. The inhibitory effect of compound 4e on HCN1 and HCN4 channel expressed in COS7 cells was less potent with IC50 of 17.2 ± 1.3 and 7.3 ± 1.2 µM, respectively. Besides, we showed that application of compound 4e (10 µM) inhibited Ih and action potential firing in acutely dissociated mouse small dorsal root ganglion neurons. Our study provides a new strategy for the design and development of potent HCN channel inhibitors.
Assuntos
Amino Álcoois/farmacologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Moduladores de Transporte de Membrana/farmacologia , Potenciais de Ação/efeitos dos fármacos , Amino Álcoois/síntese química , Amino Álcoois/química , Animais , Células COS , Chlorocebus aethiops , Humanos , Masculino , Moduladores de Transporte de Membrana/síntese química , Moduladores de Transporte de Membrana/química , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Canais de PotássioRESUMO
Doxorubicin (DOX) is a highly active anticancer drug with severe cytotoxicity, which is strongly associated with oxidative stress. Carvedilol (CAR), used as its racemate with S-CAR and R-CAR (1:1), has been previously reported to ameliorate the DOX-induced cytotoxicity. However, the main contributor from CAR of its protective effects has not been clear. Therefore, in this study, we aimed to investigate further the different effects of CAR enantiomers on DOX-induced cytotoxicity in human umbilical vein endothelial cells and rats, respectively. Results indicated that S-CAR could significantly attenuate DOX-induced cell death, apoptotic morphological changes, decrease the mitochondrial membrane potential and oxidative stress responses by increasing the superoxide dismutase and catalase activities, and decreasing malondialdehyde contents and reactive oxygen species levels via the phosphoinositide 3-kinase/AKT/endothelial nitric oxide synthase pathway in vitro. Consistent with the in vitro study, the protective effects of S-CAR on the myocardial tissues and hemodynamics were also detected in rats suffering because of DOX treatment. With the obtained results, we can first conclude that S-CAR provides superior protection to injury induced by DOX relative to that of racemic CAR and R-CAR.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Carvedilol/farmacologia , Doxorrubicina/toxicidade , Animais , Antioxidantes/metabolismo , Peso Corporal/efeitos dos fármacos , Catalase/sangue , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hemodinâmica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/sangue , Superóxido Dismutase/sangue , Análise de SobrevidaRESUMO
Colorectal cancer (CRC) is a common malignancy, most of which remain unresponsive to chemotherapy. As one of the earliest cytotoxic drugs, methotrexate (MTX) serves as an anti-metabolite and anti-folate chemotherapy for various cancers. Unfortunately, MTX resistance prevents its clinical application in cancer therapy. Thereby, overcoming the drug resistance is an alternative strategy to maximize the therapeutic efficacy of MTX in clinics. Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years. More and more emerging evidences have demonstrated that they play important regulatory roles in various biological activities and disease progression including drug resistance. In the present study, a MTX-resistant colorectal cell line HT-29 (HT-29-R) was developed, which displayed the active proliferation and shortened cell cycle. LncRNA H19 was found to be significantly upregulated in this resistant cell line. Further investigation showed that H19 knockdown sensitized the MTX resistance in HT-29-R cells while its overexpression improved the MTX resistance in the parental cells, suggesting that H19 mediate MTX resistance. The Wnt/ß-catenin signaling was activated in HT-29-R cells, and H19 knockdown suppressed this signaling in the parental cells. In conclusion, H19 mediated MTX resistance via activating Wnt/ß-catenin signaling, which help to develop H19 as a promising therapeutic target for MTX resistant CRC.
Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , RNA Longo não Codificante/genética , Via de Sinalização Wnt , Neoplasias Colorretais/genética , Células HT29 , Humanos , Metotrexato/farmacologiaRESUMO
Hpn is a small histidine-rich cytoplasmic protein from Helicobacter pylori and has been recognized as a high-risk factor for several cancers including gastric cancer, colorectal cancer, and MALT lymphoma. However, the relationship between Hpn and cancers remains elusive. In this study, we discovered that Hpn protein effectively suppressed cell growth and induced apoptosis in hepatocellular carcinoma (HCC). A two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics was performed to find the molecular targets of Hpn in HCC cells. It was identified that twelve proteins were differentially expressed, with USP5 being one of the most significantly downregulated protein. The P14ARF -P53 signaling was activated by USP5 knockdown in HCC cells. Furthermore, USP5 overexpression significantly rescued the suppressive effect of Hpn on the viability of HCC cells. In conclusion, our study suggests that Hpn plays apoptosis-inducing roles through suppressing USP5 expression and activating the P14ARF -P53 signaling. Therefore, Hpn may be a potential candidate for developing novel anti-HCC drugs.
Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas/metabolismo , Transdução de Sinais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Endopeptidases/metabolismo , Genes Supressores de Tumor , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas Oncogênicas/metabolismo , Proteômica/métodos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Benzbromarone (BBR) is effective in the treatment of gout; however, clinical findings have shown it can also cause fatal hepatic failure. Our early studies demonstrated that CYP3A catalyzed the biotransformation of BBR to epoxide intermediate(s) that reacted with sulfur nucleophiles of protein to form protein covalent binding both in vitro and in vivo. The present study attempted to define the correlation between metabolic epoxidation and hepatotoxicity of BBR by manipulating the structure of BBR. We rationally designed and synthesized three halogenated BBR derivatives, fluorinated BBR (6-F-BBR), chlorinated BBR (6-Cl-BBR), and brominated BBR (6-Br-BBR), to decrease the potential for cytochrome P450-mediated metabolic activation. Both in vitro and in vivo uricosuric activity assays showed that 6-F-BBR achieved favorable uricosuric effect, while 6-Cl-BBR and 6-Br-BBR showed weak uricosuric efficacy. Additionally, 6-F-BBR elicited much lower hepatotoxicity in mice. Fluorination of BBR offered advantage to metabolic stability in liver microsomes, almost completely blocked the formation of epoxide metabolite(s) and protein covalent binding, and attenuated hepatic and plasma glutathione depletion. Moreover, the structural manipulation did not alter the efficacy of BBR. This work provided solid evidence that the formation of the epoxide(s) is a key step in the development of BBR-induced hepatotoxicity.