RESUMO
The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5-14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 ('Leu-in' to 'Leu-out') by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats.
Assuntos
Desenho de Fármacos , Piperidinas/farmacologia , Inibidores de Proteases/farmacologia , Pirimidinas/farmacologia , Renina/antagonistas & inibidores , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Ratos Sprague-Dawley , Renina/sangue , Relação Estrutura-AtividadeRESUMO
Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We show here that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of Sec15, which interacts with Rab11, or Exo70, both components of the exocyst tethering complex, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.
Assuntos
Membrana Celular/metabolismo , Endocitose , Exocitose , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Membrana Celular/ultraestrutura , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Vesículas Transportadoras/ultraestrutura , Proteínas de Transporte Vesicular/metabolismoRESUMO
The angiotensin II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of ¹²5I-Sar¹-Ile8-AII to human angiotensin type 1 receptors with an IC50 of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of 7.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC50 values of 6.7, 5.1, 44.9, and 15.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC50 values of 242.5, 191.6, >10,000, and >10,000 nM). AZL also inhibited the accumulation of AII-induced inositol 1-phosphate (IP1) in the cell-based assay with an IC50 value of 9.2 nmol; this effect was resistant to washout (IC50 value of 81.3 nM). Olmesartan and valsartan inhibited IP1 accumulation with IC50 values of 12.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC50 value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human angiotensin type 1 receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'2 value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Oxidiazóis/metabolismo , Oxidiazóis/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Benzimidazóis/química , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Humanos , Masculino , Oxidiazóis/química , Ligação Proteica/fisiologia , Coelhos , Receptor Tipo 1 de Angiotensina/fisiologiaRESUMO
The anticoagulant and antithrombotic profiles of TAK-442, a direct factor Xa (FXa) inhibitor, were investigated. TAK-442 showed potent inhibition of human FXa (Ki = 1.8 nM) and high specificity, with a 440-fold greater selectivity than thrombin and negligible effects on trypsin, plasmin, and tissue plasminogen activator (K(i) > 30 microM). [corrected] In human plasma, TAK-442 doubled FXa-induced clotting time, prothrombin time (PT), and activated partial thromboplastin time at 0.19, 0.55, and 0.59 microM, respectively. The relative PT-prolonging potencies of TAK-442, rivaroxaban, and apixaban were 1, 2.0-2.6, and 0.46-1.3, respectively, in 4 different PT reagents. In a rabbit model of venous thrombosis, 50- and 100-micrograms/kg [corrected] TAK-442 (intravenous bolus followed by 1-hour infusion) reduced thrombus formation by 50% and 81%, with plasma anti-FXa activity of 23%-26% and 34%-38%, respectively, and only marginal prolongation of PT and activated partial thromboplastin time. Melagatran, a thrombin inhibitor, showed similar antithrombotic activity to TAK-442. However, 500-micrograms/kg [corrected TAK-442 did not affect bleeding time (BT), whereas the same dose of melagatran significantly prolonged BT by 3.6-fold compared with vehicle control. These findings suggest that TAK-442 has similar antithrombotic effects as melagatran but does not cause BT prolongation, and plasma anti-FXa activity may reliably predict its potency.
Assuntos
Anticoagulantes/uso terapêutico , Inibidores do Fator Xa , Fibrinolíticos/uso terapêutico , Pirimidinonas/uso terapêutico , Sulfonas/uso terapêutico , Trombose Venosa/tratamento farmacológico , Animais , Anticoagulantes/farmacologia , Azetidinas/farmacologia , Benzilaminas/farmacologia , Tempo de Sangramento , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Cães , Fibrinolíticos/farmacologia , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfolinas/farmacologia , Pirazóis/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Coelhos , Ratos , Ratos Sprague-Dawley , Rivaroxabana , Sulfonas/farmacologia , Tiofenos/farmacologia , Trombina/antagonistas & inibidores , Trombose Venosa/sangueRESUMO
We have recently discovered imidazo[1,5-c]imidazol-3-one derivative 1 as a potent, selective, and orally bioavailable factor Xa (FXa) inhibitor. In this study, we have synthesized metabolites of 1 and evaluated their biological activities. As a result, we identified the active metabolites S-5 and 6 with a potent FXa inhibitory activity comparable to 1 and a favorable pharmacokinetic profile in monkeys.
Assuntos
Anticoagulantes/síntese química , Fator Xa/fisiologia , Imidazóis/síntese química , Piperidinas/síntese química , Animais , Anticoagulantes/sangue , Anticoagulantes/química , Anticoagulantes/farmacocinética , Área Sob a Curva , Inibidores do Fator Xa , Humanos , Imidazóis/sangue , Imidazóis/química , Imidazóis/farmacocinética , Macaca fascicularis , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Piperidinas/sangue , Piperidinas/química , Piperidinas/farmacocinética , Espectrofotometria InfravermelhoRESUMO
The coagulation enzyme factor Xa (FXa) has been recognized as a promising target for the development of new antithrombotic agents. We previously found compound 1 to be an orally bioavailable FXa inhibitor in fasted monkeys; however, 1 showed poor bioavailability in rats and fed monkeys. To work out the pharmacokinetic problems, we focused our synthetic efforts on the chemical conversion of the 4-(imidazo[1,2- a]pyridin-5-yl)piperazine moiety of 1 to imidazolylpiperidine derivatives (fused and nonfused), which resulted in the discovery of the weakly basic imidazo[1,5- c]imidazol-3-one 3q as a potent and selective FXa inhibitor. Compound 3q showed favorable oral bioavailability in rats and monkeys under both fasted and fed conditions and antithrombotic efficacy in a rat model of venous thrombosis after oral administration, without a significant increase in bleeding time (unlike warfarin). On the basis of these promising properties, compound 3q was selected for further evaluation.
Assuntos
Anticoagulantes/síntese química , Inibidores do Fator Xa , Imidazóis/síntese química , Sulfonas/síntese química , Administração Oral , Animais , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Disponibilidade Biológica , Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Citocromo P-450 CYP3A , Ingestão de Alimentos/efeitos dos fármacos , Humanos , Imidazóis/farmacocinética , Imidazóis/farmacologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Moleculares , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonas/farmacocinética , Sulfonas/farmacologia , Trombose Venosa/prevenção & controleRESUMO
We have recently reported the discovery of orally active sulfonylalkylamide Factor Xa (FXa) inhibitors, as typified by compound 1 (FXa IC(50)=0.061 microM). Since the pyridylpiperidine moiety was not investigated in our previous study, we conducted detailed structure-activity relationship studies on this S4 binding element. This investigation led to the discovery of piperazinylimidazo[1,2-a]pyridine 2b as a novel and potent FXa inhibitor (FXa IC(50)=0.021 microM). Further modification resulted in the discovery of 2-hydroxymethylimidazo[1,2-a]pyridine 2e (FXa IC(50)=0.0090 microM), which was found to be a selective and orally bioavailable FXa inhibitor with reduced CYP3A4 inhibition.
Assuntos
Inibidores do Fator Xa , Piridinas/química , Piridinas/farmacologia , Administração Oral , Disponibilidade Biológica , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Relação Estrutura-AtividadeRESUMO
Factor Xa (FXa) is a trypsin-like serine protease involved in the coagulation cascade and has received great interest as a potential target for the development of new antithrombotic agents. Most of amidine-type FXa inhibitors reported have been found to show extremely poor oral bioavailability. Compound 1 is one of the first reported non-amidine type FXa inhibitors. To discover novel and orally active FXa inhibitors, we investigated flexible linear linkers between the 6-chloronaphthalene ring and the 1-(pyridin-4-yl)piperidine moiety of 1 and found the orally active sulfonylalkylamide 2f with an FXa IC(50) of 0.05 microM, comparable with that of 1. Further modification to reduce the CYP3A4 inhibitory activity of 2f resulted in the potent, selective, and orally active 2-methylpyridine analogue 2s (FXa IC(50) of 0.061 microM), for which the liability of CYP3A4 inhibition was significantly weakened compared to 2f. Compound 2s also showed long lasting anticoagulant activity in cynomolgus monkeys.
Assuntos
Amidas/administração & dosagem , Amidas/síntese química , Antitrombina III/administração & dosagem , Antitrombina III/síntese química , Compostos de Enxofre/administração & dosagem , Compostos de Enxofre/síntese química , Administração Oral , Alquilação , Amidas/química , Amidas/classificação , Animais , Antitrombina III/química , Antitrombina III/classificação , Reagentes de Ligações Cruzadas/química , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Fator Xa/química , Fator Xa/metabolismo , Inibidores do Fator Xa , Haplorrinos , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Compostos de Enxofre/química , Compostos de Enxofre/classificaçãoRESUMO
We recently showed that Rab11 is involved not only in formation of recycling vesicles containing the transferrin (Tfn)-transferrin receptor (TfnR) complex at perinuclear recycling endosomes but also in tethering of recycling vesicles to the plasma membrane (PM) in concert with the exocyst tethering complex. We here aimed at identifying SNARE proteins responsible for fusion of Tfn-TfnR-containing recycling vesicles with the PM, downstream of the exocyst. We showed that exocyst subunits, Sec6 and Sec8, can interact with SNAP23 and SNAP25, both of which are PM-localizing Qbc-SNAREs, and that depletion of SNAP23 and/or SNAP25 in HeLa cells suppresses fusion of Tfn-TfnR-containing vesicles with the PM, leading to accumulation of the vesicles at the cell periphery. We also found that VAMP2, an R-SNARE, is colocalized with endocytosed Tfn on punctate endosomal structures, and that its depletion in HeLa cells suppresses recycling vesicle exocytosis. These observations indicate that fusion of recycling vesicles with the PM downstream of the exocyst is mediated by SNAP23/25 and VAMP2, and provide novel insight into non-neuronal roles of VAMP2 and SNAP25.
RESUMO
We discovered a novel benzimidazole derivative, named compound (comp.) 1, with unique antiangiogenic characteristics. Comp.1 cytostatically inhibited the vascular endothelial growth factor- and basic fibroblast growth factor-induced growth of endothelial cells (50% inhibitory concentration: 29-79 nM) without a cytotoxic phase, but did not affect the growth of other types of cells up to 90 microM. Comp.1 also inhibited the tube formation derived from a rat aorta fragment, but the oral (p.o.) treatment of comp.1 (46 mg/kg, administered twice daily (b.i.d.)) did not inhibit aniogenesis in a mouse sponge model. Comp.8, an analogue of comp.1, showed a specific inhibitory effect on endothelial cell growth. Comp.8 also suppressed angiogenesis (15 mg/kg, b.i.d., p.o., 70% inhibition) in the sponge model without body weight loss.
Assuntos
Benzimidazóis/farmacologia , Endotélio Vascular/citologia , Neovascularização Patológica/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Cinética , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Ratos , Relação Estrutura-Atividade , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Veias/citologia , Veias/efeitos dos fármacos , Veias/fisiologia , Aumento de Peso/efeitos dos fármacosRESUMO
A 72-year-old male patient with Parkinson's disease referred to the Dental Hospital of Kyushu University, Fukuoka, Japan, presented with movement of an implant-supported prosthesis, slight pain, and purulent drainage from the gingiva in the anterior region of the maxilla, where implants had been placed more than 20 years earlier. He requested that the mobile implant and prosthesis be removed and was in need of implant therapy in his maxilla. In addition to Parkinson's disease the patient had a severe gag reflex, which made treatment difficult. A total of 6 treatments were required. New implants were successfully placed while the patient was under anesthesia (a combination of intravenously administered 3% prilocaine hydrochloride and vasopressin); midazolam was also administered intravenously. The results of this case indicate that use of regional anesthesia in combination with midazolam can be recommended for implant surgery in patients with Parkinson's disease. Intravenous midazolam can be considered the sedative of choice for the surgical treatment of patients suffering from systemic disease.
Assuntos
Implantes Dentários , Hipnóticos e Sedativos/administração & dosagem , Midazolam/administração & dosagem , Doença de Parkinson/complicações , Idoso , Anestésicos Intravenosos/administração & dosagem , Prótese Dentária Fixada por Implante , Falha de Restauração Dentária , Seguimentos , Humanos , Masculino , Maxila/cirurgia , Prilocaína/administração & dosagem , Reoperação , Vasoconstritores/administração & dosagem , Vasopressinas/administração & dosagemRESUMO
The small GTPase Arf6 is transiently associated with the ingressing cleavage furrow and subsequently targeted to the Flemming body during cytokinesis, suggesting its activation around the cleavage furrow. Here, we show that EFA6 (exchange factor for Arf6) localizes on the cleavage furrow through its PH domain. Time-lapse analysis showed that both EFA6 and Arf6 are transiently localized around the ingressing cleavage furrow, but only Arf6 is subsequently targeted to the Flemming body. Expression of an EFA6 mutant suppresses Arf6 recruitment onto the Flemming body. These results suggest that EFA6 participates in activation of Arf6 around the cleavage furrow during cytokinesis.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Citocinese , Proteínas do Tecido Nervoso/metabolismo , Fator 6 de Ribosilação do ADP , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina , Células HeLa , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína , Transporte Proteico , Análise de Célula Única , Imagem com Lapso de TempoRESUMO
Coagulation enzyme factor Xa (FXa) is a particularly promising target for the development of new anticoagulant agents. We previously reported the imidazo[1,5-c]imidazol-3-one derivative 1 as a potent and orally active FXa inhibitor. However, it was found that 1 predominantly undergoes hydrolysis upon incubation with human liver microsomes, and the human specific metabolic pathway made it difficult to predict the human pharmacokinetics. To address this issue, our synthetic efforts were focused on modification of the imidazo[1,5-c]imidazol-3-one moiety of the active metabolite 3a, derived from 1, which resulted in the discovery of the tetrahydropyrimidin-2(1H)-one derivative 5k as a highly potent and selective FXa inhibitor. Compound 5k showed no detectable amide bond cleavage in human liver microsomes, exhibited a good pharmacokinetic profile in monkeys, and had a potent antithrombotic efficacy in a rabbit model without prolongation of bleeding time. Compound 5k is currently under clinical development with the code name TAK-442.
Assuntos
Anticoagulantes/química , Inibidores do Fator Xa , Pirimidinonas/farmacologia , Administração Oral , Animais , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Disponibilidade Biológica , Tempo de Sangramento , Descoberta de Drogas , Haplorrinos , Humanos , Microssomos Hepáticos/metabolismo , Farmacocinética , Pirimidinonas/farmacocinética , CoelhosRESUMO
The objective of this study was to clarify the influence of pioglitazone (Pio) on proinsulin (PI) in patients with type 2 diabetes mellitus. The subjects were 55 patients with type 2 diabetes. Among them, 18, 18, and 19 patients were respectively treated with Pio alone (group P), gliclazide (Gli) alone (group G), or Pio plus Gli (group PG) for 12 weeks. Fasting blood samples were obtained before and after treatment and were used to measure fasting plasma glucose (FPG), HbA1C, immunoreactive insulin (IRI), and PI. The levels of FPG, HbA1C, and IRI showed a significant decrease after treatment with Pio in groups P and PG. Treatment with Pio also caused PI to decrease significantly (group P: from 24.7 +/- 12.9 (mean +/- SD) to 14.0 +/- 6.2 pmol/L, p < 0.01, group PG: from 24.3 +/- 11.3 to 14.4 +/- 6.5 pmol/L, p < 0.01). In group G, treatment with Gli caused FPG and HbA1C to decrease significantly, but PI showed no change (21.5 +/- 12.3 to 21.6 +/- 10.4 pmol/L, p = n.s.). In patients with type 2 diabetes, treatment with Pio achieved an improvement of glycemic control and reduced the load on the pancreatic beta cells.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Proinsulina/sangue , Tiazóis/uso terapêutico , Tiazolidinedionas , Idoso , Glicemia/análise , Quimioterapia Combinada , Jejum/sangue , Feminino , Gliclazida/uso terapêutico , Hemoglobinas Glicadas/análise , Humanos , Insulina/sangue , Masculino , Pessoa de Meia-Idade , PioglitazonaRESUMO
The relationship between blood levels of N-carboxymethyl-lysine (CML) or pentosidine and the severity of microangiopathy was investigated in patients with type 2 diabetes. Blood CML and pentosidine levels were measured by ELISA in 97 type 2 diabetics (46 men and 51 women). CML and pentosidine levels were significantly higher in patients with chronic renal failure than in those with normoalbuminuria, microalbuminuria, or macroalbuminuria (all p < 0.05). Among the diabetics without nephropathy (n = 49), blood CML levels were significantly higher in the patients who had proliferative diabetic retinopathy than in those without retinopathy or those who had background retinopathy (both p < 0.01). In contrast, blood pentosidine levels showed no significant differences among the three retinopathy groups. These findings suggest that the blood level of CML is related to the severity of both nephropathy and retinopathy, while the pentosidine level is only related to the severity of nephropathy.