RESUMO
Amyotrophic lateral sclerosis (ALS) is a major life-threatening disease caused by motor neuron degeneration. More effective treatments through drug discovery are urgently needed. Here, we established an effective high-throughput screening system using induced pluripotent stem cells (iPSCs). Using a Tet-On-dependent transcription factor expression system carried on the PiggyBac vector, motor neurons were efficiently and rapidly generated from iPSCs by a single-step induction method. Induced iPSC transcripts displayed characteristics similar to those of spinal cord neurons. iPSC-generated motor neurons carried a mutation in fused in sarcoma (FUS) and superoxide dismutase 1 (SOD1) genes and had abnormal protein accumulation corresponding to each mutation. Calcium imaging and multiple electrode array (MEA) recordings demonstrated that ALS neurons were abnormally hyperexcitable. Noticeably, protein accumulation and hyperexcitability were ameliorated by treatment with rapamycin (mTOR inhibitor) and retigabine (Kv7 channel activator), respectively. Furthermore, rapamycin suppressed ALS neuronal death and hyperexcitability, suggesting that protein aggregate clearance through the activation of autophagy effectively normalized activity and improved neuronal survival. Our culture system reproduced several ALS phenotypes, including protein accumulation, hyperexcitability, and neuronal death. This rapid and robust phenotypic screening system will likely facilitate the discovery of novel ALS therapeutics and stratified and personalized medicine for sporadic motor neuron diseases.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase-1/metabolismo , Mutação , Fenótipo , Superóxido Dismutase/metabolismoRESUMO
ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator that exhibits selectivity for S1P receptors 1 and 5. Treatment with ONO-4641 leads to a reduction in magnetic resonance imaging disease measures in patients with relapsing-remitting multiple sclerosis. The objective of this study was to explore the potential impact of ONO-4641 treatment based on its immunomodulatory effects. Severe aplastic anemia is a bone marrow (BM) failure disease typically caused by aberrant immune destruction of blood progenitors. Although the T helper type 1-mediated pathology is well described for aplastic anemia, the molecular mechanisms driving disease progression remain undefined. We evaluated the efficacy of ONO-4641 in a mouse model of aplastic anemia. ONO-4641 reduced the severity of BM failure in a dose-dependent manner, resulting in higher blood and BM cell counts. By evaluating the mode of action, we found that ONO-4641 inhibited the infiltration of donor-derived T lymphocytes to the BM. ONO-4641 also induced the accumulation of hematopoietic stem cells in the BM of model mice. These observations indicate, for the first time, that S1P receptor modulators demonstrate efficacy in the mouse model of aplastic anemia and suggest that treatment with ONO-4641 might delay the progression of aplastic anemia. SIGNIFICANCE STATEMENT: ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator selective for S1P receptors 1 and 5. In this study, we demonstrated that ONO-4641 regulates the trafficking of T lymphocytes along with hematopoietic stem and progenitor cells, leading to alleviation of pancytopenia and destruction of bone marrow in a bone marrow failure-induced mouse model mimicking human aplastic anemia.
Assuntos
Anemia Aplástica/tratamento farmacológico , Azetidinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Naftalenos/farmacologia , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Linfócitos T/efeitos dos fármacos , Anemia Aplástica/imunologia , Animais , Azetidinas/uso terapêutico , Movimento Celular , Células Cultivadas , Células-Tronco Hematopoéticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Naftalenos/uso terapêutico , Moduladores do Receptor de Esfingosina 1 Fosfato/uso terapêutico , Receptores de Esfingosina-1-Fosfato/metabolismo , Linfócitos T/fisiologiaRESUMO
Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-ß-CD, HP-γ-CD, and their homolog 2-hydroxypropyl-α-cyclodextrin (HP-α-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-ß-CD and HP-γ-CD, unlike HP-α-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-ß-CD and HP-γ-CD, unlike HP-α-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-ß-CD and HP-γ-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.
Assuntos
1-Desoxinojirimicina/análogos & derivados , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Ciclodextrinas/farmacologia , Proteína C1 de Niemann-Pick/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , 1-Desoxinojirimicina/uso terapêutico , Animais , Células CHO , Colesterol/metabolismo , Cricetulus , Lisossomos/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Esfingolipídeos/metabolismoRESUMO
OBJECTIVES: Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to cell surface receptors (S1P1-5). In this study, we examined the effect of S1P1 agonist, ONO-W061, on murine Candida albicans water-soluble fraction (CAWS)-induced vasculitis. METHODS: Mice were administered ONO-W061, and the number of peripheral blood cells was counted. Vasculitis was induced by an intraperitoneal injection of CAWS. Expression of S1P receptors and CXCL1 was analyzed by quantitative RT-PCR. ONO-W061 was orally administered, and vasculitis was evaluated histologically. Number of neutrophils, macrophages and T cells in the vasculitis tissue was counted using flow cytometry. Production of chemokines from S1P-stimulated human umbilical vein endothelial cells (HUVECs) was measured by ELISA. RESULTS: Number of peripheral blood lymphocytes was decreased by ONO-W061. Expression of CXCL1 and S1P1 was enhanced in CAWS-induced vasculitis tissue. Vasculitis score, CXCL1 and number of neutrophils in the vasculitis tissue were lower in ONO-W061-treated mice. Treatment of HUVECs with S1P upregulated the production of CXCL1 and IL-8 in vitro, and this was inhibited by ONO-W061. CONCLUSIONS: ONO-W061 significantly improved CAWS-induced vasculitis. This effect may be partly exerted through the inhibited production of chemokines by endothelial cells, which in turn could induce neutrophil recruitment into inflamed vessels.
Assuntos
Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/agonistas , Esfingosina/análogos & derivados , Vasculite/tratamento farmacológico , Animais , Candida albicans , Quimiocina CXCL1/metabolismo , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucina-8/metabolismo , Contagem de Leucócitos , Masculino , Camundongos Endogâmicos BALB C , Esfingosina/metabolismo , Vasculite/imunologia , Vasculite/metabolismoRESUMO
Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P(1) agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P(3) agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P(1) agonistic activity with excellent selectivity over hS1P(3) and in vivo PLL activity in mice.
Assuntos
Química Farmacêutica/métodos , Lisofosfolipídeos/antagonistas & inibidores , Naftalenos/química , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Esfingosina/análogos & derivados , Administração Oral , Animais , Benzeno/química , Cloro/química , Desenho de Fármacos , Humanos , Ligantes , Camundongos , Modelos Químicos , Ratos , Esfingosina/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P(1) agonistic activity as well as selectivity over S1P(3) agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P(1) receptor agonist with high selectivity against S1P(3) and enhanced efficacy in lowering peripheral lymphocyte counts in mice.
Assuntos
Naftalenos/síntese química , Propanóis/química , Receptores de Lisoesfingolipídeo/agonistas , Administração Oral , Animais , Células CHO , Cricetinae , Cricetulus , Cloridrato de Fingolimode , Humanos , Linfócitos/efeitos dos fármacos , Camundongos , Estrutura Molecular , Naftalenos/administração & dosagem , Naftalenos/farmacologia , Propanóis/administração & dosagem , Propanóis/farmacologia , Propilenoglicóis , Esfingosina/análogos & derivados , Relação Estrutura-AtividadeRESUMO
Sphingolipids play a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, little is known about the precise roles of sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, and its receptor modulation in COPD. In this study, we demonstrated that the S1P receptor modulator ONO-4641 induced the expansion of lung CD11b+Gr-1+ cells and lymphocytopenia in naive mice. ONO-4641-expanded CD11b+Gr-1+ cells showed higher arginase-1 activity, decreased T cell proliferation, and lower IFN-γ production in CD3+ T cells, similar to the features of myeloid-derived suppressor cells. ONO-4641 treatment decreased airspace enlargement in elastase-induced and cigarette smoke-induced emphysema models and attenuated emphysema exacerbation induced by post-elastase pneumococcal infection, which was also associated with an increased number of lung CD11b+Gr-1+ cells. Adoptive transfer of ONO-4641-expanded CD11b+Gr-1+ cells protected against elastase-induced emphysema. Lymphocytopenia observed in these models likely contributed to beneficial ONO-4641 effects. Thus, ONO-4641 attenuated murine pulmonary emphysema by expanding lung CD11b+Gr-1+ cell populations and inducing lymphocytopenia. The S1P receptor might be a promising target for strategies aimed at ameliorating pulmonary emphysema progression.
Assuntos
Azetidinas/uso terapêutico , Pulmão/imunologia , Naftalenos/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Enfisema Pulmonar/tratamento farmacológico , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Transferência Adotiva , Animais , Azetidinas/farmacologia , Antígeno CD11b/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Naftalenos/farmacologia , Receptores de Superfície Celular/metabolismo , Linfócitos T/imunologiaRESUMO
The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, is described. While it has been revealed that the modulation of the S1P1 receptor is an effective way to treat autoimmune diseases such as relapsing-remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P3 receptor is implicated in some undesirable effects. We carried out a structure-activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30â¯000-fold selectivity for S1P1 over S1P3 and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED50 = 0.029 mg/kg, 24 h after oral dosing).
Assuntos
Azetidinas/farmacologia , Linfócitos/efeitos dos fármacos , Naftalenos/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Administração Oral , Animais , Doenças Autoimunes/tratamento farmacológico , Azetidinas/administração & dosagem , Azetidinas/química , Azetidinas/farmacocinética , Células CHO , Cricetulus , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/administração & dosagem , Naftalenos/química , Naftalenos/farmacocinética , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
CC chemokine receptor 4 (CCR4) has been implicated as a preferential marker for T helper type 2 (Th2) cells, and is believed to be involved in the pathology of allergic diseases by controlling Th2 cell trafficking into inflamed tissues. The objective of the study was to characterize the pharmacological properties of E0001-163, a novel CCR4 antagonist. E0001-163 was tested in both in vitro chemotaxis assays as well as in vivo mouse models of CCR4 ligand-induced air pouch and antigen-induced airway inflammation by utilizing in vitro-polarized Th2 cells. In vitro, E0001-163 inhibited migratory response of human Th2-polarized cells to CCL22, a CCR4 ligand, with an IC50 value of 11.9 nM. E0001-163 significantly suppressed CCL22-induced Th2 cell trafficking into mouse air pouch in a dose-dependent manner at doses of 3 and 10mg/kg, suggesting that E0001-163 has an inhibitory effect on CCR4-mediated T cell trafficking in vivo. In addition, E0001-163 partially decreased Th2 cell trafficking and the level of IL-4 in the lungs in Th2-tansferred and ovalbumin (OVA)-challenged mice. T cell trafficking involves multiple chemokine receptors both in acute and chronic phases, and our findings suggest that CCR4, together with other chemokine receptors, may be involved in Th2 cell trafficking under disease conditions.