RESUMO
α/ßKlotho coreceptors simultaneously engage fibroblast growth factor (FGF) hormones (FGF19, FGF21 and FGF23)1,2 and their cognate cell-surface FGF receptors (FGFR1-4) thereby stabilizing the endocrine FGF-FGFR complex3-6. However, these hormones still require heparan sulfate (HS) proteoglycan as an additional coreceptor to induce FGFR dimerization/activation and hence elicit their essential metabolic activities6. To reveal the molecular mechanism underpinning the coreceptor role of HS, we solved cryo-electron microscopy structures of three distinct 1:2:1:1 FGF23-FGFR-αKlotho-HS quaternary complexes featuring the 'c' splice isoforms of FGFR1 (FGFR1c), FGFR3 (FGFR3c) or FGFR4 as the receptor component. These structures, supported by cell-based receptor complementation and heterodimerization experiments, reveal that a single HS chain enables FGF23 and its primary FGFR within a 1:1:1 FGF23-FGFR-αKlotho ternary complex to jointly recruit a lone secondary FGFR molecule leading to asymmetric receptor dimerization and activation. However, αKlotho does not directly participate in recruiting the secondary receptor/dimerization. We also show that the asymmetric mode of receptor dimerization is applicable to paracrine FGFs that signal solely in an HS-dependent fashion. Our structural and biochemical data overturn the current symmetric FGFR dimerization paradigm and provide blueprints for rational discovery of modulators of FGF signalling2 as therapeutics for human metabolic diseases and cancer.
Assuntos
Fator de Crescimento de Fibroblastos 23 , Proteoglicanas de Heparan Sulfato , Hormônios , Receptores de Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Humanos , Microscopia Crioeletrônica , Fator de Crescimento de Fibroblastos 23/química , Fator de Crescimento de Fibroblastos 23/metabolismo , Fator de Crescimento de Fibroblastos 23/ultraestrutura , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/metabolismo , Hormônios/química , Hormônios/metabolismo , Proteínas Klotho/química , Proteínas Klotho/metabolismo , Proteínas Klotho/ultraestrutura , Multimerização Proteica , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestruturaRESUMO
Cardiovascular disease (CVD) is the major cause of death in chronic kidney disease (CKD) and is associated with high circulating fibroblast growth factor (FGF)23 levels. It is unresolved whether high circulating FGF23 is a mere biomarker or pathogenically contributes to cardiomyopathy. It is also unknown whether the C-terminal FGF23 peptide (cFGF23), a natural FGF23 antagonist proteolyzed from intact FGF23 (iFGF23), retards CKD progression and improves cardiomyopathy. We addressed these questions in three murine models with high endogenous FGF23 and cardiomyopathy. First, we examined wild-type (WT) mice with CKD induced by unilateral ischemia-reperfusion and contralateral nephrectomy followed by a high-phosphate diet. These mice were continuously treated with intraperitoneal implanted osmotic minipumps containing either iFGF23 protein to further escalate FGF23 bioactivity, cFGF23 peptide to block FGF23 signaling, vehicle, or scrambled peptide as negative controls. Exogenous iFGF23 protein given to CKD mice exacerbated pathological cardiac remodeling and CKD progression, whereas cFGF23 treatment improved heart and kidney function, attenuated fibrosis, and increased circulating soluble Klotho. WT mice without renal insult placed on a high-phosphate diet and homozygous Klotho hypomorphic mice, both of whom develop moderate CKD and clear cardiomyopathy, were treated with cFGF23 or vehicle. Mice treated with cFGF23 in both models had improved heart and kidney function and histopathology. Taken together, these data indicate high endogenous iFGF23 is not just a mere biomarker but pathogenically deleterious in CKD and cardiomyopathy. Furthermore, attenuation of FGF23 bioactivity by cFGF23 peptide is a promising therapeutic strategy to protect the kidney and heart from high FGF23 activity.NEW & NOTEWORTHY There is a strong correlation between cardiovascular morbidity and high circulating fibroblast growth factor 23 (FGF23) levels, but causality was never proven. We used a murine chronic kidney disease (CKD) model to show that intact FGF23 (iFGF23) is pathogenic and contributes to both CKD progression and cardiomyopathy. Blockade of FGF23 signaling with a natural proteolytic product of iFGF23, C-terminal FGF23, alleviated kidney and cardiac histology, and function in three separate murine models of high endogenous FGF23.
Assuntos
Cardiomiopatias , Insuficiência Renal Crônica , Animais , Camundongos , Fator de Crescimento de Fibroblastos 23 , Modelos Animais de Doenças , Insuficiência Renal Crônica/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Biomarcadores , Fosfatos , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/complicaçõesRESUMO
ABSTRACT: Chronic kidney disease (CKD) is a significant global health threat that imposes a substantial burden on both individuals and societies. CKD frequently correlates with cardiovascular events, particularly left ventricular hypertrophy (LVH), which contributes to the high mortality rate associated with CKD. Fibroblast growth factor 23 (FGF23), a hormone primarily involved in regulating calcium and phosphorus metabolism, has been identified as a major risk factor for LVH in CKD patients. Elevated serum FGF23 levels are known to induce LVH and myocardial fibrosis by activating the fibroblast growth factor receptor 4 (FGFR4) signal pathway. Therefore, targeting FGFR4 and its downstream signaling pathways holds potential as a treatment strategy for cardiac dysfunction in CKD. In our current study, we have discovered that Hypericin, a key component derived from Hypericum perforatum , has the ability to alleviate CKD-related LVH by targeting the FGFR4/phospholipase C gamma 1 (PLCγ1) signaling pathway. Through in vitro experiments using rat cardiac myocyte H9c2 cells, we observed that Hypericin effectively inhibits FGF23-induced hypertrophy and fibrosis by suppressing the FGFR4/PLCγ1/calcineurin/nuclear factor of activated T-cell (NFAT3) signaling pathway. In addition, our in vivo studies using mice on a high-phosphate diet and rat models of 5/6 nephrectomy demonstrated that Hypericin has therapeutic effects against CKD-induced LVH by modulating the FGFR4/PLCγ1/calcineurin/NFAT3 signaling pathway. In conclusion, our research highlights the potential of Hypericin as a candidate for the treatment of CKD-induced cardiomyopathy. By suppressing the FGFR4/PLCγ1 signaling pathway, Hypericin shows promise in attenuating LVH and myocardial fibrosis associated with CKD.
Assuntos
Antracenos , Modelos Animais de Doenças , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos , Fibrose , Hipertrofia Ventricular Esquerda , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Perileno , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos , Insuficiência Renal Crônica , Transdução de Sinais , Animais , Perileno/análogos & derivados , Perileno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/prevenção & controle , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Ratos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Linhagem Celular , Antracenos/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Fatores de Transcrição NFATC/metabolismo , CamundongosRESUMO
The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23-FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling.
Assuntos
Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/química , Glucuronidase/metabolismo , Comunicação Parácrina , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação/genética , Líquidos Corporais/metabolismo , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Glucuronidase/genética , Heparitina Sulfato/metabolismo , Humanos , Proteínas Klotho , Ligantes , Masculino , Camundongos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , SolubilidadeRESUMO
Acute lung injury (ALI) is a diffuse lung dysfunction disease characterized by high prevalence and high mortality. Thus far, no effective pharmacological treatment has been made for ALI in clinics. Inflammation is critical to the development of ALI. Therefore, anti-inflammation may be a potential therapy strategy for ALI. Indazole-containing derivatives, representing one of the most important heterocycles in drug molecules, are endowed with a broad range of biological properties, such as anti-cancer and anti-inflammation. In the current study, we investigated the biological effects of Cyy-272, a newly synthesized indazole compound, on LPS-induced ALI both in vivo and in vitro. Results show that Cyy-272 can inhibit the release of inflammatory cytokines in LPS-stimulated macrophage and alleviate LPS induced ALI. Further experiment revealed that Cyy-272 exhibit anti-inflammation activity by inhibiting JNK phosphorylation. Overall, our studies show that an indazole derivative, Cyy-272, is effective in suppressing LPS-induced JNK activation and inflammatory signaling.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/prevenção & controle , Indazóis/uso terapêutico , Lipopolissacarídeos/toxicidade , MAP Quinase Quinase 4/antagonistas & inibidores , Lesão Pulmonar Aguda/metabolismo , Animais , Relação Dose-Resposta a Droga , Indazóis/química , Indazóis/farmacologia , MAP Quinase Quinase 4/química , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Células RAW 264.7RESUMO
Hypoferremia results as an acute phase response to infection and inflammation aiming to reduce iron availability to pathogens. Activation of toll-like receptors (TLRs), the key sensors of the innate immune system, induces hypoferremia mainly through the rise of the iron hormone hepcidin. Conversely, stimulation of erythropoiesis suppresses hepcidin expression via induction of the erythropoietin-responsive hormone erythroferrone. Iron deficiency stimulates transcription of the osteocyte-secreted protein FGF23. Here we hypothesized that induction of FGF23 in response to TLR4 activation is a potent contributor to hypoferremia and, thus, impairment of its activity may alleviate hypoferremia induced by lipopolysaccharide (LPS), a TLR 4 agonist. We used the C-terminal tail of FGF23 to impair endogenous full-length FGF23 signaling in wild-type mice, and investigated its impact on hypoferremia. Our data show that FGF23 is induced as early as pro-inflammatory cytokines in response to LPS, followed by upregulation of hepcidin and downregulation of erythropoietin (Epo) expression in addition to decreased serum iron and transferrin saturation. Further, LPS-induced hepatic and circulating hepcidin were significantly reduced by FGF23 signaling disruption. Accordingly, iron sequestration in liver and spleen caused by TLR4 activation was completely abrogated by FGF23 signaling inhibition, resulting in alleviation of serum iron and transferrin saturation deficit. Taken together, our studies highlight for the first time that inhibition of FGF23 signaling alleviates LPS-induced acute hypoferremia.
Assuntos
Hepcidinas , Ferro , Animais , Peptídeo C , Eritropoese , Fator de Crescimento de Fibroblastos 23 , Hepcidinas/genética , Inflamação/tratamento farmacológico , CamundongosRESUMO
Mechanisms of cardiomyopathy caused by obesity/hyperlipidemia are complicated. Obesity is usually associated with chronic low-grade inflammation and may lead to the onset and progression of myocardial fibrosis and remodeling. TLR4/MyD88 signaling pathway, as a key regulator of inflammation, plays an important role in the pathogenesis of obesity-induced cardiomyopathy. We previously demonstrated that LM9, a novel MyD88 inhibitor, attenuated inflammatory responses and fibrosis in obesity-induced cardiomyopathy by inhibiting the formation of TLR4/MyD88 complex. In this study, we investigated the protective effects of LM9 on obesity-induced cardiomyopathy in vitro and in vivo. We showed that LM9 (5, 10 µM) significantly attenuates palmitic acid (PA)-induced inflammation in mouse peritoneal macrophages, evidenced by decreased expression of proinflammatory genes including TNF-α, IL-6, IL-1ß, and ICAM-1. In cardiac-derived H9C2 cells, LM9 treatment suppressed PA-induced inflammation, lipid accumulation, and fibrotic responses. In addition, LM9 treatment also inhibited PA-activated TLR4/MyD88/NF-κB signaling pathway. We further revealed in HEK293 cells that LM9 treatment blocked the TLR4/MyD88 binding and MyD88 homodimer formation. In HFD-fed mice, administration of LM9 (5, 10 mg/kg, ig, every other days for 8 weeks) dose-dependently alleviated inflammation and fibrosis in heart tissues and decreased serum lipid concentration. In conclusion, this study demonstrates that MyD88 inhibitor LM9 exerts protective effects against obesity-induced cardiomyopathy, suggesting LM9 to be a promising therapeutic candidate drug for the obesity-related cardiac complications.
Assuntos
Anti-Inflamatórios/uso terapêutico , Cardiomiopatias/tratamento farmacológico , Fibrose/tratamento farmacológico , Inflamação/tratamento farmacológico , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Piperazinas/uso terapêutico , Tiazóis/uso terapêutico , Animais , Cardiomiopatias/epidemiologia , Cardiomiopatias/patologia , Dieta Hiperlipídica , Relação Dose-Resposta a Droga , Fibrose/patologia , Células HEK293 , Humanos , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Miocárdio/patologia , NF-kappa B/metabolismo , Obesidade/complicações , Ratos , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Retinal ischemia-reperfusion (I/R) injury is a common pathological process in many eye disorders. Oxidative stress and inflammation play a role in retinal I/R injury. Recent studies show that toll-like receptor 4 (TLR4) is involved in initiating sterile inflammatory response in retinal I/R. However, the molecular mechanism by which TLR4 is activated is not known. In this study, we show that retinal I/R injury involves a co-receptor of TLR4, myeloid differentiation 2 (MD2). Inhibition of MD2 prevented cell death and preserved retinal function following retinal I/R injury. We confirmed these findings using MD2 knockout mice. Furthermore, we utilized human retinal pigment epithelial cells (ARPE-19 cells) to show that oxidative stress-induced cell death as well as inflammatory response are mediated through MD2. Inhibition of MD2 through a chemical inhibitor or knockdown prevented oxidative stress-induced cell death and expression of inflammatory cytokines. Oxidative stress was found to activate TLR4 in a MD2-dependent manner via increasing the expression of high mobility group box 1. In summary, our study shows that oxidative stress in retinal I/R injury can activate TLR4 signaling via MD2, resulting in induction of inflammatory genes and retinal damage. MD2 may represent an attractive therapeutic target for retinal I/R injury.
Assuntos
Antígeno 96 de Linfócito/deficiência , Estresse Oxidativo/fisiologia , Traumatismo por Reperfusão/metabolismo , Doenças Retinianas/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Chalconas/farmacologia , Chalconas/uso terapêutico , Humanos , Antígeno 96 de Linfócito/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Distribuição Aleatória , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Doenças Retinianas/tratamento farmacológicoRESUMO
Inflammation and oxidative stress plays an important role in the development of obesity-related complications and cardiovascular disease. Benzimidazole and imidazopyridine compounds are a class of compounds with a variety of activities, including anti-inflammatory, antioxidant and anti-cancer. X22 is an imidazopyridine derivative we synthesized and evaluated previously for anti-inflammatory activity in lipopolysaccharide-stimulated macrophages. However, its ability to alleviate obesity-induced heart injury via its anti-inflammatory actions was unclear. This study was designed to evaluate the cardioprotective effects of X22 using cell culture studies and a high-fat diet rat model. We observed that palmitic acid treatment in cardiac-derived H9c2 cells induced a significant increase in reactive oxygen species, inflammation, apoptosis, fibrosis and hypertrophy. All of these changes were inhibited by treatment with X22. Furthermore, oral administration of X22 suppressed high-fat diet-induced oxidative stress, inflammation, apoptosis, hypertrophy and fibrosis in rat heart tissues and decreased serum lipid concentration. We also found that the anti-inflammatory and anti-oxidative actions of X22 were associated with Nrf2 activation and nuclear factor-kappaB (NF-κB) inhibition, respectively, both in vitro and in vivo. The results of this study indicate that X22 may be a promising cardioprotective agent and that Nrf2 and NF-κB may be important therapeutic targets for obesity-related complications.
Assuntos
Traumatismos Cardíacos/tratamento farmacológico , Traumatismos Cardíacos/prevenção & controle , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Inflamação/tratamento farmacológico , Obesidade/complicações , Estresse Oxidativo/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Dieta Hiperlipídica , Fibrose , Traumatismos Cardíacos/complicações , Traumatismos Cardíacos/patologia , Imidazóis/administração & dosagem , Imidazóis/química , Inflamação/complicações , Lipídeos/sangue , Masculino , Miocárdio/patologia , NF-kappa B/metabolismo , Obesidade/sangue , Obesidade/patologia , Ácido Palmítico , Piridinas/administração & dosagem , Piridinas/química , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Endotoxin-induced acute inflammatory diseases such as sepsis, mediated by excessive production of various proinflammatory cytokines, remain the leading cause of mortality in critically ill patients. Lipopolysaccharide (LPS), the characteristic endotoxin found in the outer membrane of Gram-negative bacteria, can induce the innate immunity system and through the myeloid differentiation protein 2 (MD2) and Toll-like receptor 4 (TLR4) complex, increase the production of inflammatory mediators. Our previous studies have found that a curcumin analog, L48H37 [1-ethyl-3,5-bis(3,4,5-trimethoxybenzylidene)piperidin-4-one], was able to inhibit LPS-induced inflammation, particularly tumor necrosis factor α and interleukin 6 production and gene expression in mouse macrophages. In this study, a series of biochemical experiments demonstrate L48H37 specifically targets MD2 and inhibits the interaction and signaling transduction of LPS-TLR4/MD2. L48H37 binds to the hydrophobic region of MD2 pocket and forms hydrogen bond interactions with Arg(90) and Tyr(102). Subsequently, L48H37 was shown to suppress LPS-induced mitogen-activated protein kinase phosphorylation and nuclear factor κB activation in macrophages; it also dose dependently inhibits the cytokine expression in macrophages and human peripheral blood mononuclear cells stimulated by LPS. In LPS-induced septic mice, both pretreatment and treatment with L48H37 significantly improved survival and protected lung injury. Taken together, this work identified a new MD2 specific inhibitor, L48H37, as a potential candidate in the treatment of sepsis.
Assuntos
Curcumina/análogos & derivados , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/antagonistas & inibidores , Sepse/tratamento farmacológico , Receptor 4 Toll-Like/efeitos dos fármacos , Animais , Curcumina/metabolismo , Curcumina/farmacologia , Citocinas/biossíntese , Diarileptanoides , Endotoxinas/toxicidade , Humanos , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Choque Séptico/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/efeitos dos fármacosRESUMO
Fibroblast growth factor 2 (FGF2) plays a critical role in tumorigenesis and progression of solid tumor and is upregulated in gastric carcinoma serum. Therefore, it is regarded as a potential therapeutic target of human gastric cancer. Suppression of bioactivities of FGF2 may contribute to human gastric cancer therapy. Herein, we obtained a novel FGF2-binding peptide derivative (named P32), which originated from a previously isolated P7 peptide with poor stability. We proved that P32, which had a half-life in human plasma up to 12 h, enhanced stability and exerted strong inhibitory effect on FGF2-induced cell proliferation and invasion in human gastric cancer cell lines. Further investigations revealed that the underlying anti-proliferation mechanisms of P32 in vitro included arresting FGF2-stimulated cells at the G0/G1 phase and reducing the activation of AKT and Erk1/2 cascades. The FGF2-binding peptide derivative P32 has improved stability, is relatively safe, and may have therapeutic potential in FGF2-driven gastric cancer.
Assuntos
Carcinogênese/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Peptídeos/metabolismo , Neoplasias Gástricas/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Invasividade Neoplásica/genética , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Ressonância de Plasmônio de SuperfícieRESUMO
Given that receptor tyrosine kinases (RTKs) have emerged as key regulators of all aspects of cancer development, including proliferation, invasion, angiogenesis and metastasis, the RTK family represents an important therapeutic target for anti-cancer drug development. Oxindole structure has been used in RTK inhibitors such as SU4984 and intedanib. In this study, two series of new heterocyclic compounds containing oxindole scaffold have been designed and synthesized, and their inhibitory activity against the proliferation of nine cancer cell lines has been evaluated. Among them, compounds 9a and 9b displayed the strongest anti-proliferative activity with the IC50s below 10µM. Flow cytometric analysis showed that the compounds 9a and 9b dose-dependently arrested the cell cycle at G0/G1 phase. Although the leading compounds SU4984 and intedanib targets FGFR1, the kinase activity test revealed that these compounds only showed slight inhibitory activity on FGFR1 kinase. Further enzymatic test aided by molecular docking simulation in the ATP-binding site demonstrated that 9a and 9b are potent inhibitors of c-Kit kinase. These compounds are worthy of further evaluation as anticancer agents.
Assuntos
Antineoplásicos/síntese química , Indóis/química , Inibidores de Proteínas Quinases/síntese química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Indóis/metabolismo , Simulação de Acoplamento Molecular , Oxindóis , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Pirróis/química , Pirróis/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , SunitinibeRESUMO
Asthma represents a significant global challenge that affects individuals across all age groups and imposes substantial social and economic burden. Due to heterogeneity of the disease, not all patients obtain benefit with current treatments. The objective of this study was to explore the impact of MD2 on the progression of asthma using L6H21, a novel MD2 inhibitor, to identify potential targets and drug candidates for asthma treatment. To establish an asthma-related murine model and evaluate the effects of L6H21, ovalbumin (OVA) was used to sensitize and challenge mice. Pathological changes were examined with various staining techniques, such as H&E staining, glycogen staining, and Masson staining. Inflammatory cell infiltration and excessive cytokine secretion were evaluated by analyzing BALF cell count, RT-PCR, and ELISA. The TLR4/MD2 complex formation, as well as the activation of the MAPK and NF-кB pathways, was examined using western blot and co-IP. Treatment with L6H21 demonstrated alleviation of increased airway resistance, lung tissue injury, inflammatory cell infiltration and excessive cytokine secretion triggered by OVA. In addition, it also ameliorated mucus production and collagen deposition. In the L6H21 treatment group, inhibition of MAPK and NF-кB activation was observed, along with the disruption of TLR4/MD2 complex formation, in contrast to the model group. Thus, L6H21 effectively reduced the formation of the MD2 and TLR4 complex induced by OVA in a dose-dependent manner. This reduction resulted in the attenuation of MAPKs/NF-κB activation, enhanced suppression of inflammatory factor secretion, reduced excessive recruitment of inflammatory cells, and ultimately mitigated airway damage. MD2 emerges as a crucial target for asthma treatment, and L6H21, as an MD2 inhibitor, shows promise as a potential drug candidate for the treatment of asthma.
Assuntos
Asma , Chalcona , Chalconas , Humanos , Camundongos , Animais , Chalcona/uso terapêutico , Ovalbumina/uso terapêutico , NF-kappa B/genética , NF-kappa B/metabolismo , Chalconas/farmacologia , Chalconas/uso terapêutico , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/uso terapêutico , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/patologia , Pulmão/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos BALB CRESUMO
Heart failure (HF) is one of the major causes of death among diabetic patients. Although studies have shown that curcumin analog C66 can remarkably relieve diabetes-associated cardiovascular and kidney complications, the role of SJ-12, SJ-12, a novel curcumin analog, in diabetic cardiomyopathy and its molecular targets are unknown. 7-week-old male C57BL/6 mice were intraperitoneally injected with single streptozotocin (STZ) (160 mg/kg) to develop diabetic cardiomyopathy (DCM). The diabetic mice were then treated with SJ-12 via gavage for two months. Body weight, fast blood glucose, cardiac utrasonography, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers were assessed. The potential target of SJ-12 was evaluated via RNA-sequencing analysis. The O-GlcNAcylation levels of SP1 were detected via immunoprecipitation. SJ-12 effectively suppressed myocardial hypertrophy and fibrosis, thereby preventing heart dysfunction in mice with STZ-induced heart failure. RNA-sequencing analysis revealed that SJ-12 exerted its therapeutic effects through the modulation of the calcium signaling pathway. Furthermore, SJ-12 reduced the O-GlcNAcylation levels of SP1 by inhibiting O-linked N-acetylglucosamine transferase (OGT). Also, SJ-12 stabilized Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a (SERCA2a), a crucial regulator of calcium homeostasis, thus reducing hypertrophy and fibrosis in mouse hearts and cultured cardiomyocytes. However, the anti-fibrotic effects of SJ-12 were not detected in SERCA2a or OGT-silenced cardiomyocytes, indicating that SJ-12 can prevent DCM by targeting OGT-dependent O-GlcNAcylation of SP1.These findings indicate that SJ-12 can exert cardioprotective effects in STZ-induced mice by reducing the O-GlcNAcylation levels of SP1, thus stabilizing SERCA2a and reducing myocardial fibrosis and hypertrophy. Therefore, SJ-12 can be used for the treatment of diabetic cardiomyopathy.
Assuntos
Diabetes Mellitus Experimental , Cardiomiopatias Diabéticas , Camundongos Endogâmicos C57BL , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/tratamento farmacológico , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Masculino , Camundongos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/tratamento farmacológico , Estreptozocina , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fibrose , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/genética , Sinalização do Cálcio/efeitos dos fármacosRESUMO
Obesity has shown a global epidemic trend. The high-lipid state caused by obesity can maintain the heart in a prolonged low-grade inflammatory state and cause ventricular remodeling, leading to a series of pathologies, such as hypertrophy, fibrosis, and apoptosis, which eventually develop into obese cardiomyopathy. Therefore, prolonged low-grade inflammation plays a crucial role in the progression of obese cardiomyopathy, making inflammation regulation an essential strategy for treating this disease. Cyy-272, an indazole derivative, is an anti-inflammatory compound independently synthesized by our laboratory. Our previous studies revealed that Cyy-272 can exert anti-inflammatory effects by inhibiting the phosphorylation and activation of C-Jun N-terminal kinase (JNK), thereby alleviating lipopolysaccharide (LPS)-induced acute lung injury (ALI). The current study aimed to evaluate the potential of Cyy-272 to mitigate the occurrence and progression of obese cardiomyopathy through the inhibition of the JNK signaling pathway. Our results indicate that the compound Cyy-272 has encouraging therapeutic effects on obesity-induced cardiac injury. It significantly inhibits inflammation in cardiomyocytes and heart tissues induced by high lipid concentrations, further alleviating the resulting hypertrophy, fibrosis, and apoptosis. Mechanistically, the protective effect of Cyy-272 on obese cardiomyopathy can be attributed to its direct inhibition of JNK protein phosphorylation. In conclusion, we identified a novel compound, Cyy-272, capable of alleviating obese cardiomyopathy and confirmed that its effect is achieved through direct inhibition of JNK.
Assuntos
Cardiomiopatias , Indazóis , Proteínas Quinases JNK Ativadas por Mitógeno , Obesidade , Animais , Obesidade/tratamento farmacológico , Obesidade/complicações , Cardiomiopatias/tratamento farmacológico , Indazóis/farmacologia , Indazóis/uso terapêutico , Indazóis/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Apoptose/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos , Fibrose , Anti-Inflamatórios/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/química , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacosRESUMO
In the title compound, C(33)H(25)F(2)NO(2), the acenaphthene ring system forms dihedral angles of 50.93â (14) and 36.89â (14)° with the benzene rings. The pyrrolidine and cyclo-penta-none rings adopt envelope (with the N atom as the flap) and twisted conformations, respectively. In the crystal, C-Hâ¯O and C-Hâ¯F inter-actions link the mol-ecules.
RESUMO
Liver fibrosis is characterised by the activation of hepatic stellate cells (HSCs) and matrix deposition. Accumulating evidence has revealed that the oncogenic protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) acts as a therapeutic target of fibrosis. Although several SHP2 inhibitors have reached early clinical trials, there are currently no FDA-approved drugs that target SHP2. In this study, we aimed to identify novel SHP2 inhibitors from an in-house natural product library to treat liver fibrosis. Out of the screened 800 compounds, a furanogermacrane sesquiterpene, linderalactone (LIN), significantly inhibited SHP2 dephosphorylation activity in vitro. Cross-validated enzymatic assays, bio-layer interferometry (BLI) assays, and site-directed mutagenesis were used to confirm that LIN directly binds to the catalytic PTP domain of SHP2. In vivo administration of LIN significantly ameliorated carbon tetrachloride (CCl4)-induced HSC activation and liver fibrosis by inhibiting the TGFß/Smad3 pathway. Thus, LIN or its derivatives could be considered potential therapeutic agents against SHP2-related diseases, such as liver fibrosis or NASH.
RESUMO
Artemether-lumefantrine is an artemisinin-based combination therapy for the treatment of malaria, which are primarily metabolized by cytochrome P450 3A4. Therapeutic difference caused by gene polymorphisms of CYP3A4 may lead to uncertain adverse side effects or treatment failure. The aim of this study was to evaluate the effect of CYP3A4 gene polymorphism on artemether-lumefantrine metabolism in vitro. Enzyme kinetics assay was performed using recombinant human CYP3A4 cell microsomes. The analytes, dihydroartimisinin and desbutyl-lumefantrine, were detected by ultra-performance liquid chromatography tandem mass spectrometry. The results demonstrated that compared to CYP3A4.1, the intrinsic clearance of CYP3A4.4, 5, 9, 16, 18, 23, 24, 28, 31-34 significantly reduced for artemether (58.5%-93.3%), and CYP3A4.17 almost loss catalytic activity. Simultaneously, CYP3A4.5, 14, 17, 24 for lumefantrine were decreased by 56.1%-99.6%, and CYP3A4.11, 15, 18, 19, 23, 28, 29, 31-34 for lumefantrine was increased by 51.7%-296%. The variation in clearance rate indicated by molecular docking could be attributed to the disparity in the binding affinity of artemether and lumefantrine with CYP3A4. The data presented here have enriched our understanding of the effect of CYP3A4 gene polymorphism on artemether-lumefantrine metabolizing. These findings serve as a valuable reference and provide insights for guiding the treatment strategy involving artemether-lumefantrine.
Assuntos
Antimaláricos , Malária Falciparum , Humanos , Antimaláricos/efeitos adversos , Artemeter/uso terapêutico , Citocromo P-450 CYP3A/genética , Simulação de Acoplamento Molecular , Combinação Arteméter e Lumefantrina/uso terapêutico , Lumefantrina/uso terapêutico , Fluorenos/efeitos adversos , Malária Falciparum/induzido quimicamente , Malária Falciparum/tratamento farmacológicoRESUMO
Acute lung injury (ALI) and sepsis, characterized by systemic inflammatory response syndrome, remain the major causes of death in severe patients. Inhibiting the release of proinflammatory cytokines is considered to be a promising method for the treatment of inflammation-related diseases. In this study, a total of 28 4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide derivatives were designed and synthesized and their anti-inflammatory activities in J774A.1 were evaluated. Among them, derivative 13a was found to significantly inhibit lipopolysaccharide (LPS)-induced expression of the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on J774A.1, THP-1 and LX-2 cells, and inhibited the activation of the NF-κB pathway. Furthermore, administration of 13ain vivo significantly improved the symptoms in LPS-induced ALI mice, including alleviation of pathological changes in the lung tissue, reduction of pulmonary edema, and inhibition of macrophage infiltration. Moreover, the administration of 13ain vivo significantly promoted survival in LPS-induced sepsis mice. 13a demonstrated favorable pharmacokinetic properties with T1/2 value of 11.8 h and F value of 36.3%. Therefore, this study has identified a novel 4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide derivative, 13a, which is an effective anti-inflammatory agent. The findings have laid a foundation for the further development of agents to treat ALI and sepsis.
Assuntos
Lesão Pulmonar Aguda , Sepse , Camundongos , Animais , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Pulmão/metabolismo , Anti-Inflamatórios/efeitos adversos , Citocinas/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sepse/induzido quimicamente , Sepse/tratamento farmacológico , Sepse/metabolismoRESUMO
BACKGROUND: NLR family pyrin domain-containing 3 (NLRP3)-mediated pyroptosis plays a key role in various acute and chronic inflammatory diseases. Targeted inhibition of NLRP3-mediated pyroptosis may be a potential therapeutic strategy for various inflammatory diseases. Ergolide (ERG) is a sesquiterpene lactone natural product derived from the traditional Chinese medicinal herb, Inula britannica. ERG has been shown to have anti-inflammatory and anti-cancer activities, but the target is remains unknown. HYPOTHESIS/PURPOSE: This study performed an in-depth investigation of the anti-inflammatory mechanism of ERG in NLRP3-mediated pyroptosis and NLPR3 inflammasome related sepsis and acute lung injury model. METHODS: ELISA and Western blot were used to determine the IL-1ß and P20 levels. Co-immunoprecipitation assays were used to detect the interaction between proteins. Drug affinity response target stability (DARTS) assays were used to explore the potential target of ERG. C57BL/6J mice were intraperitoneally injected with E. coli DH5α (2 × 109 CFU/mouse) to establish a sepsis model. Acute lung injury was induced by intratracheal administrationof lipopolysaccharide in wild-type mice and NLRP3 knockout mice with or without ERG treatment. RESULTS: We showed that ERG is an efficient inhibitor of NLRP3-mediated pyroptosis in the first and second signals of NLRP3 inflammasome activation. Furthermore, we demonstrated that ERG irreversibly bound to the NACHT domain of NLRP3 to prevent the assembly and activation of the NLRP3 inflammasome. ERG remarkably improved the survival rate of wild-type septic mice. In lipopolysaccharide-induced acute lung injury model, ERG alleviated acute lung injury of wild-type mice but not NLRP3 knockout mice. CONCLUSION: Our results revealed that the anti-pyroptosis effect of ERG are dependent on NLRP3 and NLRP3 NACHT domain is ERG's direct target. Therefore, ERG can serve as a precursor drug for the development of novel NLRP3 inhibitors to treat NLRP3 inflammasome mediated inflammatory diseases.