RESUMO
PURPOSE: To evaluate the safety and potential efficacy of LLF580, a genetically engineered variant of human fibroblast growth factor-21, for triglyceride lowering, weight loss, and hepatic fat reduction. METHODS: A multicenter, double-blind, parallel design trial in obese, mildly hypertriglyceridemic adults randomized (1:1) to LLF580 300 mg or placebo subcutaneously every 4 weeks for 3 doses. RESULTS: Of 64 randomized study participants, 61 (mean ± SD: age 45 ± 11 years, 49% male, 80/15/5% Caucasian/African American/other, body mass index 36.1 ± 3.8 kg/m2) received LLF580 (n = 30) or placebo (n = 31) at 7 research sites in the United States. LLF580 lowered serum triglycerides by 54% (least square mean placebo adjusted change from baseline), total cholesterol 7%, low-density lipoprotein cholesterol 12%, and increased high-density lipoprotein cholesterol 36% compared with placebo (all P < 0.001) over 12 weeks. Substantial reduction of liver fat of 52% over placebo (P < 0.001) was also demonstrated in the setting of improved liver function tests including alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, the composite enhanced liver fibrosis score, and N-terminal type III collagen propeptide (all P < 0.05). Insulin and C-peptide levels and insulin resistance by homeostatic model assessment for insulin resistance were all lower, and adiponectin higher with LLF580 treatment compared with placebo, whereas fasting glucose and glycated hemoglobin were unchanged. Reductions in biomarkers of bone formation without differences in markers of bone resorption were observed. LLF580 was generally safe and well tolerated, except for higher incidence of generally mild to moderate gastrointestinal adverse effects. CONCLUSIONS: In obese, mildly hypertriglyceridemic adults, LLF580 was generally safe and demonstrated beneficial effects on serum lipids, liver fat, and biomarkers of liver injury, suggesting it may be effective for treatment of select metabolic disorders including hypertriglyceridemia and nonalcoholic fatty liver disease. Assessments of longer term safety and efficacy are warranted. CLINICALTRIALS.GOV IDENTIFIER: NCT03466203.
Assuntos
Biomarcadores/sangue , Índice de Massa Corporal , Fígado Gorduroso/prevenção & controle , Fatores de Crescimento de Fibroblastos/administração & dosagem , Hipertrigliceridemia/terapia , Obesidade/fisiopatologia , Triglicerídeos/sangue , Adulto , Método Duplo-Cego , Feminino , Fatores de Crescimento de Fibroblastos/genética , Seguimentos , Humanos , Hipertrigliceridemia/genética , Hipertrigliceridemia/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Fibroblast growth factor (FGF) 21 is a member of the FGF family of proteins. The biological activity of FGF21 was first shown to induce insulin-independent glucose uptake in adipocytes through the GLUT1 transporter. Subsequently, it was shown to have effects on the liver to increase fatty acid oxidation. FGF21 treatment provides beneficial metabolic effects in both animal models and patients with obesity, type 2 diabetes mellitus (T2D) and/or fatty liver disease. In this paper, we revisited the original finding and found that insulin-independent glucose uptake in adipocytes is preserved in the presence of an insulin receptor antagonist. Using a 40-kDa PEGylated (PEG) and half-life extended form of FGF21 (FGF21-PEG), we extended these in vitro results to 2 different mouse models of diabetes. FGF21-PEG normalized plasma glucose in streptozotocin-treated mice, a model of type 1 diabetes (T1D), without restoring pancreatic ß-cell function. FGF21-PEG also normalized plasma glucose levels and improved glucose tolerance in mice chronically treated with an insulin competitive insulin receptor antagonist, a model of autoimmune/type-B insulin resistance. These data extend the pharmacological potential of FGF21 beyond the settings of T2D, fatty liver, and obesity.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Fatores de Crescimento de Fibroblastos/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Células HEK293 , Humanos , Hiperglicemia/sangue , Hiperglicemia/etiologia , Hiperglicemia/patologia , Hiperglicemia/prevenção & controle , Insulina/metabolismo , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/sangue , Obesidade/complicações , Obesidade/patologia , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/fisiologia , EstreptozocinaRESUMO
LDL cholesterol (LDL-C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low-density lipoprotein cholesterol receptor (LDL-R). LDL-R is targeted for lysosomal degradation by association with proprotein convertase subtilisin-kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor-sparing phenomena, including loss-of-function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate-determining steps for protein-ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild-type versus gain-of-function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins ("hydration sites"). We compared these results with binding kinetics data for PCSK9, full-length LDL-R ectodomain, and isolated EGF-A repeat. We propose that the fast k(on) and entropically driven thermodynamics observed for PCSK9-EGF-A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H-bonds from water to polar EGF-A groups). We further propose that the relatively fast k(off) observed for EGF-A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k(off) observed for EGF-A and LDL-R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex.
Assuntos
Simulação por Computador , Fator de Crescimento Epidérmico/química , Conformação Proteica , Serina Endopeptidases/química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Fator de Crescimento Epidérmico/genética , Humanos , Modelos Moleculares , Mutação , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores de LDL/química , Serina Endopeptidases/genética , Solventes/química , Relação Estrutura-Atividade , Termodinâmica , Água/químicaRESUMO
PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.
Assuntos
Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Linhagem Celular , Humanos , Mutação/genética , Serina/genética , Serina/metabolismo , Serina Endopeptidases/genéticaRESUMO
The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.
Assuntos
Anti-Hipertensivos/farmacologia , Hipertensão/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Regulação Alostérica , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacocinética , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Simulação de Acoplamento Molecular , Piperazinas/síntese química , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacocinética , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Descoberta de Drogas , Células HEK293 , Células HT29 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
The melanocortin 4 receptor (MC4R) plays an important role in body weight regulation and energy homeostasis. Administration of peptidic MC4R antagonists (usually by intracerebro ventricular injection) has been shown in the literature to increase body weight and/or food intake in several rodent models. We report here the identification of a novel nonpeptidic MC4R antagonist and its effects on tumor-induced weight loss in mice following peripheral administration.
Assuntos
Benzamidinas/síntese química , Emaciação/tratamento farmacológico , Imidazóis/síntese química , Neoplasias/complicações , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Administração Cutânea , Animais , Benzamidinas/química , Benzamidinas/farmacologia , Emaciação/etiologia , Imidazóis/química , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Ensaio Radioligante , Relação Estrutura-Atividade , Transplante HeterólogoAssuntos
Proteínas Ativadoras de GTPase/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Oligopeptídeos/química , Proteínas RGS/química , Proteínas Ativadoras de GTPase/química , Guanosina Difosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Cinética , Subunidades Proteicas , Proteínas RGS/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoAssuntos
Acne Vulgar/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Piperazinas/farmacologia , Piridazinas/farmacologia , Glândulas Sebáceas/efeitos dos fármacos , Pele/efeitos dos fármacos , Estearoil-CoA Dessaturase/antagonistas & inibidores , Acne Vulgar/enzimologia , Acne Vulgar/patologia , Animais , Atrofia , Inibidores Enzimáticos/química , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos , Piperazinas/química , Piridazinas/química , Glândulas Sebáceas/enzimologia , Glândulas Sebáceas/patologia , Pele/patologia , Estearoil-CoA Dessaturase/metabolismoRESUMO
The identification and optimization of a series of acylguanidine-based melanocortin-4 receptor antagonists is discussed.
Assuntos
Guanidina/química , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Compostos de Sulfidrila/química , Animais , Sítios de Ligação , Encéfalo/metabolismo , Guanidina/análogos & derivados , Guanidina/farmacologia , Concentração Inibidora 50 , Camundongos , Plasma/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/farmacologia , Fatores de TempoRESUMO
Chemokine-induced T lymphocyte recruitment to the lung is critical for allergic inflammation, but chemokine signaling pathways are incompletely understood. Regulator of G protein signaling (RGS)16, a GTPase accelerator (GTPase-activating protein) for Galpha subunits, attenuates signaling by chemokine receptors in T lymphocytes, suggesting a role in the regulation of lymphocyte trafficking. To explore the role of RGS16 in T lymphocyte-dependent immune responses in a whole-organism model, we generated transgenic (Tg) mice expressing RGS16 in CD4(+) and CD8(+) cells. rgs16 Tg T lymphocytes migrated to CC chemokine ligand 21 or CC chemokine ligand 12 injection sites in the peritoneum, but not to CXC chemokine ligand 12. In a Th2-dependent model of allergic pulmonary inflammation, CD4(+) lymphocytes bearing CCR3, CCR5, and CXCR4 trafficked in reduced numbers to the lung after acute inhalation challenge with allergen (OVA). In contrast, spleens of sensitized and challenged Tg mice contained increased numbers of CD4(+)CCR3(+) cells producing more Th2-type cytokines (IL-4, IL-5, and IL-13), which were associated with increased airway hyperreactivity. Migration of Tg lymphocytes to the lung parenchyma after adoptive transfer was significantly reduced compared with wild-type lymphocytes. Naive lymphocytes displayed normal CCR3 and CXCR4 expression and cytokine responses, and compartmentation in secondary lymphoid organs was normal without allergen challenge. These results suggest that RGS16 may regulate T lymphocyte activation in response to inflammatory stimuli and migration induced by CXCR4, CCR3, and CCR5, but not CCR2 or CCR7.
Assuntos
Quimiotaxia de Leucócito/imunologia , Inflamação/imunologia , Pulmão/patologia , Ativação Linfocitária/imunologia , Proteínas/fisiologia , Proteínas RGS/fisiologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Doença Aguda , Transferência Adotiva , Alérgenos/administração & dosagem , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Quimiotaxia de Leucócito/genética , Cruzamentos Genéticos , Citocinas/biossíntese , Citocinas/metabolismo , Feminino , Homeostase/genética , Homeostase/imunologia , Humanos , Imunização , Inflamação/genética , Inflamação/patologia , Pulmão/imunologia , Ativação Linfocitária/genética , Tecido Linfoide/crescimento & desenvolvimento , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas RGS/biossíntese , Proteínas RGS/genética , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/fisiologia , Transdução de Sinais/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Notch signaling plays a critical role in cell fate determination in many developmental systems, including the hematopoietic system. We and others have recently cloned a novel Notch ligand called Delta4. In this study, we show the effect of retrovirus-mediated ectopic expression of Delta4 in hematopoietic cells. Lethally irradiated mice transplanted with bone marrow cells expressing Delta4 initially suffered from leukopenia and thrombocytopenia. Although all lineages were affected, the deficit in B cells and platelets was the most durable and profound. A rapid expansion of CD4(+)CD8(+) cells occurred shortly after transplantation. CD4(+)CD8(+) cells progressively invaded all tissues analyzed except the thymus, which surprisingly was atrophic. CD4(+)CD8(+) cells were mainly non-Delta4-transduced cells, strongly suggesting that the disease was not cell autonomous. Around 15 weeks after transplantation, mice died from this severe lymphoproliferative disorder, which was not transplantable in late-stage disease into secondary recipients. Mice transduced with a soluble form of Delta4 behaved like control mice. Characterization of early hematopoietic development revealed that Delta4 expression impaired formation of day-12 spleen colony-forming units (CFU-Ss) and, to a greater extent, pre-CFU-Ss. No effect was observed on myeloid colony-forming cells (CFU-Cs), indicating that Delta4 specifically acted on the earliest hematopoietic stem cell compartment. These results show that constitutive expression of Delta4 in hematopoietic cells impairs the development of B cells, platelets, and early stem cells and induces a lethal lymphoproliferative disease.
Assuntos
Proteínas Sanguíneas/farmacocinética , Substâncias de Crescimento/farmacocinética , Hematopoese/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Transtornos Linfoproliferativos/etiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/efeitos dos fármacos , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/farmacologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Proteínas de Ligação ao Cálcio , Vetores Genéticos , Substâncias de Crescimento/genética , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ligantes , Transtornos Linfoproliferativos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Receptores Notch , Retroviridae/genética , Linfócitos T/efeitos dos fármacos , Distribuição Tecidual , Transdução GenéticaRESUMO
A novel series of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R) is reported. Members of this series have been identified, which exhibit sub-micromolar binding affinity for the MC4-R, functional potency <100nM, and good oral exposure in rat. Antagonists of the MC4-R are potentially useful in the therapeutic treatment of involuntary weight loss due to advanced age or disease (e.g. cancer or AIDS), an area of large, unmet medical need.