RESUMO
Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many Food and Drug Administration-approved drugs. To identify new targets for fibrosis, we used a synthetic GPCR system named designed receptors exclusively activated by designer drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq- and Gs-DREADDs abrogated the induction of TGFß-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFß To investigate endogenous GPCR modulating TGFß signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFß signaling. Among the agonists examined, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that the DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof of concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. SIGNIFICANCE STATEMENT: Fibrosis is the hallmark of many end-stage cardiometabolic diseases, and there is an unmet medical need to discover new antifibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes designed receptors exclusively activated by designer drug chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.
Assuntos
Drogas Desenhadas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Prostaglandina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Metabolic dysregulation and mitochondrial dysfunction are important features of acute and chronic tissue injury across species, and human genetics and preclinical data suggest that the master metabolic regulator 5'-adenosine monophosphate-activated protein kinase (AMPK) may be an effective therapeutic target for chronic kidney disease (CKD). We have recently disclosed a pan-AMPK activator, MK-8722, that was shown to have beneficial effects in preclinical models. In this study we investigated the effects of MK-8722 in a progressive rat model of diabetic nephropathy to determine whether activation of AMPK would be of therapeutic benefit. We found that MK-8722 administration in a therapeutic paradigm is profoundly renoprotective, as demonstrated by a reduction in proteinuria (63% decrease in MK-8722 10 mg/kg per day compared with vehicle group) and a significant improvement in glomerular filtration rate (779 and 430 µl/min per gram kidney weight in MK-8722 10 mg/kg per day and vehicle group, respectively), as well as improvements in kidney fibrosis. We provide evidence that the therapeutic effects of MK-8722 may be mediated by modulation of renal mitochondrial quality control as well by attenuating fibrotic and lipotoxic mechanisms in kidney cells. MK-8722 (10 mg/kg per day compared with vehicle group) achieved modest blood pressure reduction (10 mmHg lower for mean blood pressure) and significant metabolic improvements (decreased plasma glucose, triglyceride, and body weight) that could contribute to renoprotection. These data further validate the concept that targeting metabolic dysregulation in CKD could be a potential therapeutic approach. SIGNIFICANCE STATEMENT: We demonstrate in the present study that the pharmacological activation of AMPK using a small-molecule agent provided renoprotection and improved systemic and cellular metabolism. We further indicate that modulation of renal mitochondrial quality control probably contributed to renoprotection and was distinct from the effects of enalapril. Our findings suggest that improving renal mitochondrial biogenesis and function and attenuating fibrosis and lipotoxicity by targeting key metabolic nodes could be a potential therapeutic approach in management of CKD that could complement the current standard of care.
Assuntos
Nefropatias Diabéticas/metabolismo , Hipoglicemiantes/uso terapêutico , Imidazóis/uso terapêutico , Proteínas Quinases/metabolismo , Piridinas/uso terapêutico , Quinases Proteína-Quinases Ativadas por AMP , Idoso , Animais , Benzimidazóis , Glicemia/metabolismo , Pressão Sanguínea , Células Cultivadas , Nefropatias Diabéticas/tratamento farmacológico , Feminino , Taxa de Filtração Glomerular , Humanos , Hipoglicemiantes/farmacologia , Imidazóis/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Zucker , Triglicerídeos/sangueRESUMO
The parallel medicinal chemistry (PMC) was effectively applied to accelerate the optimization of diacylglycerol O-acyltransferase I (DGAT-1) inhibitors. Through a highly collaborative and iterative library design, synthesis and testing, a benzimidazole lead was rapidly and systematically advanced to a highly potent, selective and bioavailable DGAT1 inhibitor with the potential for further development.
Assuntos
Benzimidazóis/farmacologia , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/química , Química Farmacêutica , Diacilglicerol O-Aciltransferase/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Previously disclosed benzimidazole-based DGAT1 inhibitors containing a cyclohexane carboxylic acid moiety suffer from isomerization at the alpha position of the carboxylic acid group, generating active metabolites which exhibit DGAT1 inhibition comparable to the corresponding parent compounds. In this report, we describe the design, synthesis and profiling of benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety. Our results show that single isomer 3A maintains in vitro and in vivo inhibition against DGAT1. In contrast to previous lead compounds, 3A does not undergo isomerization during in vitro hepatocyte incubation study or in vivo mouse study.
Assuntos
Benzimidazóis/química , Ácidos Carboxílicos/química , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Animais , Benzimidazóis/metabolismo , Ácidos Carboxílicos/metabolismo , Cromatografia Líquida de Alta Pressão , Cicloexanonas/química , Diacilglicerol O-Aciltransferase/metabolismo , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Hepatócitos/química , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Isomerismo , Espectrometria de Massas , Camundongos , RatosRESUMO
Purpose To evaluate the biodistribution, metabolism, and pharmacokinetics of a new type I collagen-targeted magnetic resonance (MR) probe, CM-101, and to assess its ability to help quantify liver fibrosis in animal models. Materials and Methods Biodistribution, pharmacokinetics, and stability of CM-101 in rats were measured with mass spectrometry. Bile duct-ligated (BDL) and sham-treated rats were imaged 19 days after the procedure by using a 1.5-T clinical MR imaging unit. Mice were treated with carbon tetrachloride (CCl4) or with vehicle two times a week for 10 weeks and were imaged with a 7.0-T preclinical MR imaging unit at baseline and 1 week after the last CCl4 treatment. Animals were imaged before and after injection of 10 µmol/kg CM-101. Change in contrast-to-noise ratio (ΔCNR) between liver and muscle tissue after CM-101 injection was used to quantify liver fibrosis. Liver tissue was analyzed for Sirius Red staining and hydroxyproline content. The institutional subcommittee for research animal care approved all in vivo procedures. Results CM-101 demonstrated rapid blood clearance (half-life = 6.8 minutes ± 2.4) and predominately renal elimination in rats. Biodistribution showed low tissue gadolinium levels at 24 hours (<3.9% injected dose [ID]/g ± 0.6) and 10-fold lower levels at 14 days (<0.33% ID/g ± 12) after CM-101 injection with negligible accumulation in bone (0.07% ID/g ± 0.02 and 0.010% ID/g ± 0.004 at 1 and 14 days, respectively). ΔCNR was significantly (P < .001) higher in BDL rats (13.6 ± 3.2) than in sham-treated rats (5.7 ± 4.2) and in the CCl4-treated mice (18.3 ± 6.5) compared with baseline values (5.2 ± 1.0). Conclusion CM-101 demonstrated fast blood clearance and whole-body elimination, negligible accumulation of gadolinium in bone or tissue, and robust detection of fibrosis in rat BDL and mouse CCl4 models of liver fibrosis. © RSNA, 2017 Online supplemental material is available for this article.
Assuntos
Fibrose/patologia , Gadolínio/farmacocinética , Cirrose Hepática/diagnóstico por imagem , Fígado/patologia , Imageamento por Ressonância Magnética , Polissacarídeos Bacterianos/farmacocinética , Animais , Tetracloreto de Carbono/farmacocinética , Modelos Animais de Doenças , Fibrose/diagnóstico por imagem , Meia-Vida , Fígado/diagnóstico por imagem , Espectrometria de Massas , Camundongos , Ratos , Distribuição TecidualRESUMO
Acyl-CoA:diacylglycerol acyltransferase (DGAT)1 and DGAT2 catalyze triglyceride (TG) biosynthesis in humans. Biallelic loss-of-function mutations in human DGAT1 result in severe congenital diarrhea and protein-losing enteropathy. Additionally, pharmacologic inhibition of DGAT1 led to dose-related diarrhea in human clinical trials. Here we identify a previously unknown DGAT1 mutation in identical twins of South Asian descent. These male patients developed watery diarrhea shortly after birth, with protein-losing enteropathy and failure to thrive. Exome sequencing revealed a homozygous recessive mutation in DGAT1, c.314T>C, p.L105P. We show here that the p.L105P DGAT1 enzyme produced from the mutant allele is less abundant, resulting in partial loss of TG synthesis activity and decreased formation of lipid droplets in patient-derived primary dermal fibroblasts. Thus, in contrast with complete loss-of-function alleles of DGAT1, the p.L105P missense allele partially reduces TG synthesis activity and causes a less severe clinical phenotype. Our findings add to the growing recognition of DGAT1 deficiency as a cause of congenital diarrhea with protein-losing enteropathy and indicate that DGAT1 mutations result in a spectrum of diseases.
Assuntos
Diacilglicerol O-Aciltransferase/genética , Diarreia/congênito , Diarreia/genética , Mutação de Sentido Incorreto , Alelos , Linhagem Celular Tumoral , Pré-Escolar , Diarreia/enzimologia , Feminino , Homozigoto , Humanos , Mutação com Perda de Função , Masculino , GravidezRESUMO
SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipídeos/sangue , Proteínas de Membrana/genética , Pró-Proteína Convertase 9/genética , RNA Interferente Pequeno/genética , Receptores de LDL/genética , Animais , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hipolipemiantes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Fígado/enzimologia , Macaca mulatta , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais , Sinvastatina/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Lipase/metabolismo , Gotículas Lipídicas/enzimologia , Proteínas do Core Viral/fisiologia , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Hidrólise , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Triglicerídeos/metabolismoRESUMO
Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Animais , Carboxipeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Humanos , Camundongos , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Identifying variations in DNA that increase susceptibility to disease is one of the primary aims of genetic studies using a forward genetics approach. However, identification of disease-susceptibility genes by means of such studies provides limited functional information on how genes lead to disease. In fact, in most cases there is an absence of functional information altogether, preventing a definitive identification of the susceptibility gene or genes. Here we develop an alternative to the classic forward genetics approach for dissecting complex disease traits where, instead of identifying susceptibility genes directly affected by variations in DNA, we identify gene networks that are perturbed by susceptibility loci and that in turn lead to disease. Application of this method to liver and adipose gene expression data generated from a segregating mouse population results in the identification of a macrophage-enriched network supported as having a causal relationship with disease traits associated with metabolic syndrome. Three genes in this network, lipoprotein lipase (Lpl), lactamase beta (Lactb) and protein phosphatase 1-like (Ppm1l), are validated as previously unknown obesity genes, strengthening the association between this network and metabolic disease traits. Our analysis provides direct experimental support that complex traits such as obesity are emergent properties of molecular networks that are modulated by complex genetic loci and environmental factors.
Assuntos
Redes Reguladoras de Genes/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Síndrome Metabólica/genética , Obesidade/genética , Tecido Adiposo/metabolismo , Animais , Apolipoproteína A-II/genética , Cromossomos de Mamíferos/genética , Feminino , Desequilíbrio de Ligação , Lipase Lipoproteica/genética , Fígado/metabolismo , Escore Lod , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Síndrome Metabólica/enzimologia , Síndrome Metabólica/metabolismo , Camundongos , Obesidade/enzimologia , Obesidade/metabolismo , Fenótipo , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Locos de Características Quantitativas , Reprodutibilidade dos Testes , Proteínas Ribossômicas/genéticaRESUMO
Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Lipogênese , Fígado/metabolismo , Receptores de Glucagon/genética , Animais , Glicemia , Colesterol/sangue , Diabetes Mellitus Tipo 2/terapia , Expressão Gênica , Técnicas de Silenciamento de Genes , Lipoproteínas LDL/sangue , Masculino , Camundongos , Camundongos Obesos , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Glucagon/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Cicloexanos/química , Cicloexanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Animais , Cicloexanos/farmacocinética , Descoberta de Drogas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
The autonomic nervous system regulates fuel availability and energy storage in the liver, adipose tissue, and other organs; however, the molecular components of this neural circuit are poorly understood. We sought to identify neural populations that project from the CNS indirectly through multisynaptic pathways to liver and epididymal white fat in mice using pseudorabies virus strains expressing different reporters together with BAC transgenesis and immunohistochemistry. Neurons common to both circuits were identified in subpopulations of the paraventricular nucleus of the hypothalamus (PVH) by double labeling with markers expressed in viruses injected in both sites. The lateral hypothalamus and arcuate nucleus of the hypothalamus and brainstem regions (nucleus of the solitary tract and A5 region) also project to both tissues but are labeled at later times. Connections from these same sites to the PVH were evident after direct injection of virus into the PVH, suggesting that these regions lie upstream of the PVH in a common pathway to liver and adipose tissue (two metabolically active organs). These common populations of brainstem and hypothalamic neurons express neuropeptide Y and proopiomelanocortin in the arcuate nucleus, melanin-concentrating hormone, and orexin in the lateral hypothalamus and in the corticotrophin-releasing hormone and oxytocin in the PVH. The delineation of this circuitry will facilitate a functional analysis of the possible role of these potential command-like neurons to modulate autonomic outflow and coordinate metabolic responses in liver and adipose tissue.
Assuntos
Tecido Adiposo/patologia , Hipotálamo/patologia , Fígado/patologia , Neurônios/metabolismo , Animais , Tronco Encefálico/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/metabolismo , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Masculino , Melaninas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neuropeptídeo Y/metabolismo , Neuropeptídeos/metabolismo , Orexinas , Hormônios Hipofisários/metabolismo , Pró-Opiomelanocortina/metabolismoRESUMO
Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC(50) values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The compounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of compound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.
Assuntos
Benzimidazóis/farmacologia , Encéfalo/metabolismo , Carboxipeptidases/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Amidas/química , Animais , Transporte Biológico , Peso Corporal , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Obesidade/tratamento farmacológico , Pirrolidinas/química , Fatores de TempoRESUMO
Efforts to modify the central proline portion of lead compound 4 lead to the discovery of novel prolylcarboxypeptidase (PrCP) inhibitors. Especially, replacement with alanine afforded compound 19 displaying more potent human and mouse PrCP inhibitory activity than 4 and an overall comparable profile.
Assuntos
Alanina/química , Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Alanina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Furanos/química , Furanos/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Estabilidade de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Furanos/síntese química , Humanos , Camundongos , Camundongos Obesos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos , Animais , Butanóis/síntese química , Butanóis/química , Butanóis/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Pirrolidinas/síntese química , Pirrolidinas/química , Pirrolidinas/farmacologiaRESUMO
A series of potent inhibitors of prolylcarboxypeptidase (PrCP) was developed by modifying a lead structure that was discovered by high-throughput screening. The tert-butyl pyrrolidine was replaced by an aminocyclopentane to reduce the metabolic liabilities of the original lead. The compounds demonstrated sub-nanomolar in vitro IC(50) values, minimal activity shifts in pure plasma and improved pharmacokinetics. Complete ex vivo plasma target engagement was achieved with low brain exposure at the 20 h time point following p.o. dosing in a mouse. The results indicate that the aminocyclopentanes are useful tools for studying the therapeutic potential of peripheral (non-CNS) PrCP inhibition.
Assuntos
Aminas/farmacologia , Carboxipeptidases/antagonistas & inibidores , Ciclopentanos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos , Aminas/síntese química , Aminas/química , Animais , Ciclização , Ciclopentanos/síntese química , Ciclopentanos/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológicoRESUMO
Efforts were dedicated to develop potent and brain penetrant prolylcarboxypeptidase (PrCP) inhibitors by replacing the amide group of original leads 1 and 2 with heterocycles. Aminopyrimidines including compound 32a were identified to display good PrCP inhibitory activity (32a, IC(50)=43 nM) and impressive ability to penetrate brain in mice (brain/plasma ratio: 1.4).
Assuntos
Aminas/síntese química , Encéfalo/efeitos dos fármacos , Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacologia , Amidas/química , Aminas/química , Aminas/farmacologia , Animais , Encéfalo/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/química , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 µl of plasma.