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1.
J Med Chem ; 65(15): 10318-10340, 2022 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-35878399

RESUMO

Activation of PKG1α is a compelling strategy for the treatment of cardiovascular diseases. As the main effector of cyclic guanosine monophosphate (cGMP), activation of PKG1α induces smooth muscle relaxation in blood vessels, lowers pulmonary blood pressure, prevents platelet aggregation, and protects against cardiac stress. The development of activators has been mostly limited to cGMP mimetics and synthetic peptides. Described herein is the optimization of a piperidine series of small molecules to yield activators that demonstrate in vitro phosphorylation of vasodilator-stimulated phosphoprotein as well as antiproliferative effects in human pulmonary arterial smooth muscle cells. Hydrogen/deuterium exchange mass spectrometry experiments with the small molecule activators revealed a mechanism of action consistent with cGMP-induced activation, and an X-ray co-crystal structure with a construct encompassing the regulatory domains illustrated a binding mode in an allosteric pocket proximal to the low-affinity cyclic nucleotide-binding domain.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I , GMP Cíclico , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Humanos , Miócitos de Músculo Liso , Fosforilação , Processamento de Proteína Pós-Traducional
3.
Diabetes ; 67(2): 299-308, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097375

RESUMO

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Desenho de Fármacos , Hipoglicemiantes/uso terapêutico , Insulina Regular Humana/análogos & derivados , Lectinas Tipo C/agonistas , Lectinas de Ligação a Manose/agonistas , Receptor de Insulina/agonistas , Receptores de Superfície Celular/agonistas , Animais , Animais Endogâmicos , Ligação Competitiva , Células CHO , Cricetulus , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Hiperglicemia/prevenção & controle , Hipoglicemia/induzido quimicamente , Hipoglicemia/prevenção & controle , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacocinética , Insulina Regular Humana/efeitos adversos , Insulina Regular Humana/farmacocinética , Insulina Regular Humana/uso terapêutico , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligantes , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Taxa de Depuração Metabólica , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Suínos , Porco Miniatura
4.
PLoS One ; 12(10): e0186033, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29053717

RESUMO

GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.


Assuntos
Glucose/metabolismo , Incretinas/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animais , Células CHO , Linhagem Celular , Cricetulus , Glucagon/metabolismo , Teste de Tolerância a Glucose , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Ratos
5.
Science ; 357(6350): 507-511, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28705990

RESUMO

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cardiomegalia/induzido quimicamente , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Imidazóis/farmacologia , Piridinas/farmacologia , Animais , Benzimidazóis , Glicemia/efeitos dos fármacos , Jejum , Glicogênio/metabolismo , Hipoglicemia/induzido quimicamente , Imidazóis/efeitos adversos , Imidazóis/química , Insulina/farmacologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Piridinas/efeitos adversos , Piridinas/química
6.
Am J Physiol Endocrinol Metab ; 313(1): E37-E47, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28292762

RESUMO

G protein-coupled receptor 40 (GPR40) partial agonists lower glucose through the potentiation of glucose-stimulated insulin secretion, which is believed to provide significant glucose lowering without the weight gain or hypoglycemic risk associated with exogenous insulin or glucose-independent insulin secretagogues. The class of small-molecule GPR40 modulators, known as AgoPAMs (agonist also capable of acting as positive allosteric modulators), differentiate from partial agonists, binding to a distinct site and functioning as full agonists to stimulate the secretion of both insulin and glucagon-like peptide-1 (GLP-1). Here we show that GPR40 AgoPAMs significantly increase active GLP-1 levels and reduce acute and chronic food intake and body weight in diet-induced obese (DIO) mice. These effects of AgoPAM treatment on food intake are novel and required both GPR40 and GLP-1 receptor signaling pathways, as demonstrated in GPR40 and GLP-1 receptor-null mice. Furthermore, weight loss associated with GPR40 AgoPAMs was accompanied by a significant reduction in gastric motility in these DIO mice. Chronic treatment with a GPR40 AgoPAM, in combination with a dipeptidyl peptidase IV inhibitor, synergistically decreased food intake and body weight in the mouse. The effect of GPR40 AgoPAMs on GLP-1 secretion was recapitulated in lean, healthy rhesus macaque demonstrating that the putative mechanism mediating weight loss translates to higher species. Together, our data indicate effects of AgoPAMs that go beyond glucose lowering previously observed with GPR40 partial agonist treatment with additional potential for weight loss.


Assuntos
Regulação do Apetite/genética , Peso Corporal/genética , Ingestão de Alimentos/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Redução de Peso/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética
7.
ACS Med Chem Lett ; 8(1): 49-54, 2017 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-28105274

RESUMO

Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effective insulin sensitizers, absent of weight gain, has been a long-standing goal of diabetes research. G-protein coupled receptor 120 (GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic agonists have been associated with insulin sensitizing, anti-inflammatory, and fat metabolism effects. Herein we describe the design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance (HOMA-IR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp in diet-induced obese (DIO) mice.

8.
J Pharmacol Exp Ther ; 360(3): 466-475, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28035006

RESUMO

Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin-4 (human FXIIa inhibition constant = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against Factor Xa (FXa) and plasmin. rHA-Mut-inf binds FXIIa, but not FXII zymogen, and competitively inhibits FXIIa protease activity. Its mode of action is hence akin to typical small-molecule inhibitors. Plasma shift and aPTT studies with rHA-Mut-inf demonstrated that calculated enzyme occupancy for FXIIa in achieving a putative aPTT doubling target in human, nonhuman primate, and rabbit is more than 99.0%. The effects of rHA-Mut-inf in carotid arterial thrombosis and microembolic signal (MES) in middle cerebral artery were assessed simultaneously in rabbits. Dose-dependent inhibition was observed for both arterial thrombosis and MES. The ED50 of thrombus formation was 0.17 mg/kg i.v. rHA-Mut-inf for the integrated blood flow and 0.16 mg/kg for thrombus weight; the ED50 for MES was 0.06 mg/kg. Ex vivo aPTT tracked with efficacy. In summary, our findings demonstrated that very high enzyme occupancy will be required for FXIIa active site inhibitors, highlighting the high potency and exquisite selectivity necessary for achieving efficacy in humans. Our MES studies suggest that targeting FXIIa may offer a promising strategy for stroke prevention associated with thromboembolic events.


Assuntos
Coagulação Sanguínea , Fator XIIa/antagonistas & inibidores , Proteínas de Insetos/farmacologia , Embolia Intracraniana , Trombose Intracraniana , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/farmacologia , Animais , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Fibrinolíticos/farmacologia , Embolia Intracraniana/sangue , Embolia Intracraniana/tratamento farmacológico , Trombose Intracraniana/sangue , Trombose Intracraniana/tratamento farmacológico , Modelos Animais , Coelhos , Albumina Sérica Humana
9.
J Nucl Med ; 57(10): 1599-1606, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27230925

RESUMO

A PET tracer is desired to help guide the discovery and development of disease-modifying therapeutics for neurodegenerative diseases characterized by neurofibrillary tangles (NFTs), the predominant tau pathology in Alzheimer disease (AD). We describe the preclinical characterization of the NFT PET tracer 18F-MK-6240. METHODS: In vitro binding studies were conducted with 3H-MK-6240 in tissue slices and homogenates from cognitively normal and AD human brain donors to evaluate tracer affinity and selectivity for NFTs. Immunohistochemistry for phosphorylated tau was performed on human brain slices for comparison with 3H-MK-6240 binding patterns on adjacent brain slices. PET studies were performed with 18F-MK-6240 in monkeys to evaluate tracer kinetics and distribution in the brain. 18F-MK-6240 monkey PET studies were conducted after dosing with unlabeled MK-6240 to evaluate tracer binding selectivity in vivo. RESULTS: The 3H-MK-6240 binding pattern was consistent with the distribution of phosphorylated tau in human AD brain slices. 3H-MK-6240 bound with high affinity to human AD brain cortex homogenates containing abundant NFTs but bound poorly to amyloid plaque-rich, NFT-poor AD brain homogenates. 3H-MK-6240 showed no displaceable binding in the subcortical regions of human AD brain slices and in the hippocampus/entorhinal cortex of non-AD human brain homogenates. In monkey PET studies, 18F-MK-6240 displayed rapid and homogeneous distribution in the brain. The 18F-MK-6240 volume of distribution stabilized rapidly, indicating favorable tracer kinetics. No displaceable binding was observed in self-block studies in rhesus monkeys, which do not natively express NFTs. Moderate defluorination was observed as skull uptake. CONCLUSION: 18F-MK-6240 is a promising PET tracer for the in vivo quantification of NFTs in AD patients.


Assuntos
Isoquinolinas/química , Emaranhados Neurofibrilares , Tomografia por Emissão de Pósitrons/métodos , Animais , Autorradiografia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Isoquinolinas/metabolismo , Macaca mulatta , Masculino , Traçadores Radioativos , Radioquímica
10.
J Med Chem ; 59(10): 4778-89, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27088900

RESUMO

Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the pathologic hallmark of several neurodegenerative diseases including Alzheimer's disease. In vivo detection of NFTs using PET imaging represents a unique opportunity to develop a pharmacodynamic tool to accelerate the discovery of new disease modifying therapeutics targeting tau pathology. Herein, we present the discovery of 6-(fluoro-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine, 6 ([(18)F]-MK-6240), as a novel PET tracer for detecting NFTs. 6 exhibits high specificity and selectivity for binding to NFTs, with suitable physicochemical properties and in vivo pharmacokinetics.


Assuntos
Descoberta de Drogas , Isoquinolinas/química , Imagem Molecular , Emaranhados Neurofibrilares/patologia , Tomografia por Emissão de Pósitrons , Radioisótopos de Flúor/química , Humanos , Isoquinolinas/síntese química , Isoquinolinas/farmacocinética , Estrutura Molecular , Emaranhados Neurofibrilares/metabolismo
11.
Bioorg Med Chem Lett ; 24(3): 780-5, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24433862

RESUMO

ß-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C ß-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C ß-lactamases in vitro. It effectively restored imipenem's activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.


Assuntos
Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Cilastatina/química , Descoberta de Drogas , Inibidores Enzimáticos/química , Imipenem/química , Inibidores de beta-Lactamases , Cilastatina/farmacologia , Combinação Imipenem e Cilastatina , Cristalografia por Raios X , Combinação de Medicamentos , Farmacorresistência Bacteriana/efeitos dos fármacos , Imipenem/farmacologia , Concentração Inibidora 50 , Klebsiella/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Biológicos , Pseudomonas/efeitos dos fármacos , Relação Estrutura-Atividade
12.
Antimicrob Agents Chemother ; 56(9): 4662-70, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22710113

RESUMO

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Bifenilo/farmacologia , Depsipeptídeos/farmacologia , Glicopeptídeos/farmacologia , Glicosídeos/farmacologia , Lipopeptídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oligopeptídeos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , beta-Lactamas/farmacologia , Animais , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Compostos de Bifenilo/síntese química , Depsipeptídeos/isolamento & purificação , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/isolamento & purificação , Glicosídeos/isolamento & purificação , Humanos , Lipopeptídeos/isolamento & purificação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Família Multigênica , Oligopeptídeos/síntese química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Infecções Estafilocócicas/microbiologia , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
14.
Bioorg Med Chem Lett ; 21(14): 4363-5, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21664132

RESUMO

The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhibitors is described. A number of these analogs were potent and selective inhibitors of class C beta-lactamases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice compound 3b exhibited phamacokinetics similar to imipenem.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/química , Inibidores Enzimáticos/química , Organofosfonatos/química , Oximas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Inibidores de beta-Lactamases , Antibacterianos/síntese química , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Organofosfonatos/síntese química , Organofosfonatos/farmacologia , Oximas/síntese química , Oximas/farmacologia , Tiofenos/química , beta-Lactamases/metabolismo
15.
Bioorg Med Chem Lett ; 20(8): 2622-4, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20299220

RESUMO

4,7-Dichloro-1-benzothien-2-yl sulfonylaminomethyl boronic acid (DSABA, Compound I) was discovered as the first boronic acid-based class D beta-lactamase inhibitor. It exhibited an IC(50) of 5.6 microM against OXA-40. The compound also inhibited class A and C beta-lactamases with sub to low microM IC(50), and synergized with imipenem against Acinetobacter baumannii.


Assuntos
Antibacterianos/farmacologia , Ácidos Borônicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de beta-Lactamases , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/química , Ácidos Borônicos/química , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana
16.
Assay Drug Dev Technol ; 1(4): 521-5, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15090248

RESUMO

We describe a novel, "mix and read" immunoassay for insulin in biological samples using FMAT. Current commercial assays for insulin require multiple washing steps and can be expensive. The insulin assay described is a simple two-step, time-saving assay and amenable to robotics. The linear response for the fluorometric signal is comparable to that observed using classical ELISA and RIA. A series of mouse plasma samples were tested for insulin levels and yielded results comparable to that measured using a commercial ELISA for insulin.


Assuntos
Fluorometria/métodos , Insulina/análise , Microquímica/métodos , Animais , Ensaio de Imunoadsorção Enzimática , Fluorometria/economia , Humanos , Camundongos , Ratos
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