RESUMO
A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (â¼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (â¼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.
Assuntos
Natriuréticos/química , Bloqueadores dos Canais de Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Animais , Benzofuranos/química , Pressão Sanguínea/efeitos dos fármacos , Diuréticos/química , Diuréticos/metabolismo , Diuréticos/farmacologia , Cães , Meia-Vida , Haplorrinos , Humanos , Masculino , Natriuréticos/metabolismo , Natriuréticos/farmacologia , Piperazinas/química , Potássio/urina , Bloqueadores dos Canais de Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos , Ratos Endogâmicos SHRRESUMO
G-protein-coupled receptors (GPCRs) are modulated by many marketed drugs, and as such, they continue to be key targets for drug discovery and development. Many GPCR targets at Merck Research Laboratories (MRL) are profiled using homogenous time-resolved fluorescence (HTRF) inositol monophosphate (IP-1) cell-based functional assays using adherent cells in 384-well microplates. Due to discrepancies observed across several in vitro assays supporting lead optimization structure-activity relationship (SAR) efforts, different assay paradigms were evaluated for removing growth medium from the assay plates prior to compound addition and determination of IP-1 accumulation. Remarkably, employing the noncontact centrifugation BlueWasher method leads to left-shifted potencies across multiple structural classes and rescues "false negatives" relative to the traditional manual evacuation method. Further, assay performance is improved, with the minimum significant ratio of challenging chemotypes dropping from ~5-6 to <3. While the impact of BlueWasher on a broad range of our GPCR targets remains to be determined, for highly protein-bound small molecules, it provides a path toward improving assay reproducibility across scientists and sites as well as reducing replicates in SAR assay support.
Assuntos
Bioensaio/métodos , Células/metabolismo , Automação , Células HEK293 , Humanos , Reprodutibilidade dos TestesRESUMO
A series of biaryl chromans exhibiting potent and selective agonism for the GPR40 receptor with positive allosteric modulation of endogenous ligands (AgoPAM) were discovered as potential therapeutics for the treatment of type II diabetes. Optimization of physicochemical properties through modification of the pendant aryl rings resulted in the identification of compound AP5, which possesses an improved metabolic profile while demonstrating sustained glucose lowering.
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 , RatosRESUMO
Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Regulação Alostérica , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.
Assuntos
Benzopiranos/farmacologia , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Tiazolidinedionas/farmacologia , Animais , Benzopiranos/química , Glicemia/metabolismo , Células CHO , Cricetulus , Diabetes Mellitus Experimental/metabolismo , Avaliação Pré-Clínica de Medicamentos , Jejum/sangue , Células HEK293 , Humanos , Hipoglicemiantes/química , Insulina/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Knockout , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Tiazolidinedionas/química , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone. The most promising candidate is pyridazinone analog 32 that showed an improved functional hERG/ROMK potency ratio and preclinical PK profile. In vivo evaluation of 32 demonstrated blood pressure lowering effects in the spontaneously hypertensive rat model.
Assuntos
Canal de Potássio ERG1/metabolismo , Bloqueadores dos Canais de Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Cães , Canal de Potássio ERG1/antagonistas & inibidores , Meia-Vida , Hipertensão/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacocinética , Bloqueadores dos Canais de Potássio/uso terapêutico , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Pirimidinas/química , Ratos , Ratos Endogâmicos SHR , Compostos de Espiro/química , Relação Estrutura-Atividade , Tiadiazóis/químicaRESUMO
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/genéticaRESUMO
A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.
Assuntos
Diuréticos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Sulfonas/farmacologia , Animais , Compostos Heterocíclicos/síntese química , Ratos , Ratos Endogâmicos SHRRESUMO
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.
RESUMO
Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.
Assuntos
Oxazinas/química , Oxazinas/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Animais , Diurese/efeitos dos fármacos , Cães , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Hipertensão/tratamento farmacológico , Macaca mulatta , Oxazinas/farmacocinética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos Sprague-Dawley , Regulador Transcricional ERG/antagonistas & inibidores , Regulador Transcricional ERG/metabolismoRESUMO
The renal outer medullary potassium (ROMK) channel, located at the apical surface of epithelial cells in the thick ascending loop of Henle and cortical collecting duct, contributes to salt reabsorption and potassium secretion, and represents a target for the development of new mechanism of action diuretics. This idea is supported by the phenotype of antenatal Bartter's syndrome type II associated with loss-of-function mutations in the human ROMK channel, as well as, by cardiovascular studies of heterozygous carriers of channel mutations associated with type II Bartter's syndrome. Although the pharmacology of ROMK channels is still being developed, channel inhibitors have been identified and shown to cause natriuresis and diuresis, in the absence of any significant kaliuresis, on acute oral dosing to rats or dogs. Improvements in potency and selectivity have led to the discovery of MK-7145 [5,5'-((1R,1'R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)], a potential clinical development candidate. In spontaneously hypertensive rats, oral dosing of MK-7145 causes dose-dependent lowering of blood pressure that is maintained during the entire treatment period, and that displays additive/synergistic effects when administered in combination with hydrochlorothiazide or candesartan, respectively. Acute or chronic oral administration of MK-7145 to normotensive dogs led to dose-dependent diuresis and natriuresis, without any significant urinary potassium losses or changes in plasma electrolyte levels. Elevations in bicarbonate and aldosterone were found after 6 days of dosing. These data indicate that pharmacological inhibition of ROMK has potential as a new mechanism for the treatment of hypertension and/or congestive heart failure. In addition, Bartter's syndrome type II features are manifested on exposure to ROMK inhibitors.
Assuntos
Síndrome de Bartter/fisiopatologia , Benzofuranos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Fenótipo , Piperazinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Animais , Síndrome de Bartter/tratamento farmacológico , Benzimidazóis/farmacologia , Benzofuranos/uso terapêutico , Compostos de Bifenilo , Cães , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Células HEK293 , Humanos , Hidroclorotiazida/farmacologia , Masculino , Piperazinas/uso terapêutico , Bloqueadores dos Canais de Potássio/uso terapêutico , Ratos , Tetrazóis/farmacologiaRESUMO
ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.
RESUMO
Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.
Assuntos
Canal de Potássio ERG1/fisiologia , Compostos Heterocíclicos/farmacologia , Piperazinas/farmacologia , Compostos Heterocíclicos/química , Piperazinas/química , Relação Estrutura-AtividadeRESUMO
A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.
RESUMO
The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.
Assuntos
Diurese/efeitos dos fármacos , Diurese/fisiologia , Natriurese/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Cães , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Masculino , Natriurese/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.
Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Animais , Benzofuranos/farmacocinética , Diurese/efeitos dos fármacos , Descoberta de Drogas , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos , Ratos Sprague-Dawley , Tetrazóis/química , Tetrazóis/farmacocinética , Tetrazóis/farmacologiaRESUMO
A series of benzazepinones were synthesized and evaluated for block of Nav1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.
Assuntos
Benzazepinas/farmacologia , Neuralgia/tratamento farmacológico , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Modelos Animais de Doenças , RatosRESUMO
The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure-activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels.