RESUMEN
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.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Cardiomegalia/inducido químicamente , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Imidazoles/farmacología , Piridinas/farmacología , Animales , Bencimidazoles , Glucemia/efectos de los fármacos , Ayuno , Glucógeno/metabolismo , Hipoglucemia/inducido químicamente , Imidazoles/efectos adversos , Imidazoles/química , Insulina/farmacología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Piridinas/efectos adversos , Piridinas/químicaRESUMEN
The discovery of novel 4-hydroxy-2-(heterocyclic)pyrimidine-5-carboxamide inhibitors of hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD) is described. These are potent, selective, orally bioavailable across several species, and active in stimulating erythropoiesis. Mouse and rat studies showed hematological changes with elevations of plasma EPO and circulating reticulocytes following single oral dose administration, while 4-week q.d. po administration in rat elevated hemoglobin levels. A major focus of the optimization process was to decrease the long half-life observed in higher species with early compounds. These efforts led to the identification of 28 (MK-8617), which has advanced to human clinical trials for anemia.
Asunto(s)
Anemia/tratamiento farmacológico , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Piridazinas/farmacología , Pirimidinas/farmacología , Administración Oral , Anemia/enzimología , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Piridazinas/administración & dosificación , Piridazinas/química , Pirimidinas/administración & dosificación , Pirimidinas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Quantitative reverse transcription PCR (qRT-PCR) is a valuable tool for characterizing the effects of inhibitors on viral replication. The amplification of target viral genes through the use of specifically designed fluorescent probes and primers provides a reliable method for quantifying RNA. Due to reagent costs, use of these assays for compound evaluation is limited. Until recently, the inability to accurately dispense low volumes of qRT-PCR assay reagents precluded the routine use of this PCR assay for compound evaluation in drug discovery. Acoustic dispensing has become an integral part of drug discovery during the past decade; however, acoustic transfer of microliter volumes of aqueous reagents was time consuming. The Labcyte Echo 525 liquid handler was designed to enable rapid aqueous transfers. We compared the accuracy and precision of a qPCR assay using the Labcyte Echo 525 to those of the BioMek FX, a traditional liquid handler, with the goal of reducing the volume and cost of the assay. The data show that the Echo 525 provides higher accuracy and precision compared to the current process using a traditional liquid handler. Comparable data for assay volumes from 500 nL to 12 µL allowed the miniaturization of the assay, resulting in significant cost savings of drug discovery and process streamlining.
Asunto(s)
Tecnología Biomédica/métodos , Miniaturización/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Acústica , Evaluación Preclínica de Medicamentos/métodos , SolucionesRESUMEN
The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.
Asunto(s)
Anemia/tratamiento farmacológico , Compuestos Aza/síntesis química , Hidantoínas/síntesis química , Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Compuestos de Espiro/síntesis química , Animales , Compuestos Aza/farmacocinética , Compuestos Aza/farmacología , Perros , Canal de Potasio ERG1 , Eritropoyetina/biosíntesis , Canales de Potasio Éter-A-Go-Go/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidantoínas/farmacocinética , Hidantoínas/farmacología , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Indoles/síntesis química , Indoles/farmacocinética , Indoles/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Macaca mulatta , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Ratas , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/farmacología , Relación Estructura-Actividad , Regulación hacia ArribaRESUMEN
Structure-activity relationship (SAR) studies of 3-arylpropionic acids-a class of novel S1P(1) selective agonists-by introducing substitution to the propionic acid chain and replacing the adjacent phenyl ring with pyridine led to a series of modified 3-arylpropionic acids with enhanced half-life in rat. These analogs (e.g., cyclopropanecarboxylic acids) exhibited longer half-life in rat than did unmodified 3-arylpropionic acids. This result suggests that metabolic oxidation at the propionic acid chain, particularly at the C3 benzylic position of 3-arylpropionic acids, is probably responsible for their short half-life in rodent.
Asunto(s)
Propionatos/síntesis química , Propionatos/farmacología , Receptores de Lisoesfingolípidos/agonistas , Animales , Disponibilidad Biológica , Células CHO , Cricetinae , Cricetulus , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Semivida , Recuento de Linfocitos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
A series of 3-arylpropionic acids were synthesized as S1P1 receptor agonists. Structure-activity relationship studies on the pendant phenyl ring revealed several structural features offering selectivity of S1P1 binding against S1P2-5. These highly selective S1P1 agonists induced peripheral blood lymphocyte lowering in mice and one of them was found to be efficacious in a rat skin transplantation model, supporting that S1P1 agonism is primarily responsible for the immunosuppressive efficacy observed in preclinical animal models.
Asunto(s)
Inmunosupresores/farmacología , Fenilpropionatos/síntesis química , Fenilpropionatos/farmacología , Receptores de Lisoesfingolípidos/agonistas , Animales , Células CHO , Cricetinae , Ligandos , Recuento de Linfocitos/veterinaria , Ratones , Ratas , Trasplante de Piel , Relación Estructura-ActividadRESUMEN
Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to achieve high selectivity for a single member of the S1P family of receptors, S1P1. The optimized structure represents a highly S1P1-selective and efficacious agonist: S1P1/S1P2, S1P1/S1P3, S1P1/S1P4>10,000-fold, S1P1/S1P5>600-fold, while EC50 (S1P1) <0.2 nM. In vivo experiments are consistent with S1P1 receptor agonism alone being sufficient for achieving desired lymphocyte-lowering effect.
Asunto(s)
Inmunosupresores/síntesis química , Inmunosupresores/farmacología , Linfocitos/efectos de los fármacos , Receptores de Lisoesfingolípidos/agonistas , Animales , Células CHO , Cricetinae , Recuento de Linfocitos , Linfocitos/citología , Relación Estructura-ActividadRESUMEN
A series of 2,5-cis-disubstituted pyrrolidines were synthesized and evaluated as S1P receptor agonists. Compounds 15-21 were identified with good selectivity over S1P3 which lowered circulating lymphocytes after oral administration in mice.
Asunto(s)
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Pirrolidinas/química , Receptores de Lisoesfingolípidos/agonistas , Administración Oral , Animales , Ácidos Carboxílicos/síntesis química , Estructura Molecular , Ratas , Receptores de Lisoesfingolípidos/metabolismo , Relación Estructura-ActividadRESUMEN
A series of 2-aryl(pyrrolidin-4-yl)acetic acids were synthesized and their biological activities were evaluated as agonists of S1P receptors. These analogs were able to induce lowering of lymphocyte counts in the peripheral blood of mice and were found to have good overall pharmacokinetic properties in rat.
Asunto(s)
Acetatos/farmacología , Pirrolidinas/farmacología , Receptores de Lisoesfingolípidos/agonistas , Acetatos/síntesis química , Acetatos/química , Animales , Recuento de Linfocitos , Lisofosfolípidos/antagonistas & inhibidores , Ratones , Estructura Molecular , Unión Proteica/efectos de los fármacos , Pirrolidinas/síntesis química , Pirrolidinas/química , Ratas , Esfingosina/análogos & derivados , Esfingosina/antagonistas & inhibidores , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A class of 3,5-diphenyl-1,2,4-oxadiazole based compounds have been identified as potent sphingosine-1-phosphate-1 (S1P1) receptor agonists with minimal affinity for the S1P2 and S1P3 receptor subtypes. Analogue 26 (S1P1 IC50 = 0.6 nM) has an excellent pharmacokinetics profile in the rat and dog and is efficacious in a rat skin transplant model, indicating that S1P3 receptor agonism is not a component of immunosuppressive efficacy.
Asunto(s)
Inmunosupresores/síntesis química , Oxadiazoles/síntesis química , Receptores de Lisoesfingolípidos/agonistas , Animales , Células CHO , Cricetinae , Cricetulus , Perros , Supervivencia de Injerto , Inmunosupresores/farmacocinética , Inmunosupresores/farmacología , Recuento de Linfocitos , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Trasplante de Piel , Relación Estructura-ActividadRESUMEN
Moderately potent, selective S1P(1) receptor agonists identified from high-throughput screening have been adapted into lipophilic tails for a class of orally bioavailable amino acid-based S1P(1) agonists represented by 7. Many of the new compounds are potent S1P(1) agonists that select against the S1P(2), S1P(3), and S1P(4) (although not S1P(5)) receptor subtypes. Analogues 18 and 24 are highly orally bioavailable and possess excellent pharmacokinetic profiles in the rat, dog, and rhesus monkey.
Asunto(s)
Azetidinas/farmacología , Inmunosupresores/farmacología , Receptores de Lisoesfingolípidos/agonistas , Administración Oral , Animales , Azetidinas/farmacocinética , Disponibilidad Biológica , Células CHO , Cricetinae , Perros , Diseño de Fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Inmunosupresores/farmacocinética , Linfocitos/efectos de los fármacos , Macaca mulatta , Ratones , Ratas , Ratas Endogámicas Lew , Relación Estructura-ActividadRESUMEN
A series of conformationally constrained 3-(N-alkylamino)propylphosphonic acids were systematically synthesized and their activities as S1P receptor agonists were evaluated. Several pyrrolidine and cyclohexane analogs had S1P receptor profiles comparable to the acyclic lead compound, 3-(N-tetradecylamino)propylphosphonic acid (3), lowered circulating lymphocytes in mice after iv administration and were thus identified as being suitable for further investigations.
Asunto(s)
Diseño de Fármacos , Organofosfonatos/síntesis química , Receptores de Lisoesfingolípidos/agonistas , Animales , Células CHO , Cricetinae , Humanos , Conformación Molecular , Organofosfonatos/química , Organofosfonatos/farmacología , Relación Estructura-ActividadRESUMEN
The novel immunosuppressive agent FTY720 (1) is phosphorylated in vivo in a variety of species yielding an active metabolite that is an agonist of four of the five known G-protein-coupled sphingosine-1-phosphate (S1P) receptors. A synthesis amenable to producing gram quantities of the stereoisomeric phosphate esters, a determination of their absolute stereochemistry via an enantioselective synthesis and their characterization as S1P receptor agonists and antagonists is reported.