RESUMEN
Activation of G(i)-coupled G protein-coupled receptor (GPCRs) by their ligands leads to inhibition of adenylyl cyclase (AC) and reduction of cyclic adenosine monophosphate (cAMP) levels in cells. The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific AC activator, to increase the basal cAMP level in cells to create an assay window for ligand detection. However, there is still a need to develop a nonforskolin-based cAMP assay because of the challenges inherent in titrating the concentration of forskolin to achieve a reliable assay window, along with issues related to the cAMP-independent effects of forskolin. Herein, we describe such an assay by utilizing the endogenous activity of the calcitonin receptor in Chinese hamster ovary (CHO) cells. The calcitonin receptor is a G(s)-coupled GPCR that, when activated by calcitonin, leads to the stimulation of AC and increases cAMP in cells. Thus, we use calcitonin, instead of forskolin, to increase the basal cAMP level in CHO cells to achieve an assay window. We demonstrated that calcitonin peptides robustly increased cAMP accumulation in several CHO cell lines stably expressing well-known G(i)-coupled GPCRs, such as the Dopamine D2 receptor, the Opioid µ receptor, or the Cannabinoid receptor-1. Agonists of these G(i)-coupled GPCRs attenuated calcitonin-induced cAMP production in their receptor stable cell lines. On the other hand, antagonists and/or inverse agonists blocked the effects of their agonists on calcitonin-induced cAMP production. This calcitonin-based cAMP assay has been demonstrated to be sensitive and robust and exhibited acceptable assay windows (signal/noise ratio) and, thus, can be applied to screen for agonists and antagonists/inverse agonists of G(i)-coupled GPCRs in high-throughput screening formats.
Asunto(s)
Calcitonina/fisiología , AMP Cíclico/análisis , Receptores de Calcitonina/fisiología , Receptores Acoplados a Proteínas G/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Inhibidores de Adenilato Ciclasa , Animales , Células CHO , Técnicas de Cultivo de Célula , Colforsina/metabolismo , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Concentración 50 Inhibidora , Ligandos , Terapia Molecular Dirigida , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Ensayo de Unión Radioligante , Ratas , Receptores de Calcitonina/agonistas , Receptores de Calcitonina/análisis , Receptores de Calcitonina/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/análisis , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Polipéptido Amiloide de Islotes Pancreáticos/antagonistas & inhibidores , Receptores Opioides mu/análisis , Receptores Opioides mu/antagonistas & inhibidores , SalmónRESUMEN
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
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
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.
Asunto(s)
Inmunosupresores/síntesis química , Organofosfatos/síntesis química , Glicoles de Propileno/síntesis química , Animales , Células CHO , Cricetinae , Clorhidrato de Fingolimod , Humanos , Conformación Molecular , Esfingosina/análogos & derivadosRESUMEN
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
3-(N-Alkyl)aminopropylphosphonic acids are potent agonists of four of the five known sphingosine-1-phosphate (S1P) receptor subtypes.
Asunto(s)
Receptores Acoplados a Proteínas G/agonistas , Receptores de Lisoesfingolípidos/agonistas , Animales , Células CHO , Cricetinae , Humanos , Concentración 50 Inhibidora , Ligandos , Organofosfonatos/síntesis química , Compuestos Organofosforados/síntesis química , Radioisótopos de Fósforo , Relación Estructura-ActividadRESUMEN
Structurally modified 3-(N-benzylamino)propylphosphonic acid S1P receptor agonists that maintain affinity for S1P1, and have decreased affinity for S1P3 are efficacious, but exhibit decreased acute cardiovascular toxicity in rodents than do nonselective agonists.
Asunto(s)
Compuestos de Bencilo/farmacología , Sistema Cardiovascular/efectos de los fármacos , Miocardio/metabolismo , Compuestos Organofosforados/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Animales , Compuestos de Bencilo/química , Sitios de Unión , Células CHO , Cricetinae , Humanos , Concentración 50 Inhibidora , Cinética , Lisofosfolípidos/química , Lisofosfolípidos/farmacología , Ratones , Compuestos Organofosforados/química , Radioisótopos de Fósforo , Ratas , Receptores Acoplados a Proteínas G/metabolismo , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/farmacologíaRESUMEN
Alteration in lymphocyte trafficking and prevention of graft rejection in rodents observed on exposure to FTY720 (1) or its corresponding phosphate ester 2 can be induced by the systemic administration of potent sphingosine-1-phosphate receptor agonists exemplified by 19. The similar S1P receptor profiles of 2 and 19 coupled with their comparable potency in vivo supports a connection between S1P receptor agonism and immunosuppressive efficacy.
Asunto(s)
Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/química , Glicoles de Propileno/administración & dosificación , Glicoles de Propileno/química , Receptores de Lisoesfingolípidos/agonistas , Animales , Células CHO , Cricetinae , Clorhidrato de Fingolimod , Humanos , Factores Inmunológicos/metabolismo , Inmunosupresores/administración & dosificación , Inmunosupresores/química , Inmunosupresores/metabolismo , Inyecciones Intravenosas , Masculino , Ratones , Glicoles de Propileno/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivadosRESUMEN
It has been reported recently that the phosphorylated form of the immunomodulator FTY720 activates sphingosine 1-phosphate G protein-coupled receptors. Therefore, understanding the biology of this new class of receptors will be important in clarifying the immunological function of bioactive lysosphingolipid ligands. The S1P(4) receptor has generated interest due to its lymphoid tissue distribution. While the S1P(4) receptor binds the prototypical ligand, S1P, a survey of other lysosphingolipids demonstrated that 4D-hydroxysphinganine 1-phosphate, more commonly known as phytosphingosine 1-phosphate (PhS1P), binds to S1P(4) with higher affinity. Using radiolabeled S1P (S133P), the affinity of PhS1P for the S1P(4) receptor is 1.6nM, while that of S1P is nearly 50-fold lower (119+/-20nM). Radiolabeled PhS1P proved to be superior to S133P in routine binding assays due to improved signal-to-noise ratio. The present study demonstrates the utility of a novel radiolabeled probe, PhS133P, for in vitro studies of the S1P(4) receptor pharmacology.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Proteínas de Saccharomyces cerevisiae , Esfingosina/análogos & derivados , Esfingosina/farmacocinética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , Unión Competitiva , Células CHO , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Cinética , Ligandos , Radioisótopos de Fósforo , Receptores Lisofosfolípidos , Transactivadores/metabolismo , Dedos de ZincRESUMEN
Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.