RESUMEN
Nurr1 is a nuclear hormone receptor (NucHR) strongly implicated in the growth, maintenance, and survival of dopaminergic neurons. Nurr1 may be unable to bind ligands directly, but it forms heterodimers with other NucHRs that do. Using bioluminescence resonance energy transfer (BRET) assays to directly monitor interactions of Nurr1 with other NucHRs, we found the cancer drug bexarotene (Targretin, also LGD1069) displayed biased interactions with Nurr1-RXR heterodimers compared with RXR-RXR homodimers. Remarkably, at doses up to 100-fold lower than those effective in rodent cancer models, bexarotene rescued dopamine neurons and reversed behavioral deficits in 6-hydroxydopamine (6-OHDA) lesioned rats. Compared to the high doses used in cancer therapy, low doses of bexarotene have significantly milder side effects including a reduced increase in plasma triglycerides and less suppression of thyroid function. On the basis of extrapolations from rat to human doses, we hypothesize that low oral doses of bexarotene may provide an effective and tolerated therapy for Parkinson's disease (PD).
Asunto(s)
Conducta Animal/efectos de los fármacos , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Neuronas Dopaminérgicas/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Tetrahidronaftalenos/administración & dosificación , Administración Oral , Animales , Conducta Animal/fisiología , Bexaroteno , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Infusiones Subcutáneas , Inyecciones Intraventriculares , Masculino , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/química , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Enfermedad de Parkinson/fisiopatología , Cultivo Primario de Células , Multimerización de Proteína , Ratas , Ratas Sprague-Dawley , Tetrahidronaftalenos/uso terapéuticoRESUMEN
The recent discovery of allosteric potentiators and agonists of the muscarinic M(1) receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC(50) 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M(1) receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M(1) receptor over the M(2), M(3), M(4) and M(5) muscarinic receptor subtypes. Using GTPgammaS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M(1) receptor activation since it was not observed in M(1) knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M(1) receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M(1) selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease.
Asunto(s)
Benzoxazinas/farmacología , Cognición/efectos de los fármacos , Nootrópicos/farmacología , Receptor Muscarínico M1/agonistas , Administración Oral , Animales , Benzoxazinas/administración & dosificación , Benzoxazinas/farmacocinética , Disponibilidad Biológica , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Cognición/fisiología , Cricetinae , Cricetulus , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Muscarínicos/administración & dosificación , Agonistas Muscarínicos/farmacocinética , Agonistas Muscarínicos/farmacología , Células 3T3 NIH , Nootrópicos/administración & dosificación , Nootrópicos/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
The discovery, synthesis, and optimization of compound 1 from a high-throughput screening hit to highly potent and selective peroxisome proliferator-activated receptor delta (PPARdelta) agonists are reported. The synthesis and structure-activity relationship in this series are described in detail. On the basis of a general schematic PPAR pharmacophore model, scaffold 1 was divided into headgroup, linker, and tailgroup and successively optimized for PPAR activation using in vitro PPAR transactivation assays. A (2-methylphenoxy)acetic acid headgroup, a flexible linker, and a five-membered heteroaromatic center ring with two hydrophobic aryl substituents were required for efficient and selective PPARdelta activation. The fine-tuning of these aryl substituents led to an array of highly potent and selective compounds such as compound 38c, displaying an excellent pharmacokinetic profile in mouse. In an in vivo acute dosing model, selected members of this array were shown to induce the expression of pyruvate dehydrogenase kinase-4 (PDK4) and uncoupling protein-3 (UCP3), genes that are known to be involved in energy homeostasis and regulated by PPARdelta in skeletal muscle.
Asunto(s)
Oxazoles/farmacología , PPAR delta/agonistas , Tiazoles/farmacología , Animales , Evaluación Preclínica de Medicamentos , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estructura Molecular , Oxazoles/síntesis química , Oxazoles/química , PPAR delta/genética , Estereoisomerismo , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src-family kinases (SFKs). These type II inhibitors were optimized using a cellular Lck-dependent proliferation assay and are capable of inhibiting Lck at single-digit nanomolar concentrations. This scaffold is likely to serve a valuable template for developing potent inhibitors of a number of SFKs.
Asunto(s)
Bencimidazoles/farmacología , Descubrimiento de Drogas , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Animales , Bencimidazoles/síntesis química , Bencimidazoles/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src kinase family. Highly efficient parallel syntheses were devised to prepare analogues for SAR studies. A number of these 4-amino-6-benzimidazole-pyrimidines exhibited single-digit nanomolar IC(50)s against Lck in biochemical and cellular assays. These 4-amino-6-benzimidazole-pyrimidines represent a new class of tyrosine kinase inhibitors.
Asunto(s)
Bencimidazoles/antagonistas & inhibidores , Química Farmacéutica/métodos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirimidinas/antagonistas & inhibidores , Enfermedades Autoinmunes/tratamiento farmacológico , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/química , Solubilidad , Relación Estructura-Actividad , Familia-src Quinasas/metabolismoRESUMEN
A novel series of 2-amino-6-carboxamidobenzothiazole was discovered to have potent Lck inhibitory properties. A highly efficient chemistry was developed. Also described are the detailed SAR study and the BaF3 cell line profiling for this series.
Asunto(s)
Linfocitos B/efectos de los fármacos , Benzamidas/farmacología , Benzotiazoles/farmacología , Proliferación Celular/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Linfocitos B/citología , Benzamidas/síntesis química , Benzotiazoles/síntesis química , Sitios de Unión , Línea Celular , Ratones , Modelos Químicos , Inhibidores de Proteínas Quinasas/síntesis química , Relación Estructura-ActividadRESUMEN
We have identified a novel liver X receptor (LXR) agonist (2) that activates the LXRbeta subtype with selectivity over LXRalpha. LXRbeta selectivity was confirmed using macrophages derived from LXR mutant mice. Despite its selectivity and modest potency, the compound can induce APO-AI-dependent cholesterol efflux from macrophages with full efficacy. Our results indicate that it is possible to achieve significant LXRbeta selectivity in a small molecule while maintaining functional LXR activity.
Asunto(s)
Proteínas de Unión al ADN/agonistas , Receptores Citoplasmáticos y Nucleares/agonistas , Tiadiazoles/síntesis química , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Apolipoproteína A-I/farmacología , Línea Celular , Colesterol/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Receptores X del Hígado , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Estereoisomerismo , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/farmacologíaRESUMEN
Many companies possess a compound collection consisting of purified compounds and of unpurified products from combinatorial libraries. Using commercial and proprietary compounds as examples, this report provides clear examples of the significant impact purification can have on the activity observed for a compound and highlights the need to retest the purified compounds prior to creating structure-activity relationships. Crude mixtures made with commercial compounds led to an increase in the number of false positives in the SXR-GAL4 assay as compared with their pure and purified counterparts. An examination of proprietary compounds in an HIV assay resulted in the purification of 61 active crude synthetic mixtures. Of these 61 compounds, 32 were 5-fold less active and 2 were 5-fold more active after purification. This report details a semiautomated process developed and implemented for cherry-picking, tracking, and selectively purifying compounds found active in high-throughput screening campaigns.
Asunto(s)
Técnicas Químicas Combinatorias , Evaluación Preclínica de Medicamentos/métodos , Cromatografía Liquida , Diseño de Fármacos , Reacciones Falso Positivas , Espectrometría de Masas , Manejo de EspecímenesRESUMEN
Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M(1) muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M(1). In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M(1) but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M(1) receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.
Asunto(s)
Benzoxazinas/farmacología , Clozapina/análogos & derivados , Clozapina/farmacología , Agonistas Muscarínicos/farmacología , Piperidinas/farmacología , Receptor Muscarínico M1/agonistas , Línea Celular , Humanos , Conformación Proteica , Ensayo de Unión Radioligante , Receptor Muscarínico M1/químicaRESUMEN
Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.
Asunto(s)
Genómica , Transducción de Señal , Animales , Humanos , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
We review the literature describing constitutive activity of the five muscarinic acetylcholine receptors in native and recombinant systems and discuss the effect of constitutive activity on muscarinic pharmacology in the context of modern models of receptor activation. We include a summary of mutations found to cause constitutive activity and discuss the implications of these data for the structure, function, and activation mechanism of muscarinic receptors. Finally, we discuss the possible physiological significance of constitutive activity of muscarinic receptors, incorporating information provided by targeted deletion of each of the muscarinic subtypes.
Asunto(s)
Receptores Muscarínicos/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Unión al GTP/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genéticaRESUMEN
The primary purpose of the present series of experiments was to characterize the in vitro and in vivo pharmacology profile of 2-(4-methoxy-phenyl)-N-(4-methyl-benzyl)-N-(1-methyl-piperidin-4-yl)-acetamide hydrochloride (AC-90179), a selective serotonin (5-HT2A) receptor inverse agonist, in comparison with the antipsychotics haloperidol and clozapine. The secondary purpose was to characterize the pharmacokinetic profile of AC-90179. Like all atypical antipsychotics, AC-90179 shows high potency as an inverse agonist and competitive antagonist at 5HT2A receptors. In addition, AC-90179 exhibits antagonism at 5HT2C receptors. In contrast, AC-90179 does not have significant potency for D2 and H1 receptors that have been implicated in the dose-limiting side effects of other antipsychotic drugs. The ability of AC-90179 to block 5-HT2A receptor signaling in vivo was demonstrated by its blockade of the rate-decreasing effects of the 5-HT2A agonist, (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride, under a fixed ratio schedule of reinforcement. Similar to clozapine and haloperidol, AC-90179 attenuated phencyclidine-induced hyperactivity. Although haloperidol impaired acquisition of a simple autoshaped response and induced cataleptic-like effects at behaviorally efficacious doses, AC-90179 and clozapine did not. Furthermore, unlike haloperidol and clozapine, AC-90179 did not decrease spontaneous locomotor behavior at efficacious doses. Limited oral bioavailability of AC-90179 likely reflects rapid metabolism rather than poor absorption. Taken together, a compound with a similar pharmacological profile as AC-90179 and with increased oral bioavailability may have potential for the treatment of psychosis.
Asunto(s)
Benzamidas/farmacología , Piperidinas/farmacología , Antagonistas del Receptor de Serotonina 5-HT2 , Antagonistas de la Serotonina/farmacología , Células 3T3 , Animales , Benzamidas/efectos adversos , Benzamidas/sangre , Disponibilidad Biológica , Encéfalo/metabolismo , Células CACO-2 , Catalepsia/inducido químicamente , Permeabilidad de la Membrana Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Nariz/efectos de los fármacos , Piperidinas/efectos adversos , Piperidinas/sangre , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Antagonistas de la Serotonina/efectos adversos , Antagonistas de la Serotonina/sangreRESUMEN
The superfamily of G-protein coupled receptors (GPCRs) has more than 1000 members and is the largest family of proteins in the body. GPCRs mediate signalling of stimuli as diverse as light, ions, small molecules, peptides and proteins and are the targets for many pharmaceuticals. Most GPCR ligands are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites. In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica/fisiología , Animales , Humanos , EstereoisomerismoRESUMEN
Many naturally occurring peptides exhibit a high degree of promiscuity across G-protein coupled receptor subtypes. The degree to which this phenomenon occurs, and its physiological significance is not well characterized. In addition, many 'orphan' peptides exist for which there are no known receptors. Therefore, to identify novel interactions between biologically active peptides and G-protein coupled receptors, a library of nearly 200 peptides was screened against the human calcitonin (hCTr), human Parathyroid Hormone (PTH1R), human Corticotropin Releasing Factor (CRF1), and the human Glucagon-like peptide (GLP1) receptors using a cell-based functional assay (Receptor Selection and Amplification Technology). Functional profiling revealed that the 'orphan peptide' PHM-27 selectively activated the hCTr; no activity was observed at the PTH1, CRF1, or GLP1 receptors. PHM-27 was a potent agonist at the hCTr, with similar efficacy as human calcitonin, and a potency of 11 nM. These results were confirmed in cyclic AMP assays. Responses to calcitonin and PHM-27 could be suppressed by the antagonist salmon calcitonin (8-32). In competition binding studies, salmon calcitonin (8-32), calcitonin, and PHM-27 were each able to inhibit (125)I-calcitonin from cell membranes containing transiently expressed hCTr. These results indicate that the orphan peptide PHM-27 is a potent agonist at the hCTr.
Asunto(s)
Péptido PHI/farmacología , Receptores de Calcitonina/agonistas , Células 3T3 , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Receptores de Calcitonina/metabolismoRESUMEN
Receptors have well-conserved regions that are recognized and activated by hormones and neurotransmitters. Most drugs bind to these sites and mimic or block the action of the native ligands. Using a high-throughput functional screen, we identified a potent and selective M(1) muscarinic receptor agonist from a novel structural class. Using a series of chimeric receptors, we demonstrated that this ligand activates the receptor through a region that is not conserved among receptor subtypes, explaining its unprecedented selectivity. This region of the receptor is distinct from the conserved region that is recognized by traditional ligands. The finding that receptors for small-molecule transmitters can have multiple, structurally distinct activation sites has broad implications for the study of receptor structure/function and the potential for the discovery of novel ligands with high selectivity.
Asunto(s)
Agonistas Muscarínicos/farmacología , Piperidinas/farmacología , Receptores Muscarínicos/metabolismo , Células 3T3 , Acetilcolina/metabolismo , Animales , Sitios de Unión , Humanos , Ratones , Modelos Moleculares , Ensayo de Unión Radioligante , Receptor Muscarínico M1RESUMEN
Previous studies have found that a mutation near the junction of the sixth transmembrane domain (TM6) and the third extracellular loop of the M5 muscarinic receptor leads to constitutive activation and enhanced agonist affinity for the mutated receptor. These results were consistent with the extended ternary complex model, which predicts a correlation between agonist affinity and constitutive activity. We have introduced the homologous mutation into all five subtypes of the highly conserved muscarinic receptor family; SerThr-->TyrPro was introduced into M1 and M5, and AsnThr-->TyrPro was introduced into M2, M3, and M4. In binding assays, these mutations produced increases in affinities toward acetylcholine and carbachol that ranged from 5-fold at the M2 receptor to 15- to 20-fold at M1, M3, and M4, to 40-fold at M5. In functional assays, all five mutant receptors exhibited constitutive activity, at levels ranging between 30 and 80% of the maximal response elicited by carbachol. In every case, the muscarinic antagonist atropine inhibited this constitutive activity with high affinity. Thus, despite differences in effector coupling and in wild-type sequence at the mutation site, all five subtypes were activated by this mutation at the top of TM6. Previous studies of the M5 subtype have indicated that TM6 is a ligand-dependent switch that sets the activation state of the receptor. Based on the results of the present study, it is possible that TM6 represents a general switch for the activation of the muscarinic receptor family.