RESUMEN
The metabotropic glutamate receptor subtype 5 (mGlu5 ) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR-based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine-tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus-bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.
Asunto(s)
Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Humanos , Ligandos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACH: We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca(2+) i ) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTS: Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca(2+) i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G(670) E). CONCLUSIONS AND IMPLICATIONS: The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses.
Asunto(s)
Calcimiméticos/farmacología , Receptores Sensibles al Calcio/metabolismo , Regulación Alostérica , Compuestos de Anilina/farmacología , Calcimiméticos/química , Calcio/metabolismo , Cinacalcet , Células HEK293 , Humanos , Indoles/farmacología , Fosfatos de Inositol/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Naftalenos/farmacología , Fenetilaminas , Fosforilación , Propilaminas , Receptores Sensibles al Calcio/agonistasRESUMEN
Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu5) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca(2+) mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu5 allosteric binding site and high selectivity for mGlu5. VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel mGlu5 PAMs for the treatment of psychosis and cognitive deficits observed in individuals with schizophrenia.
Asunto(s)
Antipsicóticos/farmacología , Hipercinesia/tratamiento farmacológico , Memoria a Corto Plazo/efectos de los fármacos , Nootrópicos/farmacología , Piperazinas/farmacología , Receptor del Glutamato Metabotropico 5/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Regulación Alostérica , Animales , Antipsicóticos/química , Antipsicóticos/farmacocinética , Antipsicóticos/uso terapéutico , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Hipercinesia/metabolismo , Hipercinesia/psicología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Nootrópicos/química , Nootrópicos/farmacocinética , Nootrópicos/uso terapéutico , Piperazinas/química , Piperazinas/farmacocinética , Piperazinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5/genética , Receptores de N-Metil-D-Aspartato/genética , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , TransfecciónRESUMEN
Metabotropic glutamate receptor 5 (mGlu5) is a target for the treatment of central nervous system (CNS) disorders, such as schizophrenia and Alzheimer's disease. Furthermore, mGlu5 has been shown to play an important role in hippocampal synaptic plasticity, specifically in long-term depression (LTD) and long-term potentiation (LTP), which is thought to be involved in cognition. Multiple mGlu5-positive allosteric modulators (PAMs) have been developed from a variety of different scaffolds. Previous work has extensively characterized a common allosteric site on mGlu5, termed the MPEP (2-Methyl-6-(phenylethynyl)pyridine) binding site. However, one mGlu5 PAM, CPPHA (N-(4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl)-2-hydroxybenzamide), interacts with a separate allosteric site on mGlu5. Using cell-based assays and brain slice preparations, we characterized the interaction of a potent and efficacious mGlu5 PAM from the CPPHA series termed NCFP (N-(4-chloro-2-((4-fluoro-1,3-dioxoisoindolin-2-yl)methyl)phenyl)picolinamide). NCFP binds to the CPPHA site on mGlu5 and potentiates mGlu5-mediated responses in both recombinant and native systems. However, NCFP provides greater mGlu5 subtype selectivity than does CPPHA, making it more suitable for studies of effects on mGlu5 in CNS preparations. Of interest, NCFP does not potentiate responses involved in hippocampal synaptic plasticity (LTD/LTP), setting it apart from other previously characterized MPEP site PAMs. This suggests that although mGlu5 PAMs may have similar responses in some systems, they can induce differential effects on mGlu5-mediated physiologic responses in the CNS. Such stimulus bias by mGlu5 PAMs may complicate drug discovery efforts but would also allow for specifically tailored therapies, if pharmacological biases can be attributed to different therapeutic outcomes.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Benzamidas/metabolismo , Benzamidas/farmacología , Ftalimidas/metabolismo , Ftalimidas/farmacología , Receptores de Glutamato Metabotrópico/fisiología , Transducción de Señal/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Transducción de Señal/fisiologíaRESUMEN
A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.