RESUMO
Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.
Assuntos
Dopamina , Optogenética , Dopamina/metabolismo , Animais , Humanos , Optogenética/métodos , Camundongos , Masculino , Corpo Estriado/metabolismo , Corpo Estriado/diagnóstico por imagem , Receptores Dopaminérgicos/metabolismo , Receptores Dopaminérgicos/genética , Camundongos Endogâmicos C57BL , Regulação Alostérica , Fotometria/métodos , Células HEK293RESUMO
Dopamine (DA) D1 and D2 receptors (Rs) are critical for cognitive functioning. D1 positive allosteric modulators (D1PAMs) activate D1Rs without desensitization or an inverted U-shaped dose response curve. DETQ, [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one] is highly selective for the human D1Rs as shown in humanized D1R knock-in (hD1Ki) mice. Here, we have ascertained the efficacy of DETQ in aged [13-23-month-old (mo)] hD1Ki mice and their corresponding age-matched wild-type (WT; C57BL/6NTac) controls. We found that in aged mice, DETQ, given acutely, subchronically, and chronically, rescued both novel object recognition memory and social behaviors, using novel object recognition (NOR) and social interaction (SI) tasks, respectively without any adverse effect on body weight or mortality. We have also shown, using in vivo microdialysis, a significant decrease in basal DA and norepinephrine, increase in glutamate (Glu) and gamma-amino butyric acid (GABA) efflux with no significant changes in acetylcholine (ACh) levels in aged vs young mice. In young and aged hD1Ki mice, DETQ, acutely and subchronically increased ACh in the medial prefrontal cortex and hippocampal regions in aged hD1Ki mice without affecting Glu. These results suggest that the D1PAM mechanism is of interest as potential treatment for cognitive and social behavioral deficits in neuropsychiatric disorders including but not restricted to neurodegenerative disorders, such as Parkinson's disease.
Assuntos
Acetilcolina , Interação Social , Camundongos , Humanos , Animais , Idoso , Lactente , Camundongos Endogâmicos C57BL , Dopamina , Ácido Glutâmico , Hipocampo/metabolismo , Receptores de Dopamina D1/metabolismo , CogniçãoRESUMO
RATIONALE: Dopamine (DA) signaling through the D1 receptor has been shown to be integral to multiple aspects of cognition, including the core process of working memory. The discovery of positive allosteric modulators (PAMs) of the D1 receptor has enabled treatment modalities that may have alternative benefits to orthosteric D1 agonists arising from a synergism of action with functional D1 receptor signaling. OBJECTIVES: To investigate this potential, we have studied the effects of the novel D1 PAM DPTQ on a spatial delayed response working memory task in the rhesus monkey. Initial studies indicated that DPTQ binds to primate D1R with high affinity and selectivity and elevates spontaneous eye blink rate in rhesus monkeys in a dose-dependent manner consistent with plasma ligand exposures and central D1activation. RESULTS: Based on those results, DPTQ was tested at 2.5 mg/kg IM in the working memory task. No acute effect was observed 1 h after dosing, but performance was impaired 48 h later. Remarkably, this deficit was immediately followed by a significant enhancement in cognition over the next 3 days. In a second experiment in which DPTQ was administered on days 1 and 5, the early impairment was smaller and did not reach statistical significance, but statistically significant enhancement of performance was observed over the following week. Lower doses of 0.1 and 1.0 mg/kg were also capable of producing this protracted enhancement without inducing any transient impairment. CONCLUSIONS: DPTQ exemplifies a class of D1PAMs that may be capable of providing long-term improvements in working memory.
Assuntos
Memória de Curto Prazo , Receptores de Dopamina D1 , Animais , Receptores de Dopamina D1/metabolismo , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Memória EspacialRESUMO
An amine-containing molecule called Compound A has been reported by a group from Bristol-Myers Squibb to act as a positive allosteric modulator (PAM) at the dopamine D1 receptor. We synthesized the more active enantiomer of Compound A (BMS-A1) and compared it with the D1 PAMs DETQ and MLS6585, which are known to bind to intracellular loop 2 and the extracellular portion of transmembrane helix 7, respectively. Results from D1/D5 chimeras indicated that PAM activity of BMS-A1 tracked with the presence of D1 sequence in the N-terminal/extracellular region of the D1 receptor, a unique location compared with either of the other PAMs. In pairwise combinations, BMS-A1 potentiated the small allo-agonist activity of each of the other PAMs, while the triple PAM combination (in the absence of dopamine) produced a cAMP response about 64% of the maximum produced by dopamine. Each of the pairwise PAM combinations produced a much larger leftward shift of the dopamine EC50 than either single PAM alone. All three PAMs in combination produced a 1000-fold leftward shift of the dopamine curve. These results demonstrate the presence of three non-overlapping allosteric sites that cooperatively stabilize the same activated state of the human D1 receptor. SIGNIFICANCE STATEMENT: Deficiencies in dopamine D1 receptor activation are seen in Parkinson disease and other neuropsychiatric disorders. In this study, three positive allosteric modulators of the dopamine D1 receptor were found to bind to distinct and separate sites, interacting synergistically with each other and dopamine, with the triple combination causing a 1000-fold leftward shift of the response to dopamine. These results showcase multiple opportunities to modulate D1 tone and highlight new pharmacological approaches for allosteric modulation of G-protein-coupled receptors.
Assuntos
Dopamina , Receptores de Dopamina D1 , Humanos , Sítio Alostérico/fisiologia , Dopamina/metabolismo , Regulação Alostérica/fisiologia , Receptores de Dopamina D1/metabolismo , Receptores Acoplados a Proteínas GRESUMO
Three structurally closely related dopamine D1 receptor positive allosteric modulators (D1 PAMs) based on a tetrahydroisoquinoline (THIQ) scaffold were profiled for their CYP3A4 induction potentials. It was found that the length of the linker at the C5 position greatly affected the potentials of these D1 PAMs as CYP3A4 inducers, and the level of induction correlated well with the activation of the pregnane X receptor (PXR). Based on the published PXR X-ray crystal structures, we built a binding model specifically for these THIQ-scaffold-based D1 PAMs in the PXR ligand-binding pocket via an ensemble docking approach and found the model could explain the observed CYP induction disparity. Combined with our previously reported D1 receptor homology model, which identified the C5 position as pointing toward the solvent-exposed space, our PXR-binding model coincidentally suggested that structural modifications at the C5 position could productively modulate the CYP induction potential while maintaining the D1 PAM potency of these THIQ-based PAMs.
Assuntos
Citocromo P-450 CYP3A , Receptores de Esteroides , Receptor de Pregnano X/metabolismo , Citocromo P-450 CYP3A/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Indução EnzimáticaRESUMO
Results from recently completed clinical studies suggest the dopamine D1 receptor positive allosteric modulator (PAM) mevidalen (1) could offer unique value for lewy body dementia (LBD) patients. In nonclinical assessments, 1 was mainly eliminated by CYP3A4-mediated metabolism, therefore at the risk of being a victim of drug-drug interactions (DDI) with CYP3A4 inhibitors and inducers. An effort was initiated to identify a new D1 PAM with an improved DDI risk profile. While attempts to introduce additional metabolic pathways mediated by other CYP isoforms failed to provide molecules with an acceptable profile, we discovered that the relative contribution of CYP-mediated oxidation and UGT-mediated conjugation could be tuned to reduce the CYP3A4-mediated victim DDI risk. We have identified LY3154885 (5), a D1 PAM that possesses similar in vitro and in vivo pharmacologic properties as 1, but is metabolized mainly by UGT, predicting it could potentially offer lower victim DDI risk in clinic.
Assuntos
Citocromo P-450 CYP3A , Fármacos Neuroprotetores , Receptores de Dopamina D1/antagonistas & inibidores , Regulação Alostérica , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A , Interações Medicamentosas , Humanos , Receptores de Dopamina D1/metabolismoRESUMO
BACKGROUND: Mevidalen is a selective positive allosteric modulator (PAM) of the dopamine D1 receptor subtype. OBJECTIVE: To assess the safety and efficacy of mevidalen for treatment of cognition in patients with Lewy body dementia (LBD). METHODS: PRESENCE was a phase 2, 12-week study in participants with LBD (N = 344) randomly assigned (1:1:1:1) to daily doses of mevidalen (10, 30, or 75 mg) or placebo. The primary outcome measure was change from baseline on Cognitive Drug Research Continuity of Attention (CoA) composite score. Secondary outcomes included Alzheimer's Disease Assessment Scale-Cognitive Subscale 13 (ADAS-cog13 ), Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), and Alzheimer's Disease Cooperative Study-Clinical Global Impression of Change (ADCS-CGIC). Numerous safety measures were collected. RESULTS: Mevidalen failed to meet primary or secondary cognition endpoints. Mevidalen resulted in significant, dose-dependent improvements of MDS-UPDRS total score (sum of Parts I-III, 10 mg P < 0.05, 30 mg P < 0.05, 75 mg P < 0.01, compared to placebo). The 30 mg and 75 mg mevidalen doses significantly improved ADCS-CGIC scores compared to placebo (minimal or better improvement: 30 mg P < 0.01, 75 mg P < 0.01; moderate or better improvement: 30 mg P < 0.05, 75 mg P < 0.001). Increases in blood pressure, adverse events, and cardiovascular serious adverse events were most pronounced at the 75 mg dose. CONCLUSIONS: Mevidalen harnesses a novel mechanism of action that improves motor symptoms associated with LBD on top of standard of care while improving or not worsening non-motor symptoms associated with traditional dopaminergic therapy. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Doença de Alzheimer , Doença por Corpos de Lewy , Fármacos Neuroprotetores , Cognição , Método Duplo-Cego , Humanos , Doença por Corpos de Lewy/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêuticoRESUMO
Dopamine (DA) plays a key role in several central functions including cognition, motor activity, and wakefulness. Although efforts to develop dopamine receptor 1 (D1) agonists have been challenging, a positive allosteric modulator represents an attractive approach with potential better drug-like properties. Our previous study demonstrated an acceptable safety and tolerability profile of the dopamine receptor 1 positive allosteric modulator (D1PAM) mevidalen (LY3154207) in single and multiple ascending dose studies in healthy volunteers (Wilbraham et al., 2021). Herein, we describe the effects of mevidalen on sleep and wakefulness in humanized dopamine receptor 1 (hD1) mice and in sleep-deprived healthy male volunteers. Mevidalen enhanced wakefulness (latency to fall asleep) in the hD1 mouse in a dose dependent [3-100 mg/kg, orally (PO)] fashion when measured during the light (zeitgeber time 5) and predominantly inactive phase. Mevidalen promoted wakefulness in mice after prior sleep deprivation and delayed sleep onset by 5.5- and 15.2-fold compared with vehicle-treated animals, after the 20 and 60 mg/kg PO doses, respectively, when compared with vehicle-treated animals. In humans, mevidalen demonstrated a dose-dependent increase in latency to sleep onset as measured by the multiple sleep latency test and all doses (15, 30, and 75 mg) separated from placebo at the first 2-hour postdose time point with a circadian effect at the 6-hour postdose time point. Sleep wakefulness should be considered a translational biomarker for the dopamine receptor 1 positive allosteric modulator mechanism. SIGNIFICANCE STATEMENT: This is the first translational study describing the effects of a selective dopamine receptor 1 positive allosteric modulator (D1PAM) on sleep and wakefulness in the human dopamine receptor 1 mouse and in sleep-deprived healthy male volunteers. In both species, drug exposure correlated with sleep latency, supporting the use of sleep-wake activity as a translational central biomarker for D1PAM. Wake-promoting effects of D1PAMs may offer therapeutic opportunities in several conditions, including sleep disorders and excessive daytime sleepiness related to neurodegenerative disorders.
Assuntos
Fármacos Neuroprotetores , Vigília , Animais , Voluntários Saudáveis , Humanos , Isoquinolinas , Masculino , Camundongos , Fármacos Neuroprotetores/farmacologia , Receptores de Dopamina D1 , Sono/fisiologiaRESUMO
Mevidalen (LY3154207) is a positive allosteric modulator of the dopamine D1 receptor that enhances the affinity of dopamine for the D1 receptor. The safety, tolerability, motor effects, and pharmacokinetics of mevidalen were studied in patients with Parkinson disease. Mevidalen or placebo was given once daily for 14 days to 2 cohorts of patients (cohort 1, 75 mg; cohort 2, titration from 15 to 75 mg). For both cohorts, the median time to maximum concentration for mevidalen plasma concentration was about 2 hours, the apparent steady-state clearance was 20-25 L/h, and mevidalen plasma concentrations were similar between the 1st and 14th administration in cohort 1, indicating minimal accumulation upon repeated dosing. Mevidalen was well tolerated, and most treatment-emergent adverse events were mild. Blood pressure and pulse rate increased when taking mevidalen, but there was considerable overlap with patients taking placebo, and vital signs normalized with repeated dosing. In the Movement Disorder Society-United Parkinson's Disease Rating Scale, all patients taking mevidalen showed a better motor examination sub-score on day 6 compared to only some patients in the placebo group. These data support examining mevidalen for symptomatic treatment of patients with Parkinson disease and Lewy body dementia.
Assuntos
Fármacos Neuroprotetores , Doença de Parkinson , Humanos , Isoquinolinas/farmacocinética , Fármacos Neuroprotetores/efeitos adversos , Doença de Parkinson/tratamento farmacológico , Receptores de Dopamina D1RESUMO
Activation of the brain dopamine D1 receptor has attracted attention because of its promising role in neuropsychiatric diseases. Although efforts to develop D1 agonists have been challenging, a positive allosteric modulator (PAM), represents an attractive approach with potential better drug-like properties. Phase 1 single-ascending-dose (SAD; NCT03616795) and multiple-ascending-dose (MAD; NCT02562768) studies with the D1PAM mevidalen (LY3154207) were conducted with healthy subjects. There were no treatment-related serious adverse events (AEs) in these studies. In the SAD study, 25-200 mg administered orally showed dose-proportional pharmacokinetics (PK) and acute dose-related increases in systolic blood pressure (SBP) and diastolic blood pressure DBP) and pulse rate at doses ≥ 75 mg. AE related to central activation were seen at doses ≥ 75 mg. At 25 and 75 mg, central penetration of mevidalen was confirmed by measurement of mevidalen in cerebrospinal fluid. In the MAD study, once-daily doses of mevidalen at 15-150 mg for 14 days showed dose-proportional PK. Acute dose-dependent increases in SBP, DBP, and PR were observed on initial administration, but with repeated dosing the effects diminished and returned toward baseline levels. Overall, these findings support further investigation of mevidalen as a potential treatment for a range of neuropsychiatric disorders.
Assuntos
Dopaminérgicos , Isoquinolinas , Receptores de Dopamina D1 , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Estudos de Coortes , Dopaminérgicos/administração & dosagem , Dopaminérgicos/farmacocinética , Dopaminérgicos/farmacologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Frequência Cardíaca/efeitos dos fármacos , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacocinética , Isoquinolinas/farmacologia , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D1/metabolismoRESUMO
Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.
Assuntos
Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Colinérgicos/farmacologia , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , FosforilaçãoRESUMO
Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.
Assuntos
Isoquinolinas/farmacologia , Receptores de Dopamina D1/agonistas , Acetilcolina/metabolismo , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Cristalografia por Raios X , AMP Cíclico/metabolismo , Células HEK293 , Meia-Vida , Humanos , Isoquinolinas/química , Isoquinolinas/farmacocinética , Rim/efeitos dos fármacos , Rim/metabolismo , Locomoção/efeitos dos fármacos , Camundongos , Conformação Molecular , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Dopamina D1/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
The dopamine D1 receptor plays an important role in motor activity, reward, and cognition. Efforts to develop D1 agonists have been mixed due to poor drug-like properties, tachyphylaxis, and inverted U-shaped dose-response curves. Recently, positive allosteric modulators (PAMs) for the dopamine D1 receptor were discovered and initial pharmacological profiling has suggested that several of the above issues could be addressed with this mechanism. This paper presents an overview of key findings for DETQ (2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one), which is currently the only D1 PAM for which published in vivo data is available. In vitro studies showed selective potentiation of the human D1 receptor without significant allosteric agonist effects. Due to a species difference in affinity for DETQ, transgenic mice expressing the human D1 receptor (hD1 mice) were used in vivo. In contrast to D1 agonists, DETQ increased locomotor activity over a wide dose-range without inverted U-shaped dose response or tachyphylaxis. DETQ also reversed hypo-activity in mice with dopamine depletion due to reserpine pretreatment, suggesting potential for treatment of motor symptoms in Parkinson's disease. Potential pro-cognitive effects were supported by improved performance in the novel object recognition task, enhanced release of cortical acetylcholine and histamine, and increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB. In addition, DETQ enhanced wakefulness in EEG studies and decreased immobility in the forced-swim test. Together, these results provide support for potential utility of D1 PAMs in the treatment of several neuropsychiatric disorders. LY3154207, a close analog of DETQ, is currently in phase 2 clinical trials.
Assuntos
Agonistas de Dopamina/uso terapêutico , Transtornos Mentais/tratamento farmacológico , Receptores de Dopamina D1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Agonistas de Dopamina/química , Agonistas de Dopamina/farmacologia , Humanos , Receptores de Dopamina D1/químicaRESUMO
Diminished dopamine D1 stimulation may contribute to cognitive impairment in Alzheimer's and Parkinson's diseases, schizophrenia, and other neuropsychiatric disorders. However, orthosteric D1 receptor (D1R) agonists produce receptor desensitization and an inverted U-shaped dose-response curve, but positive allosteric modulators (PAMs) do not. We examined the cognitive effects of DETQ, a D1R PAM, in mice genetically modified to express the human D1 receptor ("hD1 mice"). Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate receptor antagonist, dosed seven days (subchronic), followed by withdrawal, produced a prolonged deficit in novel object recognition (NOR) memory, which was reversed by acute treatment with DETQ, with no evidence for an inverted U-shaped response. This was blocked by the D1R antagonist, SCH391660. Single doses of D1R agonists, SKF38393 and SKF82958, and the acetylcholinesterase inhibitor, rivastigmine, alone and the combination of subeffective doses of both DETQ and rivastigmine, also restored NOR in both subchronic PCP-treated in hD1 mice. DETQ increased cortical and hippocampal acetylcholine efflux after both acute and subchronic dosing in hD1 mice. Subchronic but not acute DETQ, inhibited glutamate and GABA efflux. DETQ-induced acetylcholine efflux was absent in subchronic PCP-treated mice, indicating that restoration of NOR in subchronic PCP-treated mice does not require cortical acetylcholine efflux. This is additional evidence that DETQ stimulates D1R without producing an inverted-U-shaped response curve and increases neurotransmitter release in the mPFC and HIP without causing tolerance. The ability of D1 PAMs to improve cognition in humans with neuropsychiatric disorders without evidence of tolerance or an inverted-U-shaped response curve needs to be established clinically.
Assuntos
Isoquinolinas/farmacologia , Receptores de Dopamina D1/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Animais , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Técnicas de Introdução de Genes , Humanos , Masculino , Memória/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Camundongos , Camundongos Transgênicos , Fenciclidina/farmacologia , Receptor 5-HT1A de Serotonina , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/fisiologia , Agonistas do Receptor 5-HT1 de Serotonina/farmacologiaRESUMO
The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a ß2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state ß2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.
Assuntos
Regulação Alostérica/fisiologia , Sítio Alostérico/fisiologia , Receptores de Dopamina D1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Linhagem Celular , Sequência Conservada/efeitos dos fármacos , Sequência Conservada/fisiologia , Dopamina/metabolismo , Células HEK293 , Humanos , Isoquinolinas/farmacologia , RatosRESUMO
DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.
Assuntos
Doenças do Sistema Nervoso/metabolismo , Transtornos Psicóticos/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Antipsicóticos/uso terapêutico , Piscadela/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopaminérgicos/uso terapêutico , Isoquinolinas/uso terapêutico , Levodopa/uso terapêutico , Macaca mulatta , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças do Sistema Nervoso/tratamento farmacológico , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Transtornos Psicóticos/tratamento farmacológico , Receptores de Dopamina D1/genética , Reserpina/uso terapêutico , Sono/efeitos dos fármacos , Vigília/efeitos dos fármacosRESUMO
Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was â¼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was â¼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (â¼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.
Assuntos
Comportamento Animal/efeitos dos fármacos , Técnicas de Introdução de Genes , Isoquinolinas/farmacologia , Locomoção/efeitos dos fármacos , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Taquifilaxia , Adamantano/análogos & derivados , Adamantano/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Benzopiranos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Isoquinolinas/efeitos adversos , Masculino , Camundongos , Transporte Proteico/efeitos dos fármacos , Receptores de Dopamina D1/agonistasRESUMO
Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide whose receptor is designated ORL1 or nociceptin receptor (NOP). We utilized a potent, selective, and orally bioavailable antagonist with documented engagement with NOP receptors in vivo to assess antidepressant- and anxiolytic-related pharmacological effects of NOP receptor blockade along with measures of cognitive and motor impingement. LY2940094 ([2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol) displayed antidepressant-like behavioral effects in the forced-swim test in mice, an effect absent in NOP -/- mice. LY2940094 also augmented the behavioral effect of fluoxetine without changing target occupancies (NOP and serotonin reuptake transporter [SERT]). LY2940094 did not have effects under a differential-reinforcement of low rate schedule. Although anxiolytic-like effects were not observed in some animal models (conditioned suppression, 4-plate test, novelty-suppressed feeding), LY2940094 had effects like that of anxiolytic drugs in three assays: fear-conditioned freezing in mice, stress-induced increases in cerebellar cGMP in mice, and stress-induced hyperthermia in rats. These are the first reports of anxiolytic-like activity with a systemically viable NOP receptor antagonist. LY2940094 did not disrupt performance in either a 5-choice serial reaction time or delayed matching-to-position assay. LY2940094 was also not an activator or suppressor of locomotion in rodents nor did it induce failures of rotarod performance. These data suggest that LY2940094 has unique antidepressant- and anxiolytic-related pharmacological effects in rodents. Clinical proof of concept data on this molecule in depressed patients have been reported elsewhere.
RESUMO
BACKGROUND: Human cerebrospinal fluid (CSF) is often acquired in Phase I clinical trials to assess the CNS penetration of new pharmacological agents and to search for biomarkers associated with PD effects. Robust methods for neurotransmitter metabolites in CSF have proven elusive, in part due to inadequate reversed phase LC retention. RESULTS: Benzoyl chloride derivatization was used to promote retention for LC-MS/MS for a panel of neurotransmitter metabolites while delivering a concise method for sample preparation. CONCLUSION: A validated assay in human CSF was obtained for 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3,4-dihydroxyphenylglycol and 5-hydroxyindoleacetic acid. This method is differentiated from other LC-MS/MS methods by delivering results in line with full regulatory expectations.
Assuntos
Benzoatos/química , Testes de Química Clínica/métodos , Neurotransmissores/líquido cefalorraquidiano , Espectrometria de Massas em Tandem , Ácido 3,4-Di-Hidroxifenilacético/líquido cefalorraquidiano , Ácido 3,4-Di-Hidroxifenilacético/normas , Animais , Cromatografia Líquida de Alta Pressão/normas , Meia-Vida , Ácido Homovanílico/líquido cefalorraquidiano , Ácido Homovanílico/normas , Humanos , Ácido Hidroxi-Indolacético/líquido cefalorraquidiano , Ácido Hidroxi-Indolacético/normas , Masculino , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/líquido cefalorraquidiano , Metoxi-Hidroxifenilglicol/normas , Neurotransmissores/química , Neurotransmissores/metabolismo , Controle de Qualidade , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/normasRESUMO
The interactions between the glutamatergic and the histaminergic systems in the brain are not fully understood. Here we studied histamine release in the medial prefrontal cortex and the posterior hypothalamus-tuberomamillary nucleus (PH-TMN) using in vivo microdialysis and electrophysiological recordings of histaminergc neurons in the PH-TMN in vivo to further address the mechanistic details of these interactions. We demonstrated that histaminergic activity was regulated by group II metabotropic glutamate receptors (mGluR 2 and 3) using systemic dosing with mGluR 2/3 agonist and antagonists and an mGluR 2 positive allosteric modulator. These interactions likely occur via direct modulation of glutamate release in the PH-TMN. The importance of circadian rhythm for histamine release was also shown using microdialysis studies with mGluR 2/3 compounds under light and dark conditions. Based on histamine release studies with NMDA and ketamine, we propose the existence of two sub-populations of NMDA receptors where one subtype is located on histaminergic cell bodies in the PH-TMN and the second on GABA-ergic neurons projecting to the PH-TMN. These subpopulations could be distinguished based on function, notably opposing actions were seen on histamine release in the medial prefrontal cortex of the rat. In summary, this paper provides evidence that the histaminergic system is closely regulated by glutamate neurons in multiple ways. In addition, this interaction depends to a great extent on the activity state of the subject.