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1.
Cell ; 167(3): 750-762.e14, 2016 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-27768894

RESUMEN

Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.


Asunto(s)
Antagonistas de Receptores de Cannabinoides/química , Morfolinas/química , Pirazoles/química , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/química , Sitios de Unión , Cannabinoides/farmacología , Cannabis/química , Cristalografía por Rayos X , Dronabinol/farmacología , Endocannabinoides/farmacología , Humanos , Ligandos , Morfolinas/síntesis química , Unión Proteica , Conformación Proteica en Hélice alfa , Pirazoles/síntesis química
2.
Nature ; 547(7664): 468-471, 2017 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-28678776

RESUMEN

The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC). Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1-agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a 'twin toggle switch' of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros-Weinstein numbering) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.


Asunto(s)
Agonistas de Receptores de Cannabinoides/química , Dronabinol/análogos & derivados , Droperidol/análogos & derivados , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/química , Sitios de Unión , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/farmacología , Cristalografía por Rayos X , Dronabinol/síntesis química , Dronabinol/química , Dronabinol/farmacología , Droperidol/síntesis química , Droperidol/química , Droperidol/farmacología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo
3.
Mol Pharmacol ; 96(5): 619-628, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31515283

RESUMEN

Cannabinoid receptor 1 (CB1) is a potential therapeutic target for the treatment of pain, obesity and obesity-related metabolic disorders, and addiction. The crystal structure of human CB1 has been determined in complex with the stabilizing antagonist AM6538. In the present study, we characterize AM6538 as a tight-binding/irreversible antagonist of CB1, as well as two derivatives of AM6538 (AM4112 and AM6542) as slowly dissociating CB1 antagonists across binding simulations and cellular signaling assays. The long-lasting nature of AM6538 was explored in vivo wherein AM6538 continues to block CP55,940-mediated behaviors in mice up to 5 days after a single injection. In contrast, the effects of SR141716A abate in mice 2 days after injection. These studies demonstrate the functional outcome of CB1 antagonist modification and open the path for development of long-lasting CB1 antagonists.


Asunto(s)
Antagonistas de Receptores de Cannabinoides/metabolismo , Antagonistas de Receptores de Cannabinoides/farmacología , Nitratos/metabolismo , Nitratos/farmacología , Piperidinas/metabolismo , Piperidinas/farmacología , Pirazoles/metabolismo , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Receptor Cannabinoide CB1/química
4.
Nature ; 481(7382): 463-8, 2012 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-22237023

RESUMEN

Exercise benefits a variety of organ systems in mammals, and some of the best-recognized effects of exercise on muscle are mediated by the transcriptional co-activator PPAR-γ co-activator-1 α (PGC1-α). Here we show in mouse that PGC1-α expression in muscle stimulates an increase in expression of FNDC5, a membrane protein that is cleaved and secreted as a newly identified hormone, irisin. Irisin acts on white adipose cells in culture and in vivo to stimulate UCP1 expression and a broad program of brown-fat-like development. Irisin is induced with exercise in mice and humans, and mildly increased irisin levels in the blood cause an increase in energy expenditure in mice with no changes in movement or food intake. This results in improvements in obesity and glucose homeostasis. Irisin could be therapeutic for human metabolic disease and other disorders that are improved with exercise.


Asunto(s)
Tejido Adiposo Pardo/citología , Tejido Adiposo Blanco/citología , Termogénesis , Transactivadores/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Ejercicio Físico/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hormonas/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Canales Iónicos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Modelos Animales , Células Musculares/metabolismo , Obesidad/sangre , Obesidad/inducido químicamente , Obesidad/prevención & control , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Condicionamiento Físico Animal/fisiología , Plasma/química , Grasa Subcutánea/citología , Grasa Subcutánea/efectos de los fármacos , Grasa Subcutánea/metabolismo , Termogénesis/efectos de los fármacos , Termogénesis/genética , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción , Proteína Desacopladora 1
5.
Mol Membr Biol ; 27(7): 260-73, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20929336

RESUMEN

The oral and central nervous systems (CNS) present a unique set of barriers to the delivery of important diagnostic and therapeutic agents. Extensive research over the past few years has enabled a better understanding of these physical and biological barriers based on tight cellular junctions and expression of active transporters and metabolizing enzymes at the luminal surfaces of the gastrointestinal (GI) tract and the blood-brain barrier (BBB). This review focuses on the recent understanding of transport across the GI tract and BBB and the development of nanotechnology-based delivery strategies that can enhance bioavailability of drugs. Multifunctional lipid nanosystems, such as oil-in-water nanoemulsions, that integrate enhancement in permeability, tissue and cell targeting, imaging, and therapeutic functions are especially promising. Based on strategic choice of edible oils, surfactants and additional surface modifiers, and different types of payloads, rationale design of multifunctional nanoemulsions can serve as a safe and effective delivery vehicle across oral and CNS barriers.


Asunto(s)
Fármacos del Sistema Nervioso Central/farmacología , Portadores de Fármacos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Emulsiones/farmacología , Nanotecnología/métodos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Disponibilidad Biológica , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Fármacos del Sistema Nervioso Central/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Portadores de Fármacos/metabolismo , Emulsiones/metabolismo , Tracto Gastrointestinal/metabolismo , Humanos , Uniones Estrechas/metabolismo
6.
Biotechnol Prog ; 37(1): e3061, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32748555

RESUMEN

Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8-/- Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)-fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX-/- and Gmds-/-, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX-/- and Gmds-/- host cells could replace Fut8-/- CHO cells for clinical manufacturing of antibody therapeutics.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Carbohidrato Epimerasas/antagonistas & inhibidores , Fucosa/metabolismo , Guanosina Difosfato/metabolismo , Hidroliasas/antagonistas & inhibidores , Cetona Oxidorreductasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Secuencia de Bases , Células CHO , Sistemas CRISPR-Cas , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Cricetinae , Cricetulus , Glicosilación , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Inmunoglobulina G/inmunología , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/metabolismo , Receptores de IgG/metabolismo
7.
Methods Enzymol ; 593: 217-235, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28750804

RESUMEN

Detailed characterization of the ligand-binding motifs and structure-function correlates of the principal GPCRs of the endocannabinoid-signaling system, the cannabinoid 1 (CB1R) and cannabinoid 2 (CB2R) receptors, is essential to inform the rational design of drugs that modulate CB1R- and CB2R-dependent biosignaling for therapeutic gain. We discuss herein an experimental paradigm termed "ligand-assisted protein structure" (LAPS) that affords a means of characterizing, at the amino acid level, CB1R and CB2R structural features key to ligand engagement and receptor-dependent information transmission. For this purpose, LAPS integrates three key disciplines and methodologies: (a) medicinal chemistry: design and synthesis of high-affinity, pharmacologically active probes as reporters capable of reacting irreversibly with particular amino acids at (or in the immediate vicinity of) the ligand-binding domain of the functionally active receptor; (b) molecular and cellular biology: introduction of discrete, conservative point mutations into the target GPCR and determination of their effect on probe binding and pharmacological activity; (c) analytical chemistry: identification of the site(s) of probe-GPCR interaction through focused, bottom-up, amino acid-level proteomic identification of the probe-receptor complex using liquid chromatography tandem mass spectrometry. Subsequent in silico methods including ligand docking and computational modeling provide supplementary data on the probe-receptor interaction as defined by LAPS. Examples of LAPS as applied to human CB2R orthosteric binding site characterization for a biarylpyrazole antagonist/inverse agonist and a classical cannabinoid agonist belonging to distinct chemical classes of cannabinergic compounds are given as paradigms for further application of this methodology to other therapeutic protein targets. LAPS is well positioned to complement other experimental and in silico methods in contemporary structural biology such as X-ray crystallography.


Asunto(s)
Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB2/química , Secuencia de Aminoácidos , Sitios de Unión , Agonistas de Receptores de Cannabinoides/química , Antagonistas de Receptores de Cannabinoides/química , Cannabinoides , Humanos , Ligandos , Modelos Moleculares , Unión Proteica
8.
Br J Pharmacol ; 174(15): 2545-2562, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28516479

RESUMEN

BACKGROUND AND PURPOSE: CB1 receptor signalling is canonically mediated through inhibitory Gαi proteins, but occurs through other G proteins under some circumstances, Gαs being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gαi blockade, CB1 /D2 receptor co-stimulation, CB1 agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB1 receptor signalling. EXPERIMENTAL APPROACH: CB1 receptors were expressed in HEK cells at different levels, and signalling characterized for cAMP by real-time BRET biosensor -CAMYEL - and for phospho-ERK by AlphaScreen. Homogenate and whole cell radioligand binding assays were performed to characterize AM6544, a novel irreversible CB1 receptor antagonist. KEY RESULTS: In HEK cells expressing high levels of CB1 receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Δ9 -THC and BAY59-3074 increased cAMP only in high-expressing cells pretreated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than the canonical inhibitory pathway. Pharmacological CB1 receptor knockdown and Gαi 1 supplementation restored canonical Gαi signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was Gαi -mediated. CONCLUSION AND IMPLICATIONS: CB1 receptor coupling to opposing G proteins is determined by both receptor and G protein expression levels, which underpins a mechanism for non-canonical signalling in a fashion consistent with Gαs signalling. CB1 receptors mediate opposite consequences in endpoints such as tumour viability depending on expression levels; our results may help to explain such effects at the level of G protein coupling.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptor Cannabinoide CB1/metabolismo , Transducción de Señal , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ligandos , Receptor Cannabinoide CB1/agonistas , Relación Estructura-Actividad
9.
J Med Chem ; 59(1): 44-60, 2016 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-26529344

RESUMEN

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.


Asunto(s)
Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB1/efectos de los fármacos , Marcadores de Afinidad , Sitio Alostérico , Animales , Arrestinas/efectos de los fármacos , Arrestinas/metabolismo , Sitios de Unión/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/antagonistas & inhibidores , Ciclohexanoles/farmacología , Descubrimiento de Drogas/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Indoles/farmacología , Ligandos , Modelos Moleculares , Compuestos de Fenilurea/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Ensayo de Unión Radioligante , Ratas , Relación Estructura-Actividad
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