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2.
Nat Methods ; 17(8): 777-787, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32661425

RESUMEN

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.


Asunto(s)
Simulación de Dinámica Molecular , Receptores Acoplados a Proteínas G/química , Programas Informáticos , Metaboloma , Modelos Moleculares , Conformación Proteica
3.
Addict Biol ; 26(5): e13017, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33559278

RESUMEN

Cocaine not only increases brain dopamine levels but also activates the sigma1 receptor (σ1 R) that in turn regulates orexigenic receptor function. Identification of interactions involving dopamine D1 (D1 R), ghrelin (GHS-R1a ), and σ1 receptors have been addressed by biophysical techniques and a complementation approach using interfering peptides. The effect of cocaine on receptor functionality was assayed by measuring second messenger, cAMP and Ca2+ , levels. The effect of acute or chronic cocaine administration on receptor complex expression was assayed by in situ proximity ligation assay. In silico procedures were used for molecular model building. σ1 R KO mice were used for confirming involvement of this receptor. Upon identification of protomer interaction and receptor functionality, a unique structural model for the macromolecular complex formed by σ1 R, D1 R, and GHS-R1a is proposed. The functionality of the complex, able to couple to both Gs and Gq proteins, is affected by cocaine binding to the σ1 R, as confirmed using samples from σ1 R-/- mice. The expression of the macromolecular complex was differentially affected upon acute and chronic cocaine administration to rats. The constructed 3D model is consistent with biochemical, biophysical, and available structural data. The σ1 R, D1 R, and GHS-R1a complex constitutes a functional unit that is altered upon cocaine binding to the σ1 R. Remarkably, the heteromer can simultaneously couple to two G proteins, thus allowing dopamine to signal via Ca2+ and ghrelin via cAMP. The anorexic action of cocaine is mediated by such complex whose expression is higher after acute than after chronic administration regimens.


Asunto(s)
Cocaína/farmacología , Hambre/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Dopamina/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Masculino , Ratones , Neuronas/efectos de los fármacos , Ratas , Receptores de Dopamina D1/metabolismo , Receptores de Ghrelina/metabolismo , Receptores sigma , Receptor Sigma-1
4.
Biophys J ; 114(6): 1285-1294, 2018 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-29590586

RESUMEN

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.


Asunto(s)
Opsinas de los Conos/metabolismo , Regeneración , Retinaldehído/metabolismo , Sitios de Unión , Opsinas de los Conos/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Termodinámica
5.
J Chem Inf Model ; 58(5): 1074-1082, 2018 05 29.
Artículo en Inglés | MEDLINE | ID: mdl-29671585

RESUMEN

The muscarinic M2 acetylcholine receptor, one of the few G-protein coupled receptors that has not only been crystallized in both active and inactive conformations but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14 µs in total) to study conformational changes triggered by the inverse agonist R-(-)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M2 receptor (PBD ID 4MQS ) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted us to identify the sequence of events in the deactivation mechanism of the M2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY2119620 when bound simultaneously with the orthosteric agonist iperoxo and find that it restricts the conformation of Trp4227.35 in a position that modulates the orientation of the Tyr4267.39 at the orthosteric-binding pocket.


Asunto(s)
Simulación de Dinámica Molecular , Receptor Muscarínico M2/metabolismo , Regulación Alostérica/efectos de los fármacos , Agonismo Inverso de Drogas , Ligandos , Conformación Proteica/efectos de los fármacos , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inhibidores
6.
Pharmacol Res Perspect ; 10(4): e00978, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35762357

RESUMEN

In this study, we report the ß1 -adrenoceptor binding kinetics of several clinically relevant ß1/2 -adrenoceptor (ß1/2 AR) agonists and antagonists. [3 H]-DHA was used to label CHO-ß1 AR for binding studies. The kinetics of ligand binding was assessed using a competition association binding method. Ligand physicochemical properties, including logD7.4 and the immobilized artificial membrane partition coefficient (KIAM ), were assessed using column-based methods. Protein Data Bank (PDB) structures and hydrophobic and electrostatic surface maps were constructed in PyMOL. We demonstrate that the hydrophobic properties of a molecule directly affect its kinetic association rate (kon ) and affinity for the ß1 AR. In contrast to our findings at the ß2 -adrenoceptor, KIAM , reflecting both hydrophobic and electrostatic interactions of the drug with the charged surface of biological membranes, was no better predictor than simple hydrophobicity measurements such as clogP or logD7.4 , at predicting association rate. Bisoprolol proved kinetically selective for the ß1 AR subtype, dissociating 50 times slower and partly explaining its higher measured affinity for the ß1 AR. We speculate that the association of positively charged ligands at the ß1 AR is curtailed somewhat by its predominantly neutral/positive charged extracellular surface. Consequently, hydrophobic interactions in the ligand-binding pocket dominate the kinetics of ligand binding. In comparison at the ß2 AR, a combination of hydrophobicity and negative charge attracts basic, positively charged ligands to the receptor's surface promoting the kinetics of ligand binding. Additionally, we reveal the potential role kinetics plays in the on-target and off-target pharmacology of clinically used ß-blockers.


Asunto(s)
Antagonistas Adrenérgicos beta , Antagonistas Adrenérgicos beta/farmacología , Cinética , Ligandos
7.
Pharmacol Res Perspect ; 10(5): e00994, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36029004

RESUMEN

G protein-coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands that bind them. Ligands exert their action via the interactions in the ligand binding pocket. We hypothesized that there is a common set of receptor interactions made by ligands of diverse structures that mediate their action and that among a large dataset of different ligands, the functionally important interactions will be over-represented. We computationally docked ~2700 known ß2AR ligands to multiple ß2AR structures, generating ca 75 000 docking poses and predicted all atomic interactions between the receptor and the ligand. We used machine learning (ML) techniques to identify specific interactions that correlate with the agonist or antagonist activity of these ligands. We demonstrate with the application of ML methods that it is possible to identify the key interactions associated with agonism or antagonism of ligands. The most representative interactions for agonist ligands involve K972.68×67 , F194ECL2 , S2035.42×43 , S2045.43×44 , S2075.46×641 , H2966.58×58 , and K3057.32×31 . Meanwhile, the antagonist ligands made interactions with W2866.48×48 and Y3167.43×42 , both residues considered to be important in GPCR activation. The interpretation of ML analysis in human understandable form allowed us to construct an exquisitely detailed structure-activity relationship that identifies small changes to the ligands that invert their pharmacological activity and thus helps to guide the drug discovery process. This approach can be readily applied to any drug target.


Asunto(s)
Descubrimiento de Drogas , Aprendizaje Automático , Receptores Adrenérgicos beta 2 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Receptores Adrenérgicos beta 2/química
8.
J Chem Theory Comput ; 15(3): 1884-1895, 2019 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-30776226

RESUMEN

Activity cliffs (ACs) are an important type of structure-activity relationship in medicinal chemistry where small structural changes result in unexpectedly large differences in biological activity. Being able to predict these changes would have a profound impact on lead optimization of drug candidates. Free-energy perturbation is an ideal tool for predicting relative binding energy differences for small structural modifications, but its performance for ACs is unknown. Here, we show that FEP can on average predict ACs to within 1.39 kcal/mol of experiment (∼1 log unit of activity). We performed FEP calculations with two different software methods: Schrödinger-Desmond FEP+ and GROMACS implementations. There was qualitative agreement in the results from the two methods, and quantitatively the error for one data set was identical, 1.43 kcal/mol, but FEP+ performed better in the second, with errors of 1.17 versus 1.90 kcal/mol. The results have far-reaching implications, suggesting well-implemented FEP calculations can have a major impact on computational drug design.


Asunto(s)
Diseño Asistido por Computadora , Diseño de Fármacos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Termodinámica , Bases de Datos de Proteínas , Humanos , Modelos Biológicos , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas/química , Programas Informáticos , Relación Estructura-Actividad
9.
Mol Neurobiol ; 56(2): 1196-1210, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29876881

RESUMEN

Despite ancient knowledge on cocaine appetite-suppressant action, the molecular basis of such fact remains unknown. Addiction/eating disorders (e.g., binge eating, anorexia, bulimia) share a central control involving reward circuits. However, we here show that the sigma-1 receptor (σ1R) mediates cocaine anorectic effects by interacting in neurons with growth/hormone/secretagogue (ghrelin) receptors. Cocaine increases colocalization of σ1R and GHS-R1a at the cell surface. Moreover, in transfected HEK-293T and neuroblastoma SH-SY5Y cells, and in primary neuronal cultures, pretreatment with cocaine or a σ1R agonist inhibited ghrelin-mediated signaling, in a similar manner as the GHS-R1a antagonist YIL-781. Results were similar in G protein-dependent (cAMP accumulation and calcium release) and in partly dependent or independent (ERK1/2 phosphorylation and label-free) assays. We provide solid evidence for direct interaction between receptors and the functional consequences, as well as a reliable structural model of the macromolecular σ1R-GHS-R1a complex, which arises as a key piece in the puzzle of the events linking cocaine consumption and appetitive/consummatory behaviors.


Asunto(s)
Cocaína/farmacología , Cuerpo Estriado/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Ghrelina/metabolismo , Neuronas/efectos de los fármacos , Ácido Oleanólico/análogos & derivados , Receptores sigma/metabolismo , Saponinas/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Neuronas/citología , Neuronas/metabolismo , Ácido Oleanólico/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Receptor Sigma-1
10.
J Clin Endocrinol Metab ; 102(7): 2433-2442, 2017 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-28419241

RESUMEN

Context: Central congenital hypothyroidism (CCH) is an underdiagnosed disorder characterized by deficient production and bioactivity of thyroid-stimulating hormone (TSH) leading to low thyroid hormone synthesis. Thyrotropin-releasing hormone (TRH) receptor (TRHR) defects are rare recessive disorders usually associated with incidentally identified CCH and short stature in childhood. Objectives: Clinical and genetic characterization of a consanguineous family of Roma origin with central hypothyroidism and identification of underlying molecular mechanisms. Design: All family members were phenotyped with thyroid hormone profiles, pituitary magnetic resonance imaging, TRH tests, and dynamic tests for other pituitary hormones. Candidate TRH, TRHR, TSHB, and IGSF1 genes were screened for mutations. A mutant TRHR was characterized in vitro and by molecular modeling. Results: A homozygous missense mutation in TRHR (c.392T > C; p.I131T) was identified in an 8-year-old boy with moderate hypothyroidism (TSH: 2.61 mIU/L, Normal: 0.27 to 4.2; free thyroxine: 9.52 pmol/L, Normal: 10.9 to 25.7) who was overweight (body mass index: 20.4 kg/m2, p91) but had normal stature (122 cm; -0.58 standard deviation). His mother, two brothers, and grandmother were heterozygous for the mutation with isolated hyperthyrotropinemia (TSH: 4.3 to 8 mIU/L). The I131T mutation, in TRHR intracellular loop 2, decreases TRH affinity and increases the half-maximal effective concentration for signaling. Modeling of TRHR-Gq complexes predicts that the mutation disrupts the interaction between receptor and a hydrophobic pocket formed by Gq. Conclusions: A unique missense TRHR defect identified in a consanguineous family is associated with central hypothyroidism in homozygotes and hyperthyrotropinemia in heterozygotes, suggesting compensatory elevation of TSH with reduced biopotency. The I131T mutation decreases TRH binding and TRHR-Gq coupling and signaling.


Asunto(s)
Hipotiroidismo Congénito/genética , Predisposición Genética a la Enfermedad , Receptores de Hormona Liberadora de Tirotropina/genética , Activación Transcripcional/genética , Niño , Simulación por Computador , Hipotiroidismo Congénito/diagnóstico , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Humanos , Masculino , Mutación Missense , Linaje , Enfermedades Raras , Pruebas de Función de la Tiroides , Tirotropina/metabolismo
11.
Sci Rep ; 7(1): 2134, 2017 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-28522847

RESUMEN

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D3R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D3R dimerization.


Asunto(s)
Antagonistas de Dopamina/farmacología , Haloperidol/farmacología , Multimerización de Proteína , Receptores de Dopamina D3/química , Espiperona/farmacología , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Receptores de Dopamina D3/antagonistas & inhibidores , Receptores de Dopamina D3/metabolismo
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