RESUMEN
BACKGROUND: Genetic heterogeneity and consanguineous marriages make recessive inherited hearing loss in Iran the second most common genetic disorder. Only two reported pathogenic variants (c.323G>C, p.Arg108Pro and c.419A>G, p.Tyr140Cys) in the S1PR2 gene have previously been linked to autosomal recessive hearing loss (DFNB68) in two Pakistani families. We describe a segregating novel homozygous c.323G>A, p.Arg108Gln pathogenic variant in S1PR2 that was identified in four affected individuals from a consanguineous five generation Iranian family. METHODS: Whole exome sequencing and bioinformatics analysis of 116 hearing loss-associated genes was performed in an affected individual from a five generation Iranian family. Segregation analysis and 3D protein modeling of the p.Arg108 exchange was performed. RESULTS: The two Pakistani families previously identified with S1PR2 pathogenic variants presented profound hearing loss that is also observed in the affected Iranian individuals described in the current study. Interestingly, we confirmed mixed hearing loss in one affected individual. 3D protein modeling suggests that the p.Arg108 position plays a key role in ligand receptor interaction, which is disturbed by the p.Arg108Gln change. CONCLUSION: In summary, we report the third overall mutation in S1PR2 and the first report outside the Pakistani population. Furthermore, we describe a novel variant that causes an amino acid exchange (p.Arg108Gln) in the same amino acid residue as one of the previously reported Pakistani families (p.Arg108Pro). This finding emphasizes the importance of the p.Arg108 amino acid in normal hearing and confirms and consolidates the role of S1PR2 in autosomal recessive hearing loss.
Asunto(s)
Sustitución de Aminoácidos , Arginina/genética , Pérdida Auditiva/genética , Receptores de Lisoesfingolípidos/genética , Adolescente , Consanguinidad , Femenino , Humanos , Irán , Masculino , Modelos Moleculares , Linaje , Unión Proteica , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Esfingosina-1-Fosfato , Secuenciación del Exoma/métodosRESUMEN
Metabolites of sphingomyelin, as well as calcium ion fluxes, have a profound role in cellular signaling in almost all cell types. In addition, metabolites of sphingomyelin often modulate calcium signaling, either directly or indirectly. This is an interesting aspect on how lipids may wield their physiological role, as calcium is probably one of the most versatile signaling molecules in the cell, and as modulation of calcium signaling has profound effects on cellular physiology. The aim of this review is to discuss the mechanisms by which metabolites of sphingomyelin, especially the sphingolipids sphingosine and sphingosine 1-phosphate (S1P), modulate calcium fluxes, and how this may affect cellular function. In addition, the pathological aspects of sphingolipid-evoked modulation of calcium fluxes will be discussed.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Susceptibilidad a Enfermedades , Esfingolípidos/metabolismo , Animales , Humanos , Lisofosfolípidos/metabolismo , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingolípidos/química , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Thirteen new sphingosine-1-phosphate receptor 1 (S1PR1) ligands were designed and synthesized by replacing azetidine-3-carboxylic acid moiety of compound 4 with new polar groups. The in vitro binding potency of these new analogs toward S1PR1 was determined. Out of 13 new compounds, four compounds 9a, 10c, 12b, and 16b displayed high S1PR1 binding potency with IC50 values of 13.2⯱â¯3.2, 14.7⯱â¯1.7, 9.7⯱â¯1.6, and 6.3⯱â¯1.3â¯nM, respectively; further binding studies of these four ligands toward S1PR2-5 suggested they are highly selective for S1PR1 over other S1PRs. The radiosynthesis of the lead radiotracer [18F]12b was achieved with good radiochemical yield (â¼14.1%), high radiochemical purity (>98%), and good specific activity (â¼54.1 GBq/µmol, decay corrected to the end of synthesis, EOS). Ex vivo autoradiography and initial biodistribution studies in rodents were performed, suggesting that [18F]12b was able to penetrate the blood-brain barrier (BBB) with high brain uptake (0.71% ID/g at 60â¯min post-injection) and no defluorination was observed. In vitro autoradiography study in brain slices of lipopolysaccharides (LPS)-induced neuroinflammation mice indicated that SEW2871, a specific S1PR1 ligand was able to reduce the uptake of [18F]12b, suggesting [18F]12b has S1PR1 specific binding. These initial results suggested that [18F]12b has potential to be an F-18 labeled radiotracer for imaging S1PR1 in the brain of the animal in vivo.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Ácido Azetidinocarboxílico/farmacocinética , Radiofármacos/farmacocinética , Receptores de Lisoesfingolípidos/metabolismo , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Ácido Azetidinocarboxílico/síntesis química , Ácido Azetidinocarboxílico/química , Relación Dosis-Respuesta a Droga , Radioisótopos de Flúor , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Ligandos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Estructura Molecular , Tomografía de Emisión de Positrones , Radiofármacos/síntesis química , Radiofármacos/química , Receptores de Lisoesfingolípidos/química , Receptores de Esfingosina-1-Fosfato , Relación Estructura-Actividad , Distribución TisularRESUMEN
Fingolimod (FTY720) is used as an immunosuppressant for multiple sclerosis. Numerous studies indicated its neuroprotective effects in stroke. However, the mechanism remains to be elucidated. This study was intended to investigate the mechanisms of phosphorylated FTY720 (pFTY720), which was the principle active molecule in regulating astrocyte-mediated inflammatory responses induced by oxygen-glucose deprivation (OGD). Results demonstrated that pFTY720 could protect astrocytes against OGD-induced injury and inflammatory responses. It significantly decreased pro-inflammatory cytokines, including high mobility group box 1 (HMGB1) and tumour necrosis factor-α (TNF-α). Further, studies displayed that pFTY720 could prevent up-regulation of Toll-like receptor 2 (TLR2), phosphorylation of phosphoinositide 3-kinase (PI3K) and nuclear translocation of nuclear factor kappa B (NFκB) p65 subunit caused by OGD. Sphingosine-1-phosphate receptor 3 (S1PR3) knockdown could reverse the above change. Moreover, administration of TLR2/4 blocker abolished the protective effects of pFTY720. Taken together, this study reveals that pFTY720 depends on S1PR3 to protect astrocytes against OGD-induced neuroinflammation, due to inhibiting TLR2/4-PI3K-NFκB signalling pathway.
Asunto(s)
Clorhidrato de Fingolimod/farmacología , Inflamación/tratamiento farmacológico , Receptores de Lisoesfingolípidos/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Animales , Astrocitos/efectos de los fármacos , Carencia Cultural , Citocinas/genética , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod/química , Proteína HMGB1/genética , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Inflamación/genética , Inflamación/patología , FN-kappa B/genética , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Cultivo Primario de Células , Ratas , Receptores de Lisoesfingolípidos/química , Transducción de Señal/efectos de los fármacos , Receptores de Esfingosina-1-Fosfato , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Sphingosine-1-phosphate (S1P) is a lipid mediator that can activate five cell membrane G protein-coupled receptors (GPCRs) which carry a variety of essential functions and are promising drug targets. S1P is composed of a polar zwitterionic head-group and a hydrophobic alkyl chain. This implies an activation mechanism of its cognate receptor that must be significantly different from what is known for prototypical GPCRs (ie receptor to small hydrophilic ligands). Here we aim to identify the structural features responsible for S1P agonism by combining molecular dynamics simulations and functional assays using S1P analogs of different alkyl chain lengths. We propose that high affinity binding involves polar interactions between the lipid head-group and receptor side chains while activation is due to hydrophobic interactions between the lipid tail and residues in a distinct binding site. We observe that ligand efficacy is directly related to alkyl chain length but also varies with receptor subtypes in correlation with the size of this binding pocket. Integrating experimental and computational data, we propose an activation mechanism for the S1P receptors involving agonist-induced conformational events that are conserved throughout class A GPCRs.
Asunto(s)
Lípidos/química , Receptores Acoplados a Proteínas G/química , Animales , Sitios de Unión , Células CHO , Cricetulus , Ligandos , Metabolismo de los Lípidos , Lisofosfolípidos/química , Lisofosfolípidos/metabolismo , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/metabolismo , Relación Estructura-ActividadRESUMEN
Activation of the first sphingosine-1-phosphate receptor (S1PR1 ) promotes permeability of the blood brain barrier, astrocyte and neuronal protection, and lymphocyte egress from secondary lymphoid tissues. Although an agonist often activates the S1PR1 , the receptor exhibits high levels of basal activity. In this study, we performed long-timescale molecular dynamics and accelerated molecular dynamics (aMD) simulations to investigate activation mechanisms of the ligand-free (apo) S1PR1 . In the aMD enhanced sampling simulations, we observed four independent events of activation, which is characterized by close interaction between Y3117.53 and Y2215.58 and increased distance between the intracellular ends of transmembrane (TM) helices 3 and 6. Although TM helices TM3, TM6, TM5 and, TM7 are associated with GPCR activation, we discovered that their movements are not necessarily correlated during activation. Instead, TM5 showed a decreased correlation with each of these regions during activation. During activation of the apo receptor, Y2215.58 and Y3117.53 became more solvated, because a water channel formed in the intracellular pocket. Additionally, a lipid molecule repeatedly entered the receptor between the extracellular ends of TM1 and TM7, providing important insights into the pathway of ligand entry into the S1PR1 .
Asunto(s)
Simulación por Computador , Simulación de Dinámica Molecular , Receptores de Lisoesfingolípidos/química , Humanos , Estructura Secundaria de Proteína , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
The bioactive lipid, sphingosine 1-phosphate (S1P) binds to a family of G protein-coupled receptors, termed S1P1-S1P5. These receptors function in, for example, the cardiovascular system to regulate vascular barrier integrity and tone, the nervous system to regulate neuronal differentiation, myelination and oligodendrocyte/glial cell survival and the immune system to regulate T- and B-cell subsets and trafficking. S1P receptors also participate in the pathophysiology of autoimmunity, inflammatory disease, cancer, neurodegeneration and others. In this review, we describe how S1P1 can form a complex with G-protein and ß-arrestin, which function together to regulate effector pathways. We also discuss the role of the S1P1-Platelet derived growth factor receptor ß functional complex (which deploys G-protein/ß-arrestin and receptor tyrosine kinase signaling) in regulating cell migration. Possible mechanisms by which different S1P-chaperones, such as Apolipoprotein M-High-Density Lipoprotein induce biological programmes in cells are also described. Finally, the role of S1P1 in health and disease and as a target for clinical intervention is appraised.
Asunto(s)
Apolipoproteínas/metabolismo , Proteínas de Unión al GTP/metabolismo , Mamíferos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , beta-Arrestinas/metabolismo , Animales , Sitios de Unión , Movimiento Celular , Humanos , Unión Proteica , Receptores de Lisoesfingolípidos/química , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de SeñalRESUMEN
Fingolimod (FTY720, Gilenya), a sphingosine-1-phosphate receptor (S1PR) modulator, is one of the first-line immunomodulatory therapies for treatment of relapsing-remitting multiple sclerosis (MS). Human S1PR1 variants have been reported to have functional heterogeneity in vitro, suggesting that S1PR1 function may influence FTY720 efficacy. In this study, we examined the influence of S1PR1 phosphorylation on response to FTY720 in neuroinflammation. We found that mice carrying a phosphorylation-defective S1pr1 gene [S1PR1(S5A) mice] were refractory to FTY720 treatment in MOG35-55-immunized and Th17-mediated experimental autoimmune encephalomyelitis (EAE) models. Long-term treatment with FTY720 induced significant lymphopenia and suppressed Th17 response in the peripheral immune system via downregulating STAT3 phosphorylation in both WT and S1PR1(S5A) mice. However, FTY720 did not effectively prevent neuroinflammation in the S1PR1(S5A) EAE mice as a result of encephalitogenic cells expressing C-C chemokine receptor 6 (CCR6). Combined treatment with FTY720 and anti-CCR6 delayed disease progression in S1PR1(S5A) EAE mice, suggesting that CCR6-mediated cell trafficking can overcome the effects of FTY720. This work may have translational relevance regarding FTY720 efficacy in MS patients and suggests that cell type-specific therapies may enhance therapeutic efficacy in MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Clorhidrato de Fingolimod/farmacología , Inmunosupresores/farmacología , Receptores de Lisoesfingolípidos/química , Animales , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple , Fosforilación , Receptores CCR6/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
Progressive hearing loss is very common in the population but we still know little about the underlying pathology. A new spontaneous mouse mutation (stonedeaf, stdf ) leading to recessive, early-onset progressive hearing loss was detected and exome sequencing revealed a Thr289Arg substitution in Sphingosine-1-Phosphate Receptor-2 (S1pr2). Mutants aged 2 weeks had normal hearing sensitivity, but at 4 weeks most showed variable degrees of hearing impairment, which became severe or profound in all mutants by 14 weeks. Endocochlear potential (EP) was normal at 2 weeks old but was reduced by 4 and 8 weeks old in mutants, and the stria vascularis, which generates the EP, showed degenerative changes. Three independent mouse knockout alleles of S1pr2 have been described previously, but this is the first time that a reduced EP has been reported. Genomic markers close to the human S1PR2 gene were significantly associated with auditory thresholds in the 1958 British Birth Cohort (n = 6099), suggesting involvement of S1P signalling in human hearing loss. The finding of early onset loss of EP gives new mechanistic insight into the disease process and suggests that therapies for humans with hearing loss due to S1P signalling defects need to target strial function.
Asunto(s)
Sustitución de Aminoácidos , Pérdida Auditiva Sensorineural/genética , Receptores de Lisoesfingolípidos/genética , Animales , Umbral Auditivo , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos , Pérdida Auditiva Sensorineural/fisiopatología , Humanos , Ratones , Persona de Mediana Edad , Receptores de Lisoesfingolípidos/química , Receptores de Esfingosina-1-Fosfato , Estría Vascular/fisiología , Secuenciación del ExomaRESUMEN
The binding process through the membrane bilayer of lipid-like ligands to a protein target is an important but poorly explored recognition process at the atomic level. In this work we succeeded in resolving the binding of the lipid inhibitor ML056 to the sphingosine-1-phosphate receptor 1 (S1P1R) using unbiased molecular dynamics simulations with an aggregate sampling of over 800 µs. The binding pathway is a multi-stage process consisting of the ligand diffusing in the bilayer leaflet to contact a "membrane vestibule" at the top of TM 7, subsequently moving from this lipid-facing vestibule to the orthosteric binding cavity through a channel formed by TMs 1 and 7 and the N-terminal of the receptor. Unfolding of the N-terminal alpha-helix increases the volume of the channel upon ligand entry, helping to reach the crystallographic pose that also corresponds to the predicted favorable pose. The relaxation timescales of the binding process show that the binding of the ligand to the "membrane vestibule" is the rate-limiting step in the multi microseconds timescale. We comment on the significance and parallels of the binding process in the context of other binding studies.
Asunto(s)
Transporte Biológico , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Ligandos , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
Recent developments in methodologies for genomic analyses have enabled a significant advance in understanding of the river buffalo genome. The S1PR1 gene has been mapped to buffalo chromosome 6 and bovine chromosome 3; this gene is of interest as it is a candidate for marbling in meat, an important economic trait. Here, we performed next generation sequencing in a buffalo BAC DNA clone and obtained a 54.5-kb sequence encompassing the entire buffalo S1PR1 gene as well as the 27 kb upstream region and the 22 kb downstream region. The gene had a total length of 4716 bp, including three exons and two introns; exons 1 and 2 were classified as non-protein-coding. In comparison with homologues from other species, the structural organization of buffalo S1PR1 was closest to that of the goat and in both species exon 2 of the gene was non-protein-coding. One hundred and nine repetitive elements were found within the buffalo gene and its boundary regions, with 50 SINE repeats being the most abundant. Alignment of S1PR1 sequences from the Murrah and Mediterranean breeds revealed two nucleotide substitutions (g.1176C>G and g.2740T>C), which represent potential SNPs that could be used in further studies of buffalo genetic structure.
Asunto(s)
Búfalos/metabolismo , Genes , Receptores de Lisoesfingolípidos/genética , Secuencia de Aminoácidos , Animales , Búfalos/genética , ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.
Asunto(s)
Cristalografía por Rayos X , Sitios de Unión , Cromatografía en Gel , Humanos , Ligandos , Modelos Moleculares , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/química , Bibliotecas de Moléculas PequeñasRESUMEN
Sphingolipids represent an important class of bioactive signaling lipids which have key roles in numerous cellular processes. Over the last few decades, the levels of bioactive sphingolipids and/or their metabolizing enzymes have been realized to be important factors involved in disease development and progression, most notably in cancer. Targeting sphingolipid-metabolizing enzymes in disease states has been the focus of many studies and has resulted in a number of pharmacological inhibitors, with some making it into the clinic as therapeutics. In order to better understand the regulation of sphingolipid-metabolizing enzymes as well as to develop much more potent and specific inhibitors, the field of sphingolipids has recently taken a turn toward structural biology. The last decade has seen the structural determination of a number of sphingolipid enzymes and effector proteins. In these terms, one of the most complete arms of the sphingolipid pathway is the sphingosine-1-phosphate (S1P) arm. The structures of proteins involved in the function and regulation of S1P are being used to investigate further the regulation of said proteins as well as in the design and development of inhibitors as potential therapeutics.
Asunto(s)
Lisofosfolípidos/metabolismo , Modelos Moleculares , Sistemas de Mensajero Secundario , Esfingosina/análogos & derivados , Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Animales , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Lisofosfolípidos/química , Moduladores del Transporte de Membrana , Conformación Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/química , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
1. Ponesimod [(R)-5-[3-chloro-4-(-2,3-dihydroxy-propoxy)-benzylidene]-2-propylimino-3-o-tolyl-thiazolidin-4-one] is an orally administered, selective S1P1 receptor modulator that blocks the egress of lymphocytes from lymphoid organs and reduces the availability of circulating effector T/B-cells. 2. The mass balance, pharmacokinetics and metabolism of 40 mg (14)C-ponesimod were investigated in six healthy male subjects. The total radioactivity in whole blood, plasma, urine, faeces and expired CO2 was determined by liquid scintillation counting. Metabolite profiling was performed by high-performance liquid chromatography and detection by mass spectrometry. 3. The majority of the radioactivity (% of administered dose) was recovered in faeces (57.3-79.6%), followed by urine (10.3-18.4%) and a small proportion in CO2 from expired air (0.6-1.9%). The average cumulative recovery (mass balance) of (14)C-associated radioactivity in faeces and urine was 77.9% of the administered dose. Unchanged ponesimod made up 25.9% of total radioactivity in faeces; none was detected in urine. Ponesimod was extensively metabolised and two pharmacologically inactive metabolites, M12 (ACT-204426) and M13 (ACT-338375), were detected in the circulation. M12 corresponded to 8.1% and M13 to 25.7% of the total drug-related radioactive exposure (AUC0-∞) in plasma. M12 was highly abundant in faeces (22.3% of total radioactivity) and to a smaller extent in urine (2.5% of total radioactivity).
Asunto(s)
Tiazoles/farmacocinética , Administración Oral , Humanos , Masculino , Persona de Mediana Edad , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Conteo por Cintilación , Tiazoles/efectos adversos , Tiazoles/química , Tiazoles/metabolismoRESUMEN
We have discovered a series of triazole/oxazole-containing 2-substituted 2-aminopropane-1,3-diol derivatives as potent and selective S1P1 agonists (prodrugs) based on pharmacophore-guided rational design. Most compounds showed high affinity and selectivity for S1P1 receptor. Compounds 19b, 19d and 19p displayed clear dose responsiveness in the lymphocyte reduction model when administered orally at doses of 0.3, 1.0, 3.0 mg/kg with reduced effect on heart rate. These three compounds were also identified to have favorable pharmacokinetic properties.
Asunto(s)
Diseño de Fármacos , Oxazoles/química , Oxazoles/farmacología , Receptores de Lisoesfingolípidos/agonistas , Triazoles/química , Triazoles/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Frecuencia Cardíaca/efectos de los fármacos , Linfocitos/efectos de los fármacos , Masculino , Modelos Moleculares , Conformación Molecular , Oxazoles/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores de Lisoesfingolípidos/química , Relación Estructura-Actividad , Triazoles/farmacocinéticaRESUMEN
G protein-coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor ß-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of ß-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.
Asunto(s)
Receptores Acoplados a Proteínas G/agonistas , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/química , Sitio Alostérico , Animales , Bioensayo/métodos , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , AMP Cíclico/química , Descubrimiento de Drogas , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente , Unión Proteica , Transporte de Proteínas , Ratas , Reproducibilidad de los Resultados , beta-Arrestinas/metabolismoRESUMEN
The sphingosine 1 phosphate receptor family has been studied widely since the initial discovery of its first member, endothelium differentiation gene 1. Since this initial discovery, the family has been renamed and the primary member of the family, the S1P1 receptor, has been targeted for a variety of disease indications and successfully drugged for the treatment of patients with relapsing multiple sclerosis. Recently, the three-dimensional structure of the S1P1 receptor has been determined by X-ray crystallography and the specifics of the sphingosine 1 phosphate ligand binding pocket mapped. Key structural features for the S1P1 receptor will be reviewed and the potential binding modes of additional pharmacologically active agents against the receptor will be analyzed in an effort to better understand the structural basis of important receptor-ligand interactions.
Asunto(s)
Receptores de Lisoesfingolípidos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Lisofosfolípidos/química , Lisofosfolípidos/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/metabolismoRESUMEN
The zwitterionic lysophospholipid Sphingosine 1-Phosphate (S1P) is a pleiotropic mediator of physiology and pathology. The synthesis, transport, and degradation of S1P are tightly regulated to ensure that S1P is present in the proper concentrations in the proper location. The binding of S1P to five G protein-coupled S1P receptors regulates many physiological systems, particularly the immune and vascular systems. Our understanding of the functions of S1P has been aided by the tractability of the system to both chemical and genetic manipulation. Chemical modulators have been generated to affect most of the known components of S1P biology, including agonists of S1P receptors and inhibitors of enzymes regulating S1P production and degradation. Genetic knockouts and manipulations have been similarly engineered to disrupt the functions of individual S1P receptors or enzymes involved in S1P metabolism. This chapter will focus on the development and utilization of these chemical and genetic tools to explore the complex biology surrounding S1P and its receptors, with particular attention paid to the in vivo findings that these tools have allowed for.
Asunto(s)
Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/genética , Animales , Técnicas Genéticas , Humanos , Lisofosfolípidos/química , Lisofosfolípidos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P(1-5). Among them, S1P(3) has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P(3). Here, aiming to identify novel S1P(3) antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2-10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Vasodilatadores/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Células Cultivadas , Técnicas In Vitro , Masculino , Ratones , Modelos Moleculares , Contracción Muscular/efectos de los fármacos , Fragmentos de Péptidos/química , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Esfingosina-1-Fosfato , Vasodilatadores/químicaRESUMEN
Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator which activates G protein-coupled sphingosine 1-phosphate receptors and thus evokes a variety of cell and tissue responses including lymphocyte trafficking, endothelial development, integrity, and maturation. We performed five all-atom 700 ns molecular dynamics simulations of the sphingosine 1-phosphate receptor 1 (S1P1) based on recently released crystal structure of that receptor with an antagonist. We found that the initial movements of amino acid residues occurred in the area of highly conserved W2696·48 in TM6 which is close to the ligand binding location. Those residues located in the central part of the receptor and adjacent to kinks of TM helices comprise of a transmission switch. Side chains movements of those residues were coupled to the movements of water molecules inside the receptor which helped in the gradual opening of intracellular part of the receptor. The most stable parts of the protein were helices TM1 and TM2, while the largest movement was observed for TM7, possibly due to the short intracellular part starting with a helix kink at P7·5°, which might be the first helix to move at the intracellular side. We show for the first time the detailed view of the concerted action of the transmission switch and Trp (W6·48) rotamer toggle switch leading to redirection of water molecules flow in the central part of the receptor. That event is a prerequisite for subsequent changes in intracellular part of the receptor involving water influx and opening of the receptor structure.