RESUMEN
Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.
Asunto(s)
Ghrelina/química , Ghrelina/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Acilación , Animales , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.
Asunto(s)
Dopamina/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Multimerización de Proteína , Receptores de Dopamina D2/química , Receptores de Ghrelina/química , Transducción de Señal , Dopamina/genética , Dopamina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Ghrelina/genética , Receptores de Ghrelina/metabolismoRESUMEN
How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Receptores de Ghrelina/química , Transferencia de Energía , Conformación ProteicaRESUMEN
In this study, we used the Martini Coarse-Grained model with no applied restraints to predict the binding mode of some peptides to G-Protein Coupled Receptors (GPCRs). Both the Neurotensin-1 and the chemokine CXCR4 receptors were used as test cases. Their ligands, NTS8-13 and CVX15 peptides, respectively, were initially positioned in the surrounding water box. Using a protocol based on Replica Exchange Molecular Dynamics (REMD), both opening of the receptors and entry of the peptides into their dedicated pockets were observed on the µs time-scale. After clustering, the most statistically representative orientations were closely related to the X-ray structures of reference, sharing both RMSD lower than 3 Å and most of the native contacts. These results demonstrate that such a model, that does not require access to tremendous computational facilities, can be helpful in predicting peptide binding to GPCRs as well as some of the receptor's conformational changes required for this key step. We also discuss how such an approach can now help to predict, de novo, the interactions of GPCRs with other intra- or extracellular peptide/protein partners.
Asunto(s)
Simulación de Dinámica Molecular , Neurotensina/metabolismo , Péptidos Cíclicos/metabolismo , Receptores CXCR4/metabolismo , Disulfuros/química , Neurotensina/química , Péptidos Cíclicos/química , Unión Proteica , Conformación Proteica , Receptores CXCR4/químicaRESUMEN
Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an openâclosed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr(160) phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.
Asunto(s)
Ciclina A/metabolismo , Ciclina D1/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ciclina A/química , Ciclina D1/química , Quinasa 2 Dependiente de la Ciclina/química , Quinasa 4 Dependiente de la Ciclina/química , Simulación de Dinámica Molecular , Movimiento (Física) , Conformación Proteica , Solventes/química , Relación Estructura-Actividad , Agua/químicaRESUMEN
The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, µ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.
Asunto(s)
Ghrelina/metabolismo , Receptores de Ghrelina/química , Receptores de Ghrelina/metabolismo , Transducción de Señal/fisiología , Arrestina/metabolismo , Arrestina/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Agonismo Inverso de Drogas , Fluorescencia , Ghrelina/farmacología , Humanos , Ligandos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/farmacología , Conformación Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ghrelina/agonistas , Relación Estructura-ActividadRESUMEN
In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ~5 kcal.mol(-1). The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2' acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P2 lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P2 in domains. This is consistent with a PI(4,5)P2 enrichment of the virus envelope as compared to the host cell membrane.
Asunto(s)
Antígenos VIH/metabolismo , Simulación de Dinámica Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/virología , Antígenos VIH/química , Humanos , Datos de Secuencia Molecular , Fosfatidilinositol 4,5-Difosfato/química , Unión Proteica , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The elastin binding protein (EBP), a spliced variant of lysosomal ß-galactosidase, is the primary receptor of elastin peptides that have been linked to emphysema, aneurysm and cancer progression. The sequences recognized by EBP share the XGXXPG consensus pattern found in numerous matrix proteins, notably in elastin where the VGVAPG motif is repeated. To delineate the elastin binding site of human EBP, we built a homology model of this protein and docked VGVAPG on its surface. Analysis of this model suggested that Gln-97 and Asp-98 were required for interaction with VGVAPG because they contribute to the definition of a pocket thought to represent the elastin binding site of EBP. Additionally, we proposed that Leu-103, Arg-107, and Glu-137 were essential residues because they could interact with VGVAPG itself. Site-directed mutagenesis experiments at these key positions validated our model. This work therefore provides the first structural data concerning the interaction of the VGVAPG with its cognate receptor. The present structural data should now allow the development of EBP-specific antagonists.
Asunto(s)
Elastina/metabolismo , Oligopéptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Chlorocebus aethiops , Cartilla de ADN , Elastina/química , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genéticaRESUMEN
After extra-cellular stimulation of G-Protein Coupled Receptors (GPCRs), GDP/GTP exchange appears as the key, rate limiting step of the intracellular activation cycle of heterotrimeric G-proteins. Despite the availability of a large number of X-ray structures, the mechanism of GDP release out of heterotrimeric G-proteins still remains unknown at the molecular level. Starting from the available X-ray structure, extensive unconstrained/constrained molecular dynamics simulations were performed on the complete membrane-anchored Gi heterotrimer complexed to GDP, for a total simulation time overcoming 500 ns. By combining Targeted Molecular Dynamics (TMD) and free energy profiles reconstruction by umbrella sampling, our data suggest that the release of GDP was much more favored on its phosphate side. Interestingly, upon the forced extraction of GDP on this side, the whole protein encountered large, collective motions in perfect agreement with those we described previously including a domain to domain motion between the two ras-like and helical sub-domains of G(α).
Asunto(s)
Biología Computacional/métodos , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Simulación de Dinámica Molecular , TermodinámicaRESUMEN
The functional properties of G protein-coupled receptors (GPCRs) are intimately associated with the different components in their cellular environment. Among them, sodium ions have been proposed to play a substantial role as endogenous allosteric modulators of GPCR-mediated signaling. However, this sodium effect and the underlying mechanisms are still unclear for most GPCRs. Here, we identified sodium as a negative allosteric modulator of the ghrelin receptor GHSR (growth hormone secretagogue receptor). Combining 23Na-nuclear magnetic resonance (NMR), molecular dynamics, and mutagenesis, we provide evidence that, in GHSR, sodium binds to the allosteric site conserved in class A GPCRs. We further leveraged spectroscopic and functional assays to show that sodium binding shifts the conformational equilibrium toward the GHSR-inactive ensemble, thereby decreasing basal and agonist-induced receptor-catalyzed G protein activation. All together, these data point to sodium as an allosteric modulator of GHSR, making this ion an integral component of the ghrelin signaling machinery.
Asunto(s)
Receptores de Ghrelina , Sodio , Regulación Alostérica , Sitio Alostérico , Ghrelina/metabolismo , Iones , Receptores de Ghrelina/metabolismo , Transducción de Señal , Sodio/metabolismoRESUMEN
Molecular dynamics with excited normal modes (MDeNM) is an enhanced sampling method for exploring conformational changes in proteins with minimal biases. The excitation corresponds to injecting kinetic energy along normal modes describing intrinsic collective motions. Herein, we developed a new automated open-source implementation, MDexciteR (https://github.com/mcosta27/MDexciteR), enabling the integration of MDeNM with two commonly used simulation programs with GPU support. Second, we generalized the method to include the excitation of principal components calculated from experimental ensembles. Finally, we evaluated whether the use of coarse-grained normal modes calculated with elastic network representations preserved the performance and accuracy of the method. The advantages and limitations of these new approaches are discussed based on results obtained for three different protein test cases: two globular and a protein/membrane system.
RESUMEN
Heterotrimeric G-proteins' activation on the intracellular side of the cell membrane is initiated by stimulation of the G-Protein Coupled Receptors (GPCRs) extra-cellular part. This two-step activation mechanism includes (1) an exchange between GDP and GTP molecules in the G(α) subunit and (2) a dissociation of the whole G(αßγ) complex into two membrane-anchored blocks, namely the isolated G(α) and G(ßγ) subunits. Although X-ray data are available for both inactive G(αßγ):GDP and active G(α):GTP complexes, intermediate steps involved in the molecular mechanism of the dissociation have not yet been addressed at the molecular level. In this study, we first built a membrane-anchored intermediate G(iαßγ):GTP complex. This model was then equilibrated by molecular dynamics simulations before the Targeted Molecular Dynamics (TMD) technique was used to force the G(α) subunit to evolve from its inactive (GDP-bound) to its active (GTP-bound) conformations, as described by available X-ray data. The TMD constraint was applied only to the G(α) subunit so that the resulting global rearrangements acting on the whole G(αßγ):GTP heterotrimer could be analyzed. We showed how these mainly local conformational changes of G(α) could initiate large domain:domain motions of the whole complex, the G(ßγ) behaving as an almost quasi-rigid block. This separation of the two G(α):GTP and G(ßγ) subunits required the loss of several interactions at the G(α):G(ßγ) interface that were reported. This study provided an atomistic view of the crucial intermediate step of the G-proteins activation, e.g., the dissociation, that could hardly be elucidated by the experiment.
Asunto(s)
Guanosina Difosfato/química , Guanosina Trifosfato/química , Proteínas de Unión al GTP Heterotriméricas/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Subunidades de Proteína/química , Cristalografía por Rayos X , Cinética , Unión Proteica , TermodinámicaRESUMEN
Nitric oxide (NO) is an important signaling molecule produced by a family of enzymes called nitric oxide synthases (NOS). Because NO is involved in various pathological conditions, the development of potent and isoform-selective NOS inhibitors is an important challenge. In the present study, the dimer of oxygenase domain of human iNOS (iNOSoxy) complexed to its natural substrate L-arginine (L-Arg) and both heme and tetrahydro-L-biopterin (BH4) cofactors was studied through multiple molecular dynamics simulations. Starting from the X-ray structure available for that complex (PDB: 1NSI ), a 16 ns equilibration trajectory was first obtained. Twelve dynamics of slow extraction of L-Arg out from the iNOSoxy active site were then performed. The steered molecular dynamics (SMD) approach was used starting from three different points of the reference trajectory for a total simulation time of 35 ns. A probable unbinding/binding pathway of L-Arg was characterized. It was suggested that a driving force directed the substrate toward the heme pocket. Key intermediate steps/residues along the access route to the active site were identified along this "funnel shape" pathway and compared to existing data. A quasi-normal mode analysis performed on the SMD data suggested that large collective motions of the protein may be involved in L-Arg binding and that opening the route to the active site in one monomer promoted an inverse, closing motion in the second monomer. Finally, our findings might help to rationalize the design of human iNOS isoform competitive inhibitors.
Asunto(s)
Arginina/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Simulación de Dinámica Molecular , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Dominio Catalítico , Bases de Datos de Proteínas , Descubrimiento de Drogas , Humanos , Movimiento , Óxido Nítrico Sintasa de Tipo II/química , Oxigenasas/química , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Especificidad por Sustrato , Agua/químicaRESUMEN
The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores de Ghrelina/química , Receptores de Ghrelina/metabolismo , Regulación Alostérica , Sitios de Unión , Membrana Celular/química , Membrana Celular/metabolismo , Cisteína/genética , Transferencia Resonante de Energía de Fluorescencia , Gangliósido G(M3)/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Mutación , Fosfatidilinositol 4,5-Difosfato/química , Conformación Proteica , Receptores de Ghrelina/genética , Transducción de SeñalRESUMEN
The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.
RESUMEN
There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.
Asunto(s)
Ghrelina , Receptores Acoplados a Proteínas G , Receptores de Ghrelina , Humanos , Ligandos , Transducción de SeñalRESUMEN
We previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a beta-turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained beta-turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1-MMP at the migration front as well as a decrease in u-PA and u-PAR expression. The cyclopeptide also altered beta1-integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125(FAK)), disorganized F-actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity.
Asunto(s)
Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Péptidos Cíclicos/farmacología , Animales , Autoantígenos/química , Western Blotting , Proliferación Celular/efectos de los fármacos , Colágeno Tipo IV/química , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Metaloproteinasa 14 de la Matriz/efectos de los fármacos , Metaloproteinasa 14 de la Matriz/metabolismo , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Receptores del Activador de Plasminógeno Tipo Uroquinasa/efectos de los fármacos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously reported the synthesis of a borononucleotide analogue of thymidine monophosphate and its association towards the formation of a new borono-linked dinucleotide. Here we describe the completion of the set of four 2'-deoxyborononucleotide analogues of natural nucleotide monophosphates, namely the previously unknown dCbn, dGbn and dAbn. These analogues were all prepared from the respective 5'-aldehydic nucleosides through a homologation/reduction sequence. The borononucleotides were subsequently obtained by either borylation (dCbn and dGbn) or cross-metathesis (CM) in the presence of the Hoveyda-Grubbs catalyst (dAbn). The reversible formation of the corresponding dinucleotides between these new analogues and uridine was studied by (1)H NMR, and semi-empirical calculations were carried out to provide bond length and electrostatic information that assess the structural similarities existing between these bioisosteres and their natural counterparts.
Asunto(s)
Boro/química , Nucleótidos de Desoxiadenina/química , Desoxicitidina Monofosfato/química , Nucleótidos de Desoxiguanina/química , Desoxirribonucleótidos/química , Desoxirribonucleótidos/síntesis química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Uridina/químicaRESUMEN
Nitrogen-in-the-ring analogues of l-fucose and l-rhamnose were prepared, which feature a spirocyclopropyl moiety in place of the methyl group of the natural sugar. The synthetic route involved a titanium-mediated aminocyclopropanation of a glycononitrile as the key step. Four new spirocyclopropyl iminosugar analogues were generated, which displayed some activity towards l-fucosidase and l-rhamnosidase.
Asunto(s)
Ciclopropanos/síntesis química , Inhibidores Enzimáticos/síntesis química , Fucosa/análogos & derivados , Ramnosa/análogos & derivados , Compuestos de Espiro/síntesis química , Ciclopropanos/química , Ciclopropanos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fucosa/síntesis química , Fucosa/química , Fucosa/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Rotación Óptica , Ramnosa/síntesis química , Ramnosa/química , Ramnosa/farmacología , Espectrometría de Masa por Ionización de Electrospray , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , alfa-L-Fucosidasa/antagonistas & inhibidores , alfa-L-Fucosidasa/metabolismoRESUMEN
Through their coupling to G proteins, G Protein-Coupled Receptors (GPCRs) trigger cellular responses to various signals. Some recent experiments have interestingly demonstrated that the G protein can also act on the receptor by favoring a closed conformation of its orthosteric site, even in the absence of a bound agonist. In this work, we explored such an allosteric modulation by performing extensive molecular dynamics simulations on the adenosine A2 receptor (A2AR) coupled to the Mini-Gs protein. In the presence of the Mini-Gs, we confirmed a restriction of the receptor's agonist binding site that can be explained by a modulation of the intrinsic network of contacts of the receptor. Of interest, we observed similar effects with the C-terminal helix of the Mini-Gs, showing that the observed effect on the binding pocket results from direct local contacts with the bound protein partner that cause a rewiring of the whole receptor's interaction network.