RESUMEN
A series of tricyclic cannabinoids incorporating a heteroaroyl group at C3 were prepared as probes to explore the binding site(s) of the CB1 and CB2 receptors. This relatively unexplored structural motif is shown to be CB2 selective with K(i) values at low nanomolar concentrations when the heteroaromatic group is 3-benzothiophenyl (41) or 3-indolyl (50). When photoactivated, the lead compound 41 was shown to successfully label the CB2 receptor through covalent attachment at the active site while 50 failed to label. The benzothiophenone moiety may be a photoactivatable moiety suitable for selective labeling.
Asunto(s)
Benzopiranos/química , Cannabinoides/química , Ligandos , Receptor Cannabinoide CB2/química , Tiofenos/química , Benzopiranos/síntesis química , Sitios de Unión , Cannabinoides/metabolismo , Células HEK293 , Humanos , Unión Proteica , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Rayos UltravioletaRESUMEN
A structure-based drug discovery method is described that incorporates target flexibility through the use of an ensemble of protein conformations. The approach was applied to fatty acid amide hydrolase (FAAH), a key deactivating enzyme in the endocannabinoid system. The resultant dynamic pharmacophore models are rapidly able to identify known FAAH inhibitors over drug-like decoys. Different sources of FAAH conformational ensembles were explored, with both snapshots from molecular dynamics simulations and a group of X-ray structures performing well. Results were compared to those from docking and pharmacophore models generated from a single X-ray structure. Increasing conformational sampling consistently improved the pharmacophore models, emphasizing the importance of incorporating target flexibility in structure-based drug design.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Animales , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , RatasRESUMEN
Little direct information is available regarding the influence of membrane environment on transmembrane (TM) G-protein-coupled receptor (GPCR) conformation and dynamics. The human CB1 cannabinoid receptor (hCB1) is a prominent GPCR pharmacotherapeutic target in which helix 7 appears critical to ligand recognition. We have chemically synthesized a hCB1 peptide corresponding to a segment of TM helix 7 and the entire contiguous helix 8 domain (fourth cytoplasmic loop) and reconstituted it in defined phospholipid-bilayer model membranes. Using an NMR-based strategy combined with molecular dynamics simulations, we provide the first direct experimental description of the orientation of hCB1 helix 7 in phospholipid membranes of varying thickness and the mechanism by which helix-7 conformation adjusts to avoid hydrophobic mismatch. Solid-state (15)N NMR data show that hCB1 helices 7 and 8 reconstituted into phospholipid bilayers are oriented in a TM and in-plane (i.e., parallel to the phospholipid membrane surface) fashion, respectively. TM helix orientation is influenced by the thickness of the hydrophobic membrane bilayer as well as the interaction of helix 8 with phospholipid polar headgroups. Molecular dynamics simulations show that a decrease in phospholipid chain-length induces a kink at P394 in TM helix 7 to avoid hydrophobic mismatch. Thus, the NP(X)nY motif found in hCB1 and highly conserved throughout the GPCR superfamily is important for flexing helix 7 to accommodate bilayer thickness. Dynamic modulation of hCB1-receptor TM helix conformation by its membrane environment may have general relevance to GPCR structure and function.
Asunto(s)
Receptor Cannabinoide CB1/química , Secuencia de Aminoácidos , Fenómenos Biofísicos , Dicroismo Circular , Dimiristoilfosfatidilcolina/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Membrana Dobles de Lípidos/química , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fosfatidilcolinas/química , Estructura Secundaria de Proteína , Receptor Cannabinoide CB1/genética , TermodinámicaRESUMEN
Monoacylglycerol lipase (MGL) is primarily responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both cannabinoid receptors. Increased tissue 2-AG levels consequent to MGL inhibition are considered therapeutic against pain, inflammation, and neurodegenerative disorders. However, the lack of MGL structural information has hindered the development of MGL-selective inhibitors. Here, we detail a fully refined homology model of MGL which preferentially identifies MGL inhibitors over druglike noninhibitors. We include for the first time insight into the active-site geometry and potential hydrogen-bonding interactions along with molecular dynamics simulations describing the opening and closing of the MGL helical-domain lid. Docked poses of both the natural substrate and known inhibitors are detailed. A comparison of the MGL active-site to that of the other principal endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, demonstrates key differences which provide crucial insight toward the design of selective MGL inhibitors as potential drugs.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Inhibidores Enzimáticos/farmacología , Glicéridos/metabolismo , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/química , Secuencia de Aminoácidos , Endocannabinoides , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Monoacilglicerol Lipasas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations indicate that the reaction of native HEWL with its natural substrate involves a covalent intermediate, in contrast to the 'textbook' mechanism for this seminal enzyme.
Asunto(s)
Muramidasa/metabolismo , Teoría Cuántica , Clara de Huevo , Modelos Moleculares , Especificidad por Sustrato , TermodinámicaRESUMEN
To explore the hydrophobic groove subsite within the CB1 cannabinoid receptor we have designed and synthesized a group of tail-substituted anandamide analogs. Our design involves the introduction of aryl or heterocyclic ring as terminal substituents that are connected to the last cis-arachidonyl double bond through aliphatic chains of variable lengths. Our results indicate that there are strict stereochemical requirements for the interaction of such analogs with the CB1 receptor. The optimal pharmacophore includes the phenyl, p-substituted phenyl, or 3-furyl substituents attached to the cis-double bond through a four methylene chain.
Asunto(s)
Ácidos Araquidónicos/síntesis química , Cannabinoides/química , Alcamidas Poliinsaturadas/síntesis química , Receptor Cannabinoide CB1/agonistas , Ácidos Araquidónicos/química , Ácidos Araquidónicos/farmacología , Endocannabinoides , Estructura Molecular , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/farmacología , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
α/ß-Hydrolase domain-containing 6 (ABHD6) represents a potentially attractive therapeutic target for indirectly potentiating 2-arachidonoylglycerol signaling; however, the enzyme is currently largely uncharacterized. Here, we describe a five element, ligand-based pharmacophore model along with a refined homology model of ABHD6. Following a virtual screen of a modest database, both the pharmacophore and homology models were found to be highly predictive, preferentially identifying ABHD6 inhibitors over drug-like non-inhibitors. The models yield insight into the features required for optimal ligand binding to ABHD6 and the atomic structure of the binding site. In combination, the two models should be very helpful not only in high-throughput virtual screening, but also in lead optimization, and will facilitate the development of novel, selective ABHD6 inhibitors as potential drugs.
Asunto(s)
Ligandos , Monoacilglicerol Lipasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Sitios de Unión , Bases de Datos Factuales , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Monoacilglicerol Lipasas/metabolismo , Estructura Terciaria de Proteína , Curva ROC , Alineación de SecuenciaRESUMEN
The membrane-associated serine hydrolase, monoacylglycerol lipase (MGL), is a well-recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure-function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge-neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (Vmax) and substrate affinity (Km)]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL-nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid-domain helix α4 and neighboring helix α6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate-binding channel. Covalent modification of membrane-associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane-associated topology of active and inhibited, covalently-modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Monoacilglicerol Lipasas/química , Monoacilglicerol Lipasas/metabolismo , Fosfolípidos/metabolismo , Humanos , Cinética , Membrana Dobles de Lípidos/química , Espectrometría de Masas , Simulación de Dinámica Molecular , Monoacilglicerol Lipasas/antagonistas & inhibidores , Fosfolípidos/química , Conformación Proteica/efectos de los fármacosRESUMEN
In previous studies, compound 1 (AM411), a 3-(1-adamantyl) analogue of the phytocannabinoid (-)-Δ(8)-tetrahydrocannabinol (Δ(8)-THC), was shown to have improved affinity and selectivity for the CB1 receptor. In this work, we further explored the role of the 1-adamantyl group at the C-3 position in a series of tricyclic cannabinoid analogues modified at the 9-northern aliphatic hydroxyl (NAH) position. Of these, 9-hydroxymethyl hexahydrocannabinol 11 (AM4054) exhibited high CB1 affinity and full agonist profile. In the cAMP assay, the 9-hydroxymethyl cannabinol analogue 24 (AM4089) had a partial agonist profile, with high affinity and moderate selectivity for rCB1 over hCB2. In vivo results in rat models of hypothermia and analgesia were congruent with in vitro data. Our in vivo data indicate that 3-(1-adamantyl) substitution, within NAH cannabinergics, imparts improved pharmacological profiles when compared to the corresponding, traditionally used 3-dimethylheptyl analogues and identifies 11 and 24 as potentially useful in vivo CB1 cannabinergic probes.
Asunto(s)
Adamantano/síntesis química , Adamantano/farmacología , Agonistas de Receptores de Cannabinoides/síntesis química , Cannabinoides/síntesis química , Cannabinoides/farmacología , Receptor Cannabinoide CB1/efectos de los fármacos , Analgésicos/síntesis química , Analgésicos/farmacología , Animales , Arrestina/metabolismo , Temperatura Corporal/efectos de los fármacos , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Hipotermia/inducido químicamente , Modelos Moleculares , Dimensión del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB2/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine ß-lactone (N-Cbz-serine ß-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the ß-subunit, confirming a suggested mechanism of hNAAA inactivation by the ß-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Inhibidores Enzimáticos/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Amidohidrolasas/química , Amidohidrolasas/genética , Dominio Catalítico , Células HEK293 , Humanos , Isotiocianatos/química , Isotiocianatos/farmacología , Cinética , Lactonas/farmacología , Modelos Moleculares , Serina/análogos & derivados , Serina/farmacología , Tetrazoles/farmacologíaRESUMEN
Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH, we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs.
Asunto(s)
Alcanosulfonatos/farmacología , Amidohidrolasas/antagonistas & inhibidores , Encéfalo/efectos de los fármacos , Palmitatos/farmacología , Fenoles/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Ensayo de Unión Radioligante , Ratas , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-ActividadRESUMEN
In pursuit of a more detailed understanding of the structural requirements for the key side chain cannabinoid pharmacophore, we have extended our SAR to cover a variety of conformationally modified side chains within the 9-keto and 9-hydroxyl tricyclic structures. Of the compounds described here, those with a seven-atom long side chain substituted with a cyclopentyl ring at C1' position have very high affinities for both CB1 and CB2 (0.97 nM < K(i) < 5.25 nM), with no preference for either of the two receptors. However, presence of the smaller cyclobutyl group at the C1' position leads to an optimal affinity and selectivity interaction with CB1. Thus, two of the C1'-cyclobutyl analogues, namely, (6aR,10aR)-3-(1-hexyl-cyclobut-1-yl)-6,6a,7,8,10,10a-hexahydro-1-hydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran-9-one and (6aR,9R,10aR)-3-(1-hexyl-cyclobut-1-yl)-6a,7,8,9,10,10a-hexahydro-6,6-dimethyl-6H-dibenzo[b,d]pyran-1,9 diol (7e-ß, AM2389), exhibited remarkably high affinities (0.84 and 0.16 nM, respectively) and significant selectivities (16- and 26-fold, respectively) for CB1. Compound 7e-ß was found to exhibit exceptionally high in vitro and in vivo potency with a relatively long duration of action.
Asunto(s)
Analgésicos/síntesis química , Benzopiranos/síntesis química , Cannabinol/análogos & derivados , Cannabinol/síntesis química , Receptor Cannabinoide CB1/agonistas , Analgesia , Analgésicos/química , Analgésicos/farmacología , Animales , Benzopiranos/química , Benzopiranos/farmacología , Cannabinol/química , Cannabinol/farmacología , Línea Celular , Femenino , Hipotermia Inducida , Técnicas In Vitro , Ratones , Modelos Moleculares , Prosencéfalo/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB2/agonistas , Bazo/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Sinaptosomas/metabolismoRESUMEN
Structure-activity relationship studies of classical cannabinoid analogues have established that the C3 aliphatic side chain plays a pivotal role in determining cannabinergic potency. In earlier work, we provided evidence for the presence of subsites within the CB1 and CB2 cannabinoid receptor binding domains that can accommodate bulky conformationally defined substituents at the C3 alkyl side chain pharmacophore of classical cannabinoids. We have now extended this work with the synthesis of a series of Delta (8)-THC analogues in which bornyl substituents are introduced at the C3 position. Our results indicate that, for optimal interactions with both CB1 and CB2 receptors, the bornyl substituents need to be within close proximity of the tricyclic core of Delta (8)-THC and that the conformational space occupied by the C3 substituents influences CB1/CB2 receptor subtype selectivity.
Asunto(s)
Compuestos de Boro/síntesis química , Cannabinoides/química , Dronabinol/síntesis química , Animales , Compuestos de Boro/química , Compuestos de Boro/clasificación , Dronabinol/química , Dronabinol/clasificación , Ligandos , Ratones , Modelos Moleculares , Estructura Molecular , Ratas , Receptores de Cannabinoides/química , Receptores de Cannabinoides/metabolismo , Relación Estructura-ActividadRESUMEN
Escherichia coli dihydrofolate reductase (DHFR) is a long-standing target for enzyme studies. The influence of protein motion on its catalytic cycle is significant, and the conformation of the M20 loop is of particular interest. We present receptor-based pharmacophore models-an equivalent of solvent-mapping of binding hotspots-based on ensembles of protein conformations from molecular dynamics simulations of DHFR.NADPH in both the closed and open conformation of the M20 loop. The optimal models identify DHFR inhibitors over druglike non-inhibitors; furthermore, high-affinity inhibitors of E. coli DHFR are preferentially identified over general DHFR inhibitors. As expected, models resulting from simulations with DHFR in the productive conformation with a closed M20 loop have better performance than those from the open-loop simulations. Model performance improves with increased dynamic sampling, indicating that including a greater degree of protein flexibility can enhance the quest for potent inhibitors. This was compared to the limited conformational sampling seen in crystal structures, which were suboptimal for this application.
Asunto(s)
Diseño de Fármacos , Escherichia coli/enzimología , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Cristalografía por Rayos X , Bases de Datos de Proteínas , Activación Enzimática/efectos de los fármacos , Ligandos , Modelos Moleculares , Conformación MolecularRESUMEN
In structure-based drug discovery, researchers would like to identify all possible scaffolds for a given target. However, techniques that push the boundaries of chemical space could lead to many false positives or inhibitors that lack specificity for the target. Is it possible to broadly identify the appropriate chemical space for the inhibitors and yet maintain target specificity? To address this question, we have turned to dihydrofolate reductase (DHFR), a well-studied metabolic enzyme of pharmacological relevance. We have extended our multiple protein structure (MPS) method for receptor-based pharmacophore models to use multiple X-ray crystallographic structures. Models were created for DHFR from human and Pneumocystis carinii. These models incorporate a fair degree of protein flexibility and are highly selective for known DHFR inhibitors over drug-like non-inhibitors. Despite sharing a highly conserved active site, the pharmacophore models reflect subtle differences between the human and P. carinii forms, which identify species-specific, high-affinity inhibitors. We also use structures of DHFR from Candida albicans as a counter example. The available crystal structures show little flexibility, and the resulting models give poorer performance in identifying species-specific inhibitors. Therapeutic success for this system may depend on achieving species specificity between the related human host and these key fungal targets. The MPS technique is a promising advance for structure-based drug discovery for DHFR and other proteins of biomedical interest.
Asunto(s)
Diseño de Fármacos , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/metabolismo , Docilidad , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Five nonpeptide, small-molecule inhibitors of the human MDM2-p53 interaction are presented, and each inhibitor represents a new scaffold. The most potent compound exhibited a Ki of 110 +/- 30 nM. These compounds were identified using our multiple protein structure (MPS) method which incorporates protein flexibility into a receptor-based pharmacophore model that identifies appropriate hotspots of binding. Docking the inhibitors with an induced-fit docking protocol suggested that the inhibitors mimicked the three critical binding residues of p53 (Phe19, Trp23, and Leu26). Docking also predicted a new orientation of the scaffolds that more fully fills the binding cleft, enabling the inhibitors to take advantage of additional hydrogen-bonding possibilities not explored by other small molecule inhibitors. One inhibitor in particular was proposed to probe the hydrophobic core of the protein by taking advantage of the flexibility of the binding cleft floor. These results show that the MPS technique is a promising advance for structure-based drug discovery and that the method can truly explore broad chemical space efficiently in the quest to discover potent, small-molecule inhibitors of protein-protein interactions. Our MPS technique is one of very few ensemble-based techniques to be proven through experimental verification of the discovery of new inhibitors.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Unión Competitiva , Análisis por Conglomerados , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Modelos Químicos , Conformación Molecular , Distribución Normal , Péptidos/química , Unión Proteica , Conformación Proteica , Programas InformáticosRESUMEN
Modeling methods allow the identification and analysis of determinants of reactivity and specificity in enzymes. The reaction between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) is widely used as a standard activity assay for glutathione S-transferases (GSTs). It is important to understand the causes of differences between catalytic GST isoenzymes and the effects of mutations and genetic polymorphisms. Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations have been performed here to investigate the addition of the glutathione anion to CDNB in the wild-type M1-1 GST isoenzyme from rat and in three single point mutant (Tyr6Phe, Tyr115Phe, and Met108Ala) M1-1 GST enzymes. We have developed a specifically parameterized QM/MM method (AM1-SRP/CHARMM22) to model this reaction by fitting to experimental heats of formation and ionization potentials. Free energy profiles were obtained from molecular dynamics simulations of the reaction using umbrella sampling and weighted histogram analysis techniques. The reaction in solution has also been simulated and is compared to the enzymatic reaction. The free energies are in excellent agreement with experimental results. Overall the results of the present study show that QM/MM reaction pathway analysis provides detailed insight into the chemistry of GST and can be used to obtain mechanistic insight into the effects of specific mutations on this catalytic process.