RESUMO
Kainate receptors play a crucial role in mediating synaptic transmission within the central nervous system. However, the lack of selective pharmacological tool compounds for the GluK3 subunit represents a significant challenge in studying these receptors. Recently presented compound 1 stands out as a potent antagonist of GluK3 receptors, exhibiting nanomolar affinity at GluK3 receptors and strongly inhibiting glutamate-induced currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with Kb values of 65 and 39 nM, respectively. This study presents the synthesis of two potent GluK3-preferring iodine derivatives of compound 1, serving as precursors for radiolabelling. Furthermore, we demonstrate the optimisation of dehalogenation conditions using hydrogen and deuterium, resulting in [2H]-1, and demonstrate the efficient synthesis of the radioligand [3H]-1 with a specific activity of 1.48 TBq/mmol (40.1 Ci/mmol). Radioligand binding studies conducted with [3H]-1 as a radiotracer at GluK1, GluK2, and GluK3 receptors expressed in Sf9 and rat P2 membranes demonstrated its potential applicability for selectively studying native GluK3 receptors in the presence of GluK1 and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-blocking ligands.
Assuntos
Ácido Glutâmico , Receptores de Ácido Caínico , Ratos , Animais , Humanos , Trítio , Deutério , Células HEK293 , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/metabolismo , Receptores de AMPA/química , Receptores de AMPA/metabolismoRESUMO
Kainate receptors belong to the family of glutamate receptors ion channels, which are responsible for the majority of rapid excitatory synaptic transmission in the central nervous system. The therapeutic potential of kainate receptors is still poorly understood, which is also due to the lack of potent and subunit-selective pharmacological tools. In search of selective ligands for the GluK3 kainate receptor subtype, a series of quinoxaline-2,3-dione analogues was synthesized and pharmacologically characterized at selected recombinant ionotropic glutamate receptors. Among them, compound 28 was found to be a competitive GluK3 antagonist with submicromolar affinity and unprecedented high binding selectivity, showing a 400-fold preference for GluK3 over other homomeric receptors GluK1, GluK2, GluK5 and GluA2. Furthermore, in functional assays performed for selected metabotropic glutamate receptor subtypes, 28 did not show agonist or antagonist activity. The molecular determinants underlying the observed affinity profile of 28 were analyzed using molecular docking and molecular dynamics simulations performed for individual GluK1 and GluK3 ligand-binding domains.
Assuntos
Receptores de Ácido Caínico , Ligantes , Simulação de Acoplamento Molecular , Domínios Proteicos , Receptores de Ácido Caínico/metabolismo , Receptor de GluK3 CainatoRESUMO
A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as at cloned homomeric kainate receptors GluK1-GluK3. Among them, six compounds bound to GluK1 receptor subtypes with reasonable affinity (Ki values in the range of 4.9-7.5µM). A structure-activity relationship (SAR) for the obtained series, focused mainly on the pharmacological effect of structural modifications in the 4- and 5-position of the phenylalanine ring, was established. To illustrate the results, molecular docking of the synthesized series to the X-ray structure of GluK1 ligand binding core was performed. The influence of individual substituents at the phenylalanine ring for both the affinity and selectivity at AMPA, GluK1 and GluK3 receptors was analyzed, giving directions for future studies.
Assuntos
Desenho de Fármacos , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Receptores de Ácido Caínico/metabolismo , Animais , Cristalografia por Raios X , Ligantes , Simulação de Acoplamento Molecular , Ratos , Receptores de Ácido Caínico/antagonistas & inibidores , Receptores de Ácido Caínico/química , Relação Estrutura-Atividade , Receptor de GluK3 CainatoRESUMO
In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal structures of GluK1 in complex with related ligands, the compounds were designed to explore possible interactions with non-conserved residues outside the glutamate ligand binding site and challenge the water binding network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl-substituted urea (compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that this preference may be attributed to contacts between the NH of the urea substituent and non-conserved Ser741 and Ser761 residues.
Assuntos
Desenho de Fármacos , Fenilalanina/farmacologia , Receptores de Ácido Caínico/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Fenilalanina/análogos & derivados , Fenilalanina/síntese química , Fenilalanina/química , Receptores de Ácido Caínico/metabolismo , Relação Estrutura-Atividade , Receptor de GluK3 CainatoRESUMO
Kainate receptors are a class of ionotropic glutamate receptors that respond to the excitatory neurotransmitter glutamate in the central nervous system and play an important role in the development of neurodegenerative disorders and the regulation of synaptic function. In the current study, we investigated the structure- activity relationship of the series of quinoxaline-2,3-diones substituted at N1, 6, and 7 positions, as ligands of kainate homomeric receptors GluK1-3 and GluK5. Pharmacological characterization showed that all derivatives obtained exhibited micromolar affinity at GluK3 receptors with Ki values in the range 0.1-4.4â µM range. The antagonistic properties of the selected analogues: N-(7-fluoro-6-iodo-2,3-dioxo-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide, N-(7-(1H-imidazol-1-yl)-6-iodo-2,3-dioxo-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide and N-(7-(1H-imidazol-1-yl)-2,3-dioxo-6-(phenylethynyl)-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide at GluK3 receptors, were confirmed by an intracellular calcium imaging assay. To correlate inâ vitro affinity data with structural features of the synthesized compounds and to understand the impact of the substituent in N1 position on ability to form additional protein-ligand interactions, molecular modeling and docking studies were carried out. Experimental solubility studies using UV spectroscopy detection have shown that 7-imidazolyl-6-iodo analogues with a sulfamoylbenzamide moiety at the N1 position are the best soluble compounds in the series, with molar solubility in TRISS buffer at pHâ 9 more than 3-fold higher compared to NBQX, a known AMPA/kainate antagonist.
Assuntos
Ácido Caínico , Receptores de Ácido Caínico , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/metabolismo , Quinoxalinas/farmacologia , Solubilidade , Relação Estrutura-AtividadeRESUMO
Targeting the protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) is a potential therapeutic strategy to control diseases involving oxidative stress. Here, six classes of known small-molecule Keap1-Nrf2 PPI inhibitors were dissected into 77 fragments in a fragment-based deconstruction reconstruction (FBDR) study and tested in four orthogonal assays. This gave 17 fragment hits of which six were shown by X-ray crystallography to bind in the Keap1 Kelch binding pocket. Two hits were merged into compound 8 with a 220-380-fold stronger affinity (Ki = 16 µM) relative to the parent fragments. Systematic optimization resulted in several novel analogues with Ki values of 0.04-0.5 µM, binding modes determined by X-ray crystallography, and enhanced microsomal stability. This demonstrates how FBDR can be used to find new fragment hits, elucidate important ligand-protein interactions, and identify new potent inhibitors of the Keap1-Nrf2 PPI.
Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , Cristalografia por Raios X , Estabilidade de Medicamentos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
On the basis of X-ray structures of ionotropic glutamate receptor constructs in complex with amino acid-based AMPA and kainate receptor antagonists, a series of rigid as well as flexible biaromatic alanine derivatives carrying selected hydrogen bond acceptors and donors have been synthesized in order to investigate the structural determinants for receptor selectivity between AMPA and the GluR5 subtype of kainate receptors. Compounds selective for either GluR5 or AMPA receptors were identified. One particular substituent position appeared to be of special importance for control of ligand selectivity. Using molecular modeling the observed structure-activity relationships at AMPA and GluR5 receptors were deduced.
Assuntos
Neurotransmissores/química , Fenilalanina/análogos & derivados , Receptores de AMPA/antagonistas & inibidores , Receptores de Ácido Caínico/antagonistas & inibidores , Animais , Sítios de Ligação , Simulação por Computador , Ligantes , Neurotransmissores/síntese química , Neurotransmissores/farmacologia , Fenilalanina/síntese química , Fenilalanina/farmacologia , Ratos , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
Among the ionotropic glutamate receptors, the physiological role of kainate receptors is less well understood. Although ligands with selectivity toward the kainate receptor subtype GluK1 are available, tool compounds with selectivity at the remaining kainate receptor subtypes are sparse. Here, we have synthesized a series of quinoxaline-2,3-diones with substitutions in the N1-, 6-, and 7-position to investigate the structure-activity relationship (SAR) at GluK1-3 and GluK5. Pharmacological characterization at native and recombinant kainate and AMPA receptors revealed that compound 37 had a GluK3-binding affinity ( Ki) of 0.142 µM and 8-fold preference for GluK3 over GluK1. Despite lower binding affinity of 22 at GluK3 ( Ki = 2.91 µM), its preference for GluK3 over GluK1 and GluK2 was >30-fold. Compound 37 was crystallized with the GluK1 ligand-binding domain to understand the SAR. The X-ray structure showed that 37 stabilized the protein in an open conformation, consistent with an antagonist binding mode.
Assuntos
Quinoxalinas/farmacologia , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Relação Estrutura-Atividade , Animais , Modelos Moleculares , Domínios Proteicos/fisiologia , Receptores de Ácido Caínico/antagonistas & inibidoresRESUMO
Selective pharmacological tool compounds are invaluable for understanding the functions of the various ionotropic glutamate receptor subtypes. For the kainate receptors, these compounds are few. Here we have synthesized nine novel quinoxaline-2,3-diones with substitutions in the 7-position to investigate the structure-activity relationship at kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Compound 11 exhibited the highest binding affinity across GluK1-3 while having selectivity toward kainate vs AMPA receptors. Compound 11 potently inhibited glutamate evoked currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with Kb values of 65 and 39 nM, respectively. The binding mode of 11 in the ligand binding domain of GluK1 was investigated by X-ray crystallography, revealing that 11 stabilizes the receptor in an open conformation, consistent with its demonstrated antagonism. Furthermore, 11 was tested for analgesic effects in the mouse tail flick test where it significantly increased tail flick latency at doses where 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]-quinoxaline-7-sulfonamide (NBQX) was ineffective.
Assuntos
Analgésicos/metabolismo , Cristalografia por Raios X/métodos , Antagonistas de Aminoácidos Excitatórios/metabolismo , Quinoxalinas/metabolismo , Receptores de Ácido Caínico/antagonistas & inibidores , Receptores de Ácido Caínico/metabolismo , Analgésicos/química , Analgésicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/química , Antagonistas de Aminoácidos Excitatórios/farmacologia , Células HEK293 , Humanos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Quinoxalinas/química , Quinoxalinas/farmacologia , Ratos , Receptores de Ácido Caínico/química , Relação Estrutura-AtividadeRESUMO
AMPA-type ionotropic glutamate receptors generally display high stereoselectivity in agonist binding. However, the stereoisomers of 2-amino-3-(4-hydroxy-1,2,5-thiadiazol-3-yl)propionic acid (TDPA) have similar enantiopharmacology. To understand this observation, we have determined the X-ray structures of ( R)-TDPA and ( S)-TDPA in complex with the ligand-binding core of iGluR2 and investigated the binding pharmacology at AMPA and kainate receptors. Both enantiomers induce full domain closure in iGluR2 but adopt different conformations when binding to the receptor, which may explain the similar enantiopharmacology.
Assuntos
Alanina/análogos & derivados , Modelos Moleculares , Receptores de AMPA/agonistas , Receptores de AMPA/química , Tiadiazóis/química , Alanina/química , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Ensaio Radioligante , Receptores de AMPA/genética , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Diclofenac is widely used in the treatment of, for example, arthritis and muscle pain. The use of diclofenac has been associated with hepatotoxicity, which has been linked to the formation of reactive metabolites. Diclofenac can be metabolized to 4'-OH- and 5-OH-diclofenac, both of which are able to form quinone imines capable of reacting with, for example, GSH and nucleophilic groups in proteins. Electrochemistry has been shown to be a suitable tool for mimicking some types of oxidative drug metabolism and for studying the formation of reactive metabolites. In these studies, the electrochemical oxidation of diclofenac to a +16 Da metabolite was shown to be identical to a synthetic standard of 5-OH-diclofenac. Furthermore, two different experimental designs were investigated with respect to the electrochemical oxidation of 4'-OH- and 5-OH-diclofenac. In the first approach, the oxidized sample was collected in an aqueous solution of GSH, whereas in the other approach, GSH was added to the sample before the oxidation was performed. From these electrochemical oxidations, a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac were observed and characterized by MS/MS. This allowed the development of sensitive LC-MS methods in order to detect the GSH conjugates from in vivo (rat bile) and in vitro (human liver microsomes (HLM), rat liver microsomes (RLM), and rat hepatocytes) samples. A wide range of mono-, di-, and triglutathionyl conjugates were detected in the in vitro and in vivo samples. It was also observed that 5-OH-diclofenac formed GSH conjugates with RLM and HLM without addition of NADPH, whereas GSH conjugate formation of 4'-OH-diclofenac was NADPH-dependent. This indicated that 5-OH-diclofenac was prone to auto-oxidation. The oxidation potentials of the two hydroxy metabolites were determined by cyclic voltammetry. A difference of 69 mV was observed between the two oxidation potentials, which in part may explain the extent of auto-oxidation for 5-OH-diclofenac. In conclusion, it was shown that electrochemical oxidation was capable of mimicking the metabolic hydroxylation of diclofenac to 5-OH-diclofenac. Furthermore, electrochemical oxidation was used to generate a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac and a number of these conjugates were also detected in metabolism studies with microsomes (HLM/RLM) and freshly isolated rat hepatocytes, and in vivo in rat bile.
Assuntos
Diclofenaco/química , Diclofenaco/farmacologia , Animais , Células Cultivadas/efeitos dos fármacos , Diclofenaco/síntese química , Eletroquímica , Glutationa/química , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oxirredução , Ratos , Espectrometria de Massas em TandemRESUMO
Four 2-substituted Tet-AMPA [Tet = tetrazolyl, AMPA = 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid] analogues were characterized functionally at the homomeric AMPA receptors GluR1i, GluR2Qi, GluR3i, and GluR4i in a Fluo-4/Ca2+ assay. Whereas 2-Et-Tet-AMPA, 2-Pr-Tet-AMPA, and 2-iPr-Tet-AMPA were nonselective GluR agonists, 2-Bn-Tet-AMPA exhibited a 40-fold higher potency at GluR4i than at GluR1i. Examination of homology models of the S1-S2 domains of GluR1 and GluR4 containing 2-Bn-Tet-AMPA suggested four nonconserved residues in a region adjacent to the orthosteric site as possible determinants of the GluR4i/GluR1i selectivity. In a mutagenesis study, doubly mutating M686V/I687A in GluR1i in combination with either D399S or E683A increased both the potency and the maximal response of 2-Bn-Tet-AMPA at this receptor to levels similar to those elicited by the agonist at GluR4i. The dependence of the novel selectivity profile of 2-Bn-Tet-AMPA upon residues located outside of the orthosteric site underlines the potential for developing GluR subtype selective ligands by designing compounds with substituents that protrude beyond the (S)-Glu binding pocket.
Assuntos
Isoxazóis/química , Propionatos/química , Receptores de AMPA/agonistas , Tetrazóis/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/análogos & derivados , Compostos de Anilina , Animais , Sítios de Ligação , Linhagem Celular , Feminino , Corantes Fluorescentes , Humanos , Técnicas In Vitro , Isoxazóis/farmacologia , Modelos Moleculares , Mutação , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Propionatos/farmacologia , Ratos , Receptores de AMPA/genética , Receptores de AMPA/fisiologia , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Relação Estrutura-Atividade , Tetrazóis/farmacologia , Termodinâmica , Xantenos , Xenopus , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Replacement of the methyl group of the AMPA receptor agonist 2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA) with a benzyl group provided the first AMPA receptor agonist, compound 7, capable of discriminating GluR2-4 from GluR1 by its more than 10-fold preference for the former receptor subtypes. An X-ray crystallographic analysis of this new analogue in complex with the GluR2-S1S2J construct shows that accommodation of the benzyl group creates a previously unobserved pocket in the receptor, which may explain the remarkable pharmacological profile of compound 7.
Assuntos
Receptores de AMPA/agonistas , Tetrazóis/síntese química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/análogos & derivados , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Técnicas In Vitro , Insetos , Modelos Moleculares , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Ensaio Radioligante , Ratos , Receptores de AMPA/química , Receptores de AMPA/fisiologia , Estereoisomerismo , Relação Estrutura-Atividade , Sinaptossomos/metabolismo , Tetrazóis/química , Tetrazóis/farmacologia , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/síntese química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
A series of racemic unnatural amino acids was rationally designed on the basis of recently published X-ray structures of the GluA2 LBD with bound phenylalanine-based antagonists. Twelve new diaryl- or aryl/heteroaryl-substituted phenylalanine derivatives were synthesized and evaluated in vitro in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)-2-amino-3-(3'-hydroxy-5-(1H-pyrazol-4-yl)-[1,1'-biphenyl]-3-yl)propanoic acid 7e, showed the binding affinity of 4.6 µm for native AMPA receptors and over fourfold lower affinity for kainic acid receptors. Furthermore, 7e was evaluated at recombinant homomeric rat GluA2 and GluA3 receptors. Recently reported X-ray structures 5CBR and 5CBS, representing two distinct antagonist binding modes, were used as templates for molecular docking of the synthesized series. Binding data supported with molecular modeling confirmed that aryl/heteroaryl-substituted phenylalanine analogues effectively bind to AMPA receptors with low micromolar affinity and high selectivity over native NMDA and kainate receptors. These properties make 7e a promising lead for the further development of new AMPA receptor ligands.
Assuntos
Fenilalanina/metabolismo , Receptores de AMPA/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Cristalografia por Raios X , Ligantes , Conformação Molecular , Fenilalanina/análogos & derivados , Estrutura Terciária de Proteína , Ratos , Receptores de AMPA/química , Receptores de AMPA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
In order to map out molecular determinants for the competitive blockade of AMPA receptor subtypes, a series of racemic aryl-substituted phenylalanines was synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors. Most of the compounds showed micromolar affinity and preference for AMPA receptors. Individual stereoisomers of selected compounds were further evaluated at recombinant homomeric rat GluA2 and GluA3 receptors. The most potent compound, (-)-2-amino-3-(6-chloro-2',5'-dihydroxy-5-nitro-[1,1'-biphenyl]-3-yl)propanoic acid, the expected R-isomer showing Ki of 1.71 µM at the GluA2 subtype, was found to competitively antagonize GluA2(Q)i receptors in TEVC electrophysiological experiments (Kb = 2.13 µM). Molecular docking experiments allowed us to compare two alternative antagonist binding modes for the synthesized phenylalanines at the GluA2 binding core, showing the direction for further structural modifications.
Assuntos
Fenilalanina/farmacologia , Receptores de AMPA/antagonistas & inibidores , Animais , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Fenilalanina/síntese química , Fenilalanina/química , Ratos , Relação Estrutura-AtividadeRESUMO
Three 4-substituted 1,2,5-oxadiazol-3-ols containing aminoalkyl substituents (analogues and homologues of gamma-aminobutyric acid (GABA)) were synthesized to investigate the hydroxy-1,2,5-oxadiazolyl moiety as a bioisoster for a carboxyl group at GABA receptors. The pK(a) values of the target compounds were close to those of GABA. At GABA(A) receptors of cultured cerebral cortical neurons, weak agonist and partial agonist profiles were identified, demonstrating the 4-hydroxy-1,2,5-oxadiazol-3-yl unit to be a nonclassical carboxyl group bioisoster.
Assuntos
Agonistas de Receptores de GABA-A , Agonistas dos Receptores de GABA-B , Oxidiazóis/síntese química , Ácido gama-Aminobutírico/análogos & derivados , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ligação Competitiva , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Antagonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-B , Humanos , Técnicas In Vitro , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxidiazóis/química , Oxidiazóis/farmacologia , Técnicas de Patch-Clamp , Ratos , Relação Estrutura-Atividade , Ácido gama-Aminobutírico/síntese química , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/farmacologiaRESUMO
A series of racemic aryl-substituted phenylalanines was synthesized and evaluated in vitro at recombinant rat GluA1-3, at GluK1-3, and at native AMPA receptors. The individual enantiomers of two target compounds, (RS)-2-amino-3-(3,4-dichloro-5-(5-hydroxypyridin-3-yl)phenyl)propanoic acid 37 and (RS)-2-amino-3-(3'-hydroxybiphenyl-3-yl)propanoic acid 38, were characterized. (S)-37 and (R)-38 were identified as the only biologically active isomers, both being antagonists at GluA2 receptors with Kb of 1.80 and 3.90 µM, respectively. To address this difference in enantiopharmacology, not previously seen for amino acid-based AMPA receptor antagonists, X-ray crystal structures of both eutomers in complex with the GluA2 ligand binding domain were solved. The cocrystal structures of (S)-37 and (R)-38 showed similar interactions of the amino acid parts but unexpected and different orientations and interactions of the biaromatic parts of the ligands inside the binding site, with (R)-38 having a binding mode not previously identified for amino acid-based antagonists.
Assuntos
Antagonistas de Aminoácidos Excitatórios/síntese química , Antagonistas de Aminoácidos Excitatórios/farmacologia , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Receptores de AMPA/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Técnicas In Vitro , Modelos Moleculares , Simulação de Acoplamento Molecular , Fenilalanina/síntese química , Ratos , Receptores de AMPA/efeitos dos fármacos , Receptores de Ácido Caínico/efeitos dos fármacos , Proteínas Recombinantes , Relação Estrutura-Atividade , Xenopus laevisRESUMO
The synthesis and pharmacological characterization of 1- and 2-alkyltetrazolyl analogues of (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA), a highly potent and selective agonist at AMPA receptors, are presented. A shorter and more convergent synthetic route than previously described, employing a new method for introducing the amino acid moiety, was developed for these derivatives. The 2-substituted isomers were selective agonists, and their activity correlated inversely with the size of the substituent. Structural explanations of the structure-activity relationship are provided.
Assuntos
Receptores de AMPA/agonistas , Receptores de AMPA/metabolismo , Tetrazóis/síntese química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/análogos & derivados , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/síntese química , Animais , Encéfalo/metabolismo , Técnicas In Vitro , Modelos Moleculares , Ensaio Radioligante , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Sinaptossomos/metabolismo , Tetrazóis/farmacologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Continued efforts into the discovery of ligands that target ionotropic glutamate receptors (iGluRs) are important for studies of the physiological roles of the various iGluR subtypes as well as for the search for drugs that can be used in the treatment of diseases of the central nervous system. A new series of phenylalanine derivatives that target iGluRs was reported to bind AMPA receptors. Herein we report our studies of these compounds at the kainate receptors GluK1-3. Several compounds bind with micromolar affinity at GluK1 and GluK3, but do not bind GluK2. The crystal structure of the most potent compound in the ligand binding domain of GluK1 revealed different modes of binding to GluK1 and GluA2, due primarily to residues Ser741 (GluK1) and Met729 (GluA2). The compound was shown to be slightly more potent at GluK1 than at AMPA receptors and to induce a domain closure similar to that observed in GluK1 structures with partial agonists.
Assuntos
Fenilalanina/análogos & derivados , Receptores de Ácido Caínico/antagonistas & inibidores , Animais , Sítios de Ligação , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Fenilalanina/síntese química , Fenilalanina/farmacologia , Estrutura Terciária de Proteína , Receptores de Ácido Caínico/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/fisiologia , Receptor de GluK3 CainatoRESUMO
In order to map out molecular determinants for competitive blockade of AMPA receptor subtypes, a series of 2-carboxyethylphenylalanine derivatives has been synthesized and pharmacologically characterized in vitro. One compound in this series, (RS)-3h, showed micromolar affinity for GluA1(o) and GluA2(R)(o) receptors with an approximately 4-fold preference for GluA1/2 vs GluA3/4. In TEVC electrophysiological experiments (RS)-3h competitively antagonized GluA2(Q)(i) receptors. The X-ray structure of the active enantiomer (S)-3h in complex with GluA2-S1S2J showed a domain closure around 8°. Even though the nitro and the carboxyethyl groups of (S)-3h were both anchored to Tyr702 through a water H-bond network, these interactions only induced weak subtype selectivity. In spite of the fact that (S)-3h induced a domain closure close to that observed for partial agonists, it did not produce agonist responses at GluA2 receptors under nondesensitizing conditions. 2-Carboxyethylphenylalanine derivatives provide a new synthetic scaffold for the introduction of substituents that could lead to AMPA receptor subtype-selective ligands.