RESUMO
In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Histamínicos/classificação , Receptores Histamínicos/metabolismo , Animais , Descoberta de Drogas , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Agonistas dos Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/metabolismo , Humanos , Cinética , Ligantes , Luciferases/metabolismo , Mimetismo Molecular , Conformação Proteica , Ensaio Radioligante , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/metabolismoRESUMO
This study focuses on the design, synthesis, molecular modeling and biological evaluation of a novel group of alkyl-1,3,5-triazinyl-methylpiperazines. New compounds were synthesized and their affinities for human histamine H4 receptor (hH4R) were evaluated. Among them, 4-(cyclohexylmethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (14) exhibited hH4R affinity with a Ki of 160â¯nM and behaved as antagonist in functional assays: the cellular aequorin-based assay (IC50â¯=â¯32â¯nM) and [35S]GTPγS binding assay (pKbâ¯=â¯6.67). In addition, antinociceptive activity of 14in vivo was observed in Formalin test (in mice) and in Carrageenan-induced acute inflammation test (in rats).
Assuntos
Analgésicos/farmacologia , Inflamação/tratamento farmacológico , Receptores Histamínicos H4/antagonistas & inibidores , Triazinas/farmacologia , Analgésicos/síntese química , Analgésicos/química , Animais , Carragenina , Relação Dose-Resposta a Droga , Formaldeído , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Ligantes , Camundongos , Estrutura Molecular , Ratos , Receptores Histamínicos H4/metabolismo , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/químicaRESUMO
The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Corantes Fluorescentes/farmacologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Receptores Histamínicos H3/metabolismo , Imagem Individual de Molécula , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HEK293 , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/química , Humanos , Ligantes , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A series of 4-pyridylpiperazine derivatives with varying regulatory region substituents proved to be potent histamine H3 receptor (H3R) ligands in the nanomolar concentration range. The most influential modification that affected the affinity toward the H3R appeared by introducing electron-withdrawing moieties into the distal aromatic ring. In order to finally discuss the influence of the characteristic 4-pyridylpiperazine moiety on H3R affinity, two Ciproxifan analogues 2 and 3 with a slight modification in their basic part were obtained. The replacement of piperazine in 3 with piperidine in compound 2, led to slightly reduced affinity towards the H3R (Ki = 3.17 and 7.70 nM, respectively). In fact, 3 showed the highest antagonistic properties among all compounds in this series, hence affirming our previous assumptions, that the 4-pyridylpiperazine moiety is the key element for suitable interaction with the human histamine H3 receptor. While its structural replacement to piperidine is also tolerated for H3R binding, the heteroaromatic 4-pyridyl moiety seems to be essential for proper ligand-receptor interaction. The putative protein-ligand interactions responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at the H3R, as well as drug-like properties of ligands were evaluated using in vitro methods. Moreover, pharmacological in vivo test results of compound 9 (structural analogue of Abbott's A-331440) clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound.
Assuntos
Fármacos Antiobesidade/síntese química , Antagonistas dos Receptores Histamínicos H3/síntese química , Receptores Histamínicos H3/metabolismo , Animais , Fármacos Antiobesidade/farmacologia , Peso Corporal , Relação Dose-Resposta a Droga , Feminino , Antagonistas dos Receptores Histamínicos H3/farmacologia , Humanos , Imidazóis/química , Ligantes , Modelos Moleculares , Piperazina/química , Piperidinas/química , Ligação Proteica , Ratos Wistar , Sequências Reguladoras de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Comprehensively characterized fluorescent probes for the histamine H3 receptor (H3R) and especially for the H4R orthologs [e.g., human (h) and mouse (m)] are highly needed as versatile complementary tools to radioligands. In view of fluorescent probes for BRET-based binding studies and for localizing the H4R in live cells, we synthesized and biologically characterized Py-5-labeled histamine derivatives. The most notable compound was UR-DEBa242 (26, 1-[4-(1H-Imidazol-4-yl)butyl]-4-{(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl}-2,6-dimethylpyridinium hydrotrifluoroacetate trifluoroacetate), acting as a partial agonist at the hH3R [pEC50 (reporter gene) 8.77] and as an inverse agonist/antagonist at the h/mH4Rs [pIC50 (reporter gene) 8.76/7.08; pIC50/pKb (ß-arrestin2) 7.81/7.30]. In confocal microscopy, 26 proved suitable for hH4R localization and trafficking studies in live cells. BRET-based binding at the NLuc-hH3,4Rs/mH4R [pKd 8.78/7.75/7.18, comparable to binding constants from radioligand binding/flow cytometry; fast association/dissociation (â¼2 min)] revealed 26 as a useful molecular tool to determine hH3,4Rs/mH4R binding affinities of ligands binding to these receptors.