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1.
J Med Chem ; 64(15): 11695-11708, 2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34309390

RESUMEN

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.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia , Colorantes Fluorescentes/farmacología , Antagonistas de los Receptores Histamínicos H3/farmacología , Receptores Histamínicos H3/metabolismo , Imagen Individual de Molécula , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Células HEK293 , Antagonistas de los Receptores Histamínicos H3/síntesis química , Antagonistas de los Receptores Histamínicos H3/química , Humanos , Ligandos , Estructura Molecular , Relación Estructura-Actividad
2.
Eur J Med Chem ; 213: 113041, 2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33261900

RESUMEN

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.


Asunto(s)
Fármacos Antiobesidad/síntesis química , Antagonistas de los Receptores Histamínicos H3/síntesis química , Receptores Histamínicos H3/metabolismo , Animales , Fármacos Antiobesidad/farmacología , Peso Corporal , Relación Dosis-Respuesta a Droga , Femenino , Antagonistas de los Receptores Histamínicos H3/farmacología , Humanos , Imidazoles/química , Ligandos , Modelos Moleculares , Piperazina/química , Piperidinas/química , Unión Proteica , Ratas Wistar , Secuencias Reguladoras de Ácidos Nucleicos , Relación Estructura-Actividad
3.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-33182741

RESUMEN

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.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores Histamínicos/clasificación , Receptores Histamínicos/metabolismo , Animales , Descubrimiento de Drogas , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Agonistas de los Receptores Histamínicos/metabolismo , Antagonistas de los Receptores Histamínicos/metabolismo , Humanos , Cinética , Ligandos , Luciferasas/metabolismo , Imitación Molecular , Conformación Proteica , Ensayo de Unión Radioligante , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/metabolismo
4.
J Med Chem ; 63(10): 5297-5311, 2020 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32420741

RESUMEN

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 (∼2 min)] revealed 26 as a useful molecular tool to determine hH3,4Rs/mH4R binding affinities of ligands binding to these receptors.


Asunto(s)
Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Receptores Histamínicos H3/análisis , Receptores Histamínicos H3/metabolismo , Receptores Histamínicos H4/análisis , Receptores Histamínicos H4/metabolismo , Animales , Células HEK293 , Humanos , Células Sf9
5.
Bioorg Med Chem ; 27(7): 1254-1262, 2019 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-30792106

RESUMEN

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 160 nM and behaved as antagonist in functional assays: the cellular aequorin-based assay (IC50 = 32 nM) and [35S]GTPγS binding assay (pKb = 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).


Asunto(s)
Analgésicos/farmacología , Inflamación/tratamiento farmacológico , Receptores Histamínicos H4/antagonistas & inhibidores , Triazinas/farmacología , Analgésicos/síntesis química , Analgésicos/química , Animales , Carragenina , Relación Dosis-Respuesta a Droga , Formaldehído , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Ligandos , Ratones , Estructura Molecular , Ratas , Receptores Histamínicos H4/metabolismo , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/química
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