RESUMEN
Influenza is an infectious disease that represents an important public health burden, with high impact on the global morbidity, mortality, and economy. The poor protection and the need of annual updating of the anti-influenza vaccine, added to the rapid emergence of viral strains resistant to current therapy make the need for antiviral drugs with novel mechanisms of action compelling. In this regard, the viral RNA polymerase is an attractive target that allows the design of selective compounds with reduced risk of resistance. In previous studies we showed that the inhibition of the polymerase acidic protein-basic protein 1 (PA-PB1) interaction is a promising strategy for the development of anti-influenza agents. Starting from the previously identified 3-cyano-4,6-diphenyl-pyridines, we chemically modified this scaffold and explored its structure-activity relationships. Noncytotoxic compounds with both the ability of disrupting the PA-PB1 interaction and antiviral activity were identified, and their mechanism of target binding was clarified with molecular modeling simulations.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Piridinas/síntesis química , Piridinas/farmacología , Proteínas Virales/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Perros , Diseño de Fármacos , Células HEK293 , Humanos , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Modelos Moleculares , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacosRESUMEN
VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [(3)H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [(3)H]VUF10166 displayed saturable binding with a Kd of 0.18 nM. Kinetic measurements gave monophasic association (6.25 × 10(7) M(-1) min(-1)) and dissociation (0.01 min(-1)) rates that yielded a similar Kd value (0.16 nM). At 5-HT3AB receptors two association (6.15 × 10(-7), 7.23 M(-1) min(-1)) and dissociation (0.024, 0.162 min(-1)) rates were seen, yielding Kd values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (Kd = 0.74 nM) and competition (Ki = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT3 receptor-selective orthosteric ligands (5-HT, allosetron, d-tubocurarine, granisetron, mCPBG, MDL72222, quipazine), but not by non-competitive antagonists (bilobalide, ginkgolide B, picrotoxin) or competitive ligands of other Cys-loop receptors (ACh, bicuculline, glycine, gabazine). To explore VUF10166 ligand-receptor interactions we used in silico modelling and docking, and tested the predictions using site directed mutagenesis. The data suggest that VUF10166 adopts a similar orientation to 5-HT3 receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.
Asunto(s)
Piperidinas/metabolismo , Quinoxalinas/metabolismo , Radiofármacos/metabolismo , Receptores de Serotonina 5-HT3/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Piperidinas/síntesis química , Quinoxalinas/síntesis química , Ensayo de Unión Radioligante , Radiofármacos/síntesis química , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/genética , Alineación de Secuencia , TritioRESUMEN
Until recently, discriminating between homomeric 5-HT3A and heteromeric 5-HT3AB receptors was only possible with ligands that bind in the receptor pore. This study describes the first series of ligands that can discriminate between these receptor types at the level of the orthosteric binding site. During a recent fragment screen, 2-chloro-3-(4-methylpiperazin-1-yl)quinoxaline (VUF10166) was identified as a ligand that displays an 83-fold difference in [(3)H]granisetron binding affinity between 5-HT3A and 5-HT3AB receptors. Fragment hit exploration, initiated from VUF10166 and 3-(4-methylpiperazin-1-yl)quinoxalin-2-ol, resulted in a series of compounds with higher affinity at either 5-HT3A or 5-HT3AB receptors. These ligands reveal that a single atom is sufficient to change the selectivity profile of a compound. At the extremes of the new compounds were 2-amino-3-(4-methylpiperazin-1-yl)quinoxaline, which showed 11-fold selectivity for the 5-HT3A receptor, and 2-(4-methylpiperazin-1-yl)quinoxaline, which showed an 8.3-fold selectivity for the 5-HT3AB receptor. These compounds represent novel molecular tools for studying 5-HT3 receptor subtypes and could help elucidate their physiological roles.
Asunto(s)
Quinoxalinas/farmacología , Receptores de Serotonina 5-HT3/metabolismo , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Quinoxalinas/química , Receptores de Serotonina 5-HT3/química , Relación Estructura-ActividadRESUMEN
Smaller stones with a wide variety of colors make a higher resolution mosaic. In much the same way, smaller chemical entities that are structurally diverse are better able to interrogate protein binding sites. This feature article describes the construction of a diverse fragment library and an analysis of the screening of six representative protein targets belonging to three diverse target classes (G protein-coupled receptors ADRB2, H1R, H3R, and H4R, the ligand-gated ion channel 5-HT3R, and the kinase PKA) using chemogenomics approaches. The integration of experimentally determined bioaffinity profiles across related and unrelated protein targets and chemogenomics analysis of fragment binding and protein structure allow the identification of: (i) unexpected similarities and differences in ligand binding properties, and (ii) subtle ligand affinity and selectivity cliffs. With a wealth of fragment screening data being generated in industry and academia, such approaches will contribute to a more detailed structural understanding of ligand-protein interactions.
Asunto(s)
Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Descubrimiento de Drogas , Preparaciones Farmacéuticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina 5-HT3/metabolismo , Sitios de Unión , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Células HEK293 , Humanos , Ligandos , Unión Proteica , Receptores Acoplados a Proteínas G/química , Receptores de Serotonina 5-HT3/química , Bibliotecas de Moléculas PequeñasRESUMEN
GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA(A) receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.
Asunto(s)
Benzodiazepinas/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Benzodiazepinas/metabolismo , Sitios de Unión , Biopolímeros , Cristalografía por Rayos X , Canales Iónicos/química , Ligandos , Modelos Moleculares , Receptores de GABA-A/metabolismo , XenopusRESUMEN
The 5-HT3 receptor, a pentameric ligand-gated ion channel (pLGIC), is an important therapeutic target. During a recent fragment screen, 6-chloro-N-methyl-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (1) was identified as a 5-HT3 hit fragment. Here we describe the synthesis and structure-activity relationships (SAR) of a series of (iso)quinoline and quinazoline compounds that were synthesized and screened for 5-HT3 R affinity using a [³H]granisetron displacement assay. These studies resulted in the discovery of several high affinity ligands of which compound 22 showed the highest affinity (pK(i) > 10) for the 5-HT3 receptor. The observed SAR is in agreement with established pharmacophore models for 5-HT3 ligands and is used for ligand-receptor binding mode prediction using homology modeling and in silico docking approaches.
Asunto(s)
Isoquinolinas/síntesis química , Quinazolinas/síntesis química , Quinolinas/síntesis química , Receptores de Serotonina 5-HT3/metabolismo , Unión Competitiva , Simulación por Computador , Diseño de Fármacos , Células HEK293 , Humanos , Isoquinolinas/química , Isoquinolinas/farmacología , Ligandos , Modelos Moleculares , Quinazolinas/química , Quinazolinas/farmacología , Quinolinas/química , Quinolinas/farmacología , Ensayo de Unión Radioligante , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A fragment library was screened against the G protein-coupled histamine H(4) receptor (H(4)R) and the ligand-gated ion channel serotonin 5-HT(3A) (5-HT(3A)R). Interestingly, significant overlap was found between H(4)R and 5-HT(3A)R hit sets. The data indicates that dual active H(4)R and 5 HT(3A)R fragments have a higher complexity than the selective compounds which has important implications for chemical genomics approaches. The results of our fragment-based library screening study illustrate similarities in ligand recognition between H(4)R and 5-HT(3A)R and have important consequences for selectivity profiling in ongoing drug discovery efforts on H(4)R and 5-HT(3A)R. The affinity profiles of our fragment screening studies furthermore match the chemical properties of the H(4)R and 5-HT(3A)R binding sites and can be used to define molecular interaction fingerprints to guide the in silico prediction of protein-ligand interactions and structure.
Asunto(s)
Compuestos Orgánicos/farmacología , Receptores Histamínicos/metabolismo , Receptores de Serotonina 5-HT3/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Estructura Molecular , Compuestos Orgánicos/química , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
Drug discovery requires a simple, rapid, and cost-effective method for the early identification of novel leads and elimination of poor candidates. Here we present an experimental design that fulfils these criteria, using a ligand-gated ion channel expressed in a mammalian cell line, whose function can be probed using a voltage-sensitive dye. The experimental design is novel, as it uses the same screen to identify hit fragments and to characterize them as agonists or antagonists. The results were independently validated using radioligand binding, although the new technique has several advantages over radioligand methods. A number of novel high-affinity ligands were found. The method is broadly applicable to a wide range of receptor types including ligand-gated ion channels (LGICs), voltage-gated ion channels (VGICs), and G protein-coupled receptors (GPCRs), all of which are important drug targets.
Asunto(s)
Descubrimiento de Drogas/métodos , Canales Iónicos Activados por Ligandos/efectos de los fármacos , Tecnología Farmacéutica/métodos , Línea Celular , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Fluorometría , Expresión Génica , Granisetrón/metabolismo , Células HEK293 , Humanos , Canales Iónicos Activados por Ligandos/agonistas , Canales Iónicos Activados por Ligandos/antagonistas & inhibidores , Receptores de Serotonina 5-HT3/efectos de los fármacos , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Serotonina/administración & dosificación , Antagonistas de la Serotonina/metabolismo , Transfección , TritioRESUMEN
Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.
Asunto(s)
Aminas/farmacología , Imipramina/farmacología , Indenos/farmacología , Receptores de Quimiocina/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Aminas/síntesis química , Aminas/química , Humanos , Imipramina/análogos & derivados , Imipramina/química , Indenos/síntesis química , Indenos/química , Ligandos , Estructura Molecular , Receptores de Quimiocina/agonistas , Relación Estructura-Actividad , Proteínas Virales/agonistasRESUMEN
G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral drugs.