RESUMEN
INTRODUCTION: Determination of the intrinsic efficacy of ligands at the 5-HT1A receptor is important for selecting drug candidates, e.g. in the case of schizophrenia where partial agonism is a favorable property shared by different atypical antipsychotics. METHODS: Using seven ligands with different intrinsic efficacies and rat hippocampus synaptosomes, we compared critically three "functional" binding assays based on the ternary complex model that considers that the activated conformation of the receptor is the one coupled to G-protein. RESULTS: The Ki ratio method, based on the difference of affinity of the competing drug when using an antagonist vs. an agonist as radioligand, discriminated the ligands according to their intrinsic efficacies, with values from 77 for the full agonist 5-hydroxytryptamine to 0.09 for the inverse agonist WAY 100,635. The GTP-shift method, based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay, was equally effective in classifying the drugs according to their intrinsic efficacy. The lower sensibility of the GTP-shift assay was investigated and explained by the different ionic conditions used in the two assays and the way competition curves were analyzed. Albeit more direct, the assay based on agonist-stimulated [(35)S]-GTPγS binding to G proteins was more expensive and of greater variability in our hands. DISCUSSION: We conclude that the GTP-shift procedure described herein for 5-HT1A receptors may expedite drug discovery efforts by predicting at the same time the affinity and intrinsic efficacy of ligands through a simple, rapid and economic ligand binding assay.
Asunto(s)
Antipsicóticos/farmacología , Unión Competitiva/fisiología , Receptor de Serotonina 5-HT1A/metabolismo , Animales , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ligandos , Masculino , Ratas , Ratas Wistar , Esquizofrenia/tratamiento farmacológicoRESUMEN
In an attempt to increase the affinity of our antipsychotic lead compound LASSBio-579 (1-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)-4-phenylpiperazine; (2)) for the 5-HT(2A) receptor, we synthesized five new N-phenylpiperazine derivatives using a linear synthetic route and the homologation strategy. The binding profile of these compounds was evaluated for a series of dopaminergic, serotonergic and alpha-adrenergic receptors relevant for schizophrenia, using classical competition assays. Increasing the length of the spacer between the functional groups of (2) proved to be appropriated since the affinity of these compounds increased 3-10-fold for the 5-HT(2A) receptor, with no relevant change in the affinity for the D2-like and 5-HT(1A) receptors. A GTP-shift assay also indicated that the most promising derivative (1-(4-(1-(4-chlorophenyl)-1H-pyrazol-4-yl) butyl)-4-phenylpiperazine) (LASSBio-1635) (6) has the expected efficacy at the 5-HT(2A) receptors, acting as an antagonist. Intraperitoneal administration of (6) prevented apomorphine-induced climbing behavior and ketamine-induced hyperlocomotion in mice, in a dose dependent manner. Together, these results show that (6) could be considered as a new antipsychotic lead compound.
Asunto(s)
Antipsicóticos/síntesis química , Antipsicóticos/farmacología , Diseño de Fármacos , Piperazinas/química , Piperazinas/síntesis química , Piperazinas/farmacología , Animales , Antipsicóticos/química , Antipsicóticos/uso terapéutico , Conducta Animal/efectos de los fármacos , Técnicas de Química Sintética , Relación Dosis-Respuesta a Droga , Locomoción/efectos de los fármacos , Masculino , Ratones , Piperazinas/uso terapéutico , Receptor de Serotonina 5-HT2A/metabolismo , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologíaRESUMEN
BACKGROUND: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases with an important role in physiological and pathological remodeling. Their activity is regulated by tissue inhibitors of matrix metalloproteinases (TIMPs). Excess MMPs and myeloperoxidase (MPO) activity have been associated with loss of tooth supporting tissues in periodontal disease (PD). We investigate the changes in salivary MMP-8, MMP-9, TIMP-1, TIMP-2, and MPO concentrations during PD treatment and compare results with plasma levels. METHODS: MMP-8, MMP-9, TIMP-1 and TIMP-2 were analyzed by ELISA. Gelatinolytic activity of MMP-9 forms was determined by zymography, and the MPO activity was determined by colorimetric assay. RESULTS: Subjects were divided into 2 groups: PD and control, which were further divided into 2 subgroups each, namely PD before (PB) and after 3 months (PA) of non-surgical periodontal therapy, and healthy volunteers at baseline (CB) and 3months after baseline (CA). Subgroup PA presented lower gelatinolytic activity and MMP-8 and TIMP-2 concentrations in the saliva compared with PB (p<0.05). The MPO activity was higher in PB compared with CB (p<0.05). There were significant correlations between the gelatinolytic activity of the saliva and MMP-8 and MMP-9 plasma levels. There was a significant correlation between plasma and saliva TIMP-2 levels. CONCLUSION: These results suggest attenuation of some inflammatory markers in the saliva and plasma after PD treatment. Moreover, correlations between salivary and plasma levels exist for some of these markers.