RESUMEN
GSK-3ß, IKK-ß, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid ß (Aß) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3ß plays the most crucial role. In this study, we present compound 62, a novel, remarkably potent, competitive GSK-3ß inhibitor (IC50 = 8 nM, Ki = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.
Asunto(s)
Enfermedad de Alzheimer , Glucógeno Sintasa Quinasa 3 beta , Quinasa I-kappa B , Tiazoles , Quinasas Asociadas a rho , Proteínas tau , Proteínas tau/metabolismo , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Tiazoles/farmacología , Tiazoles/química , Humanos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Ratones , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Línea Celular , Antiinflamatorios/farmacología , Antiinflamatorios/química , Microglía/efectos de los fármacos , Microglía/metabolismo , Óxido Nítrico/metabolismo , Lipopolisacáridos , Agregado de Proteínas/efectos de los fármacos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismoRESUMEN
As Alzheimer's disease (AD) is a neurodegenerative disease with a complex pathogenesis, the exploration of multi-target drugs may be an effective strategy for AD treatment. Multifunctional small molecular agents can be obtained by connecting two or more active drugs or privileged pharmacophores by multicomponent reactions (MCRs). In this paper, two series of polysubstituted pyrazine derivatives with multifunctional moieties were designed as anti-AD agents and synthesized by Passerini-3CR and Ugi-4CR. Since the oxidative stress plays an important role in the pathological process of AD, the antioxidant activities of the newly synthesized compounds were first evaluated. Subsequently, selected active compounds were further screened in a series of AD-related bioassays, including Aß1-42 self-aggregation and deaggregation, BACE-1 inhibition, metal chelation, and protection of SH-SY5Y cells from H2O2-induced oxidative damage. Compound A3B3C1 represented the best one with multifunctional potencies. Mechanism study showed that A3B3C1 acted on Nrf2/ARE signaling pathway, thus increasing the expression of related antioxidant proteins NQO1 and HO-1 to normal cell level. Furthermore, A3B3C1 showed good in vitro human plasma and liver microsome stability, indicating a potential for further development as multifunctional anti-AD agent.
Asunto(s)
Enfermedad de Alzheimer , Neuroblastoma , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Humanos , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Antioxidantes/uso terapéutico , Peróxido de Hidrógeno/farmacología , Inhibidores de la Colinesterasa/farmacología , Estrés Oxidativo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Diseño de Fármacos , Acetilcolinesterasa/metabolismoRESUMEN
Neurodegeneration leading to Alzheimer's disease results from a complex interplay of a variety of processes including misfolding and aggregation of amyloid beta and tau proteins, neuroinflammation or oxidative stress. Therefore, to address more than one of these, drug discovery programmes focus on the development of multifunctional ligands, preferably with disease-modifying and symptoms-reducing potential. Following this idea, herein we present the design and synthesis of multifunctional ligands and biological evaluation of their 5-HT6 receptor affinity (radioligand binding assay), cholinesterase inhibitory activity (spectroscopic Ellman's assay), antioxidant activity (ABTS assay) and metal-chelating properties, as well as a preliminary ADMET properties evaluation. Based on the results we selected compound 14 as a well-balanced and potent 5-HT6 receptor ligand (Ki = 22 nM) and human BuChE inhibitor (IC50 = 16 nM) with antioxidant potential expressed as a reduction of ABTS radicals by 35% (150 µM). The study also revealed additional metal-chelating properties of compounds 15 and 18. The presented compounds modulating Alzheimer's disease-related processes might be further developed as multifunctional ligands against the disease.
Asunto(s)
Enfermedad de Alzheimer , Inhibidores de la Colinesterasa , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Antioxidantes/química , Antioxidantes/farmacología , Butirilcolinesterasa/metabolismo , Quelantes/química , Quelantes/farmacología , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Diseño de Fármacos , Humanos , Ligandos , Receptores de Serotonina/metabolismo , Serotonina , Relación Estructura-ActividadRESUMEN
The lack of an effective treatment makes Alzheimer's disease a serious healthcare problem and a challenge for medicinal chemists. Herein we report interdisciplinary research on novel multifunctional ligands targeting proteins and processes involved in the development of the disease: BuChE, 5-HT6 receptors and ß-amyloid aggregation. Structure-activity relationship analyses supported by crystallography and docking studies led to the identification of a fused-type multifunctional ligand 50, with remarkable and balanced potencies against BuChE (IC50 = 90 nM) and 5-HT6R (Ki = 4.8 nM), and inhibitory activity against Aß aggregation (53% at 10 µM). In in vitro ADME-Tox and in vivo pharmacokinetic studies compound 50 showed good stability in the mouse liver microsomes, favourable safety profile and brain permeability with the brain to plasma ratio of 6.79 after p.o. administration in mice, thus being a promising candidate for in vivo pharmacology studies and a solid foundation for further research on effective anti-AD therapies.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Desarrollo de Medicamentos , Fármacos Neuroprotectores/farmacología , Receptores de Serotonina/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Electrophorus , Células Hep G2 , Caballos , Humanos , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Agregado de Proteínas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Multifunctional ligands as an essential variant of polypharmacology are promising candidates for the treatment of multi-factorial diseases like Alzheimer's disease. Based on clinical evidence and following the paradigm of multifunctional ligands we have rationally designed and synthesized a series of compounds targeting processes involved in the development of the disease. The biological evaluation led to the discovery of two compounds with favorable pharmacological characteristics and ADMET profile. Compounds 17 and 35 are 5-HT6R antagonists (Ki = 13 nM and Ki = 15 nM respectively) and cholinesterase inhibitors with distinct mechanisms of enzyme inhibition. Compound 17, a tacrine derivative is a reversible inhibitor of acetyl- and butyrylcholinesterase (IC50 = 8 nM and IC50 = 24 nM respectively), while compound 35 with rivastigmine-derived phenyl N-ethyl-N-methylcarbamate fragment is a selective, pseudo-irreversible inhibitor of butyrylcholinesterase (IC50 = 455 nM). Both compounds inhibit aggregation of amyloid ß in vitro (75% for compound 17 and 68% for 35 at 10 µM) moreover, compound 35 is a potent tau aggregation inhibitor in cellulo (79%). In ADMET in vitro studies both compounds showed acceptable metabolic stability on mouse liver microsomes (28% and 60% for compound 17 and 35 respectively), no or little effect on CYP3A4 and 2D6 up to a concentration of 10 µM and lack of toxicity on HepG2 cell line (IC50 values of 80 and 21 µM, for 17 and 35 respectively). Based on the pharmacological characteristics and favorable pharmacokinetic properties, we propose compounds 17 and 35 as an excellent starting point for further optimization and in-depth biological studies.
Asunto(s)
Inhibidores de la Colinesterasa/farmacología , Descubrimiento de Drogas , Indoles/farmacología , Acetilcolinesterasa/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Animales , Butirilcolinesterasa/metabolismo , Proliferación Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Electrophorus , Células Hep G2 , Caballos , Humanos , Indoles/síntesis química , Indoles/química , Ligandos , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Agregado de Proteínas/efectos de los fármacos , Receptores de Serotonina/metabolismo , Relación Estructura-Actividad , Proteínas tau/antagonistas & inhibidores , Proteínas tau/metabolismoRESUMEN
Herein we report in vitro metabolic stability in human liver microsomes (HLMs), interactions with cytochrome P450 isoenzymes (CYP3A4, CYP2D6, and CYP2C9), and cytotoxicity analyses on HEK-293, HepG2, Huh7, and WTIIB cell lines of our most recent multitarget directed ligands PF9601N, ASS234, and contilisant. Based on these results, we conclude that (1) PF9601N and contilisant are metabolically stable in the HLM assay, in contrast to the very unstable ASS234; (2) CYP3A4 activity was decreased by PF9601N at all the tested concentrations and by ASS234 and contilisant only at the highest concentration; CYP2D6 activity was reduced by ASS234 at 1, 10, and 25 µM and by PF9601N at 10 and 25 µM, whereas contilisant increased its activity at the same concentrations; CYP2C9 was inhibited by the three compounds; (3) contilisant did not affect cell viability in the widest range of concentrations: up to 10 µM on HEK-293 cells, up to 30 µM on Huh7 cells, up to 50 µM on HepG2 cells, and up to 30 or 100 µM on WTIIB cells. Based on these results, we selected contilisant as a metabolically stable and nontoxic lead compound for further studies in Alzheimer's disease therapy.
Asunto(s)
Inhibidores de la Monoaminooxidasa , Enfermedades Neurodegenerativas , Simulación por Computador , Células HEK293 , Humanos , Microsomas Hepáticos , Inhibidores de la Monoaminooxidasa/farmacologíaRESUMEN
The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds.