RESUMEN
Despite extensive efforts in the development of drugs for complex neurodegenerative diseases, treatment often remains challenging or ineffective, and hence new treatment strategies are necessary. One approach is the design of multi-target drugs, which can potentially address the complex nature of disorders such as Alzheimer's disease. We report a method for high throughput virtual screening aimed at identifying new dual target hit molecules. One of the identified hits, N,N-dimethyl-1-(4-(3-methyl-[1,2,4]triazolo[4,3-a]pyrimidin-6-yl)phenyl)ethan-1-amine (Ý;mir-2), has dual-activity as an acetylcholinesterase (AChE) inhibitor and as an α7 nicotinic acetylcholine receptor (α7 nAChR) agonist. Using computational chemistry methods, parallel and independent screening of a virtual compound library consisting of 3,848,234 drug-like and commercially available molecules from the ZINC15 database, resulted in an intersecting set of 57 compounds, that potentially possess activity at both of the two protein targets. Based on ligand efficiency as well as scaffold and molecular diversity, 16 of these compounds were purchased for in vitro validation by Ellman's method and two-electrode voltage-clamp electrophysiology. Ý;mir-2 was shown to exhibit the desired activity profile (AChE IC50 = 2.58 ± 0.96 µM; α7 nAChR activation = 7.0 ± 0.9% at 200 µM) making it the first reported compound with this particular profile and providing further evidence of the feasibility of in silico methods for the identification of novel multi-target hit molecules.
Asunto(s)
Acetilcolinesterasa/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Inhibidores de la Colinesterasa/aislamiento & purificación , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Acetilcolinesterasa/química , Acetilcolinesterasa/ultraestructura , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Inhibidores de la Colinesterasa/química , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Terapia Molecular Dirigida , Conformación Proteica/efectos de los fármacos , Relación Estructura-Actividad , Interfaz Usuario-Computador , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/ultraestructuraRESUMEN
Plants are constantly challenged in their natural environment by a range of changing conditions. We investigated the acclimation processes and adaptive plant responses to various long-term mild changes and compared them directly within one experimental set-up. Arabidopsis thaliana plants were grown in hydroponic culture for 10 d under controlled abiotic stress (15°C, 25°C, salt and osmotic) and in nutrient deficiency (nitrate and phosphate). Plant growth was monitored and proteomic experiments were performed. Resource allocation between tissues altered during the plants' response. The growth patterns and induced changes of the proteomes indicated that the underlying mechanisms of the adaptation processes are highly specific to the respective environmental condition. Our results indicated differential regulation of response to salt and osmotic treatment, while the proteins in the changed temperature regime showed an inverse, temperature-sensitive control. There was a high correlation of protein level between the nutrient-deficient treatments, but the enriched pathways varied greatly. The proteomic analysis also revealed new insights into the regulation of proteins specific to the shoot and the root. Our investigation revealed unique strategies of plant acclimation to the different applied treatments on a physiological and proteome level, and these strategies are quite distinct in tissues below and above ground.
Asunto(s)
Aclimatación/fisiología , Arabidopsis/fisiología , Ambiente , Proteómica/métodos , Proteínas de Arabidopsis/metabolismo , Biomasa , Análisis por Conglomerados , Ontología de Genes , Ósmosis , Desarrollo de la Planta , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , Proteoma/metabolismo , Temperatura , Factores de TiempoRESUMEN
Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.