RESUMEN
Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Aß42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognition-enhancing anti-AD lead.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Aminoquinolinas/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/metabolismo , Aminoquinolinas/síntesis química , Aminoquinolinas/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Simulación de Dinámica Molecular , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Proteínas tau/antagonistas & inhibidores , Proteínas tau/metabolismoRESUMEN
A combination of flash chromatography, solid phase extraction, high-performance liquid chromatography, and in vitro bioassays was used to isolate phytocomponents endowed with anticholinesterase activity in extract from Phyllanthus muellarianus. Phytocomponents responsible for the anti-cholinesterase activity of subfractions PMF1 and PMF4 were identified and re-assayed to confirm their activity. Magnoflorine was identified as an active phytocomponent from PMF1 while nitidine was isolated from PMF4. Magnoflorine was shown to be a selective inhibitor of human butyrylcholinesterase-hBChE (IC50 = 131 ± 9 µM and IC50 = 1120 ± 83 µM, for hBuChE and human acetylcholinesterase-hAChE, respectively), while nitidine showed comparable inhibitory potencies against both enzymes (IC50 = 6.68 ± 0.13 µM and IC50 = 5.31 ± 0.50 µM, for hBChE and hAChE, respectively). When compared with the commercial anti-Alzheimer drug galanthamine, nitidine was as potent as galanthamine against hAChE and one order of magnitude more potent against hBuChE. Furthermore, nitidine also showed significant, although weak, antiaggregating activity towards amyloid-ß self-aggregation.
Asunto(s)
Acetilcolinesterasa , Butirilcolinesterasa/química , Inhibidores de la Colinesterasa , Simulación del Acoplamiento Molecular , Phyllanthus/química , Corteza de la Planta/química , Extractos Vegetales/química , Acetilcolinesterasa/química , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/aislamiento & purificación , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/química , Humanos , Estructura MolecularRESUMEN
The multifactorial nature of Alzheimer's disease (AD) is a reason for the lack of effective drugs as well as a basis for the development of "multi-target-directed ligands" (MTDLs). As cases increase in developing countries, there is a need of new drugs that are not only effective but also accessible. With this motivation, we report the first sustainable MTDLs, derived from cashew nutshell liquid (CNSL), an inexpensive food waste with anti-inflammatory properties. We applied a framework combination of functionalized CNSL components and well-established acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) tacrine templates. MTDLs were selected based on hepatic, neuronal, and microglial cell toxicity. Enzymatic studies disclosed potent and selective AChE/BChE inhibitors (5, 6, and 12), with subnanomolar activities. The X-ray crystal structure of 5 complexed with BChE allowed rationalizing the observed activity (0.0352 nM). Investigation in BV-2 microglial cells revealed antineuroinflammatory and neuroprotective activities for 5 and 6 (already at 0.01 µM), confirming the design rationale.
Asunto(s)
Ligandos , Fármacos Neuroprotectores/química , Extractos Vegetales/química , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Anacardium/química , Anacardium/metabolismo , Sitios de Unión , Butirilcolinesterasa/química , Butirilcolinesterasa/metabolismo , Dominio Catalítico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Diseño de Fármacos , Humanos , Lipopolisacáridos/farmacología , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Simulación de Dinámica Molecular , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Nueces/química , Nueces/metabolismo , Relación Estructura-Actividad , Tacrina/química , Tacrina/metabolismoRESUMEN
SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (KD =42 and 84â µM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3-HSP90 binding was confirmed (KD =13â µM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Sitio Alostérico , Sitios de Unión , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Proteínas HSP90 de Choque Térmico/química , N-Metiltransferasa de Histona-Lisina/química , Humanos , Cinética , Ligandos , Simulación de Dinámica Molecular , Piperidinas/química , Piperidinas/metabolismo , Unión Proteica , EstereoisomerismoRESUMEN
The combination of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin results in compounds with nanomolar potencies toward human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that retain or improve the antioxidant properties of capsaicin. Crystal structures of their complexes with AChE and BChE revealed the molecular basis for their high potency. Brain penetration was confirmed by biodistribution studies in C57BL6 mice, with one compound (5i) displaying better brain/plasma ratio than donepezil. Chronic treatment of 10 month-old APP/PS1 mice with 5i (2 mg/kg, i.p., 3 times per week, 4 weeks) rescued learning and memory impairments, as measured by three different behavioral tests, delayed the Alzheimer-like pathology progression, as suggested by a significantly reduced Aß42/Aß40 ratio in the hippocampus, improved basal synaptic efficacy, and significantly reduced hippocampal oxidative stress and neuroinflammation. Compound 5i emerges as an interesting anti-Alzheimer lead with beneficial effects on cognitive symptoms and on some underlying disease mechanisms.
Asunto(s)
Acetilcolinesterasa/metabolismo , Antioxidantes/metabolismo , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/química , Acetilcolinesterasa/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Antioxidantes/química , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Butirilcolinesterasa/química , Inhibidores de la Colinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estrés Oxidativo/efectos de los fármacos , Relación Estructura-Actividad , Distribución TisularRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder associated with cholinergic dysfunction, provoking memory loss and cognitive dysfunction in elderly patients. The cholinergic hypothesis provided over the years with molecular targets for developing palliative treatments for AD, acting on the cholinergic system, namely, acetylcholinesterase and α7 nicotinic acetylcholine receptor (α7 nAChR). In our synthetic work, we used "click-chemistry" to synthesize two Multi Target Directed Ligands (MTDLs) MB105 and MB118 carrying tacrine and quinuclidine scaffolds which are known for their anticholinesterase and α7 nAChR agonist activities, respectively. Both, MB105 and MB118, inhibit human acetylcholinesterase and human butyrylcholinesterase in the nanomolar range. Electrophysiological recordings on Xenopus laevis oocytes expressing human α7 nAChR showed that MB105 and MB118 acted as partial agonists of the referred nicotinic receptor, albeit, with different potencies despite their similar structure. The different substitution at C-3 on the 2,3-disubstituted quinuclidine scaffold may account for the significantly lower potency of MB118 compared to MB105. Electrophysiological recordings also showed that the tacrine precursor MB320 behaved as a competitive antagonist of human α7 nAChR, in the micromolar range, while the quinuclidine synthetic precursor MB099 acted as a partial agonist. Taken all together, MB105 behaved as a partial agonist of α7 nAChR at concentrations where it completely inhibited human acetylcholinesterase activity paving the way for the design of novel MTDLs for palliative treatment of AD.
Asunto(s)
Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidores , Acetilcolinesterasa , Alquinos/química , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Barrera Hematoencefálica/efectos de los fármacos , Butirilcolinesterasa/metabolismo , Catálisis , Inhibidores de la Colinesterasa/síntesis química , Química Clic , Cobre , Reacción de Cicloadición , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad , Tacrina/química , Tacrina/farmacología , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3ß and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC50 values toward GSK-3ß, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 µM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas tau/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Fármacos del Sistema Nervioso Central/efectos adversos , Fármacos del Sistema Nervioso Central/química , Fármacos del Sistema Nervioso Central/farmacología , Dicroismo Circular , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Transferencia Resonante de Energía de Fluorescencia , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Células Hep G2 , Humanos , Microscopía de Fuerza Atómica , Terapia Molecular Dirigida/métodos , Ácido Ocadaico/toxicidad , Fosforilación/efectos de los fármacos , Ratas , Relación Estructura-Actividad , Porcinos , Tiazolidinedionas/química , Proteínas tau/antagonistas & inhibidoresRESUMEN
BACKGROUND: Alzheimer´s disease, a progressive and degenerative disorder of the brain, is the most common cause of dementia among the elderly. To face its multifactorial nature, the use of single compounds that can simultaneously modulate different targets involved in the neurodegenerative cascade has emerged as an interesting therapeutic approach. OBJECTIVE: This work investigated the ability of uleine, the major indole alkaloid purified from stem barks of the Brazilian medicinal plant Himatanthus lancifolius, to interact with crucial Alzheimer´s disease disruptive targets associated with two of its major neurodegenerative pathways: acetylcholinesterase and butyrylcholinesterase (cholinergic pathway) and ß-secretase and ß-amyloid peptide (amyloidogenic pathway). METHODS: Uleine's capacity to inhibit human acetylcholinesterase and butyrylcholinesterase enzymes was determined measuring the difference between reaction rates with and without uleine monitored at 412 nm using 5,5'- dithiobis-(2- nitrobenzoic acid) as colorimetric agent. FRET based assay was used to evaluate ß-secretase inhibition using DABCYL- Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-EDANS as substrate and ß-amyloid peptide spontaneous aggregation assay was performed using the thioflavin T spectroscopy assay. Cell viability and toxicity experiments with PC12 and SH-SY5Y cell lines were performed using the MTT colorimetric assay. RESULTS: Uleine demonstrated strong inhibitory activities for both cholinesterases (IC50 279.0±4.5 and 24.0±1.5 µM, respectively) and ß-secretase (IC50 180±22 nM). Above all, uleine significantly inhibited the self-aggregation of amyloid- ß peptide and was not toxic for PC12 or SH-SY5Y neuronal cells. CONCLUSION: These data show for the first time that the natural compound uleine has a novel, multieffective ability to decelerate or even inhibit the development of Alzheimer´s disease.
Asunto(s)
Alcaloides/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Hidrocarburos Aromáticos con Puentes/farmacología , Fármacos Neuroprotectores/farmacología , Acetilcolinesterasa/metabolismo , Alcaloides/toxicidad , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Biomarcadores/metabolismo , Hidrocarburos Aromáticos con Puentes/toxicidad , Butirilcolinesterasa/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Células HEK293 , Humanos , Fármacos Neuroprotectores/toxicidad , Corteza de la Planta/química , Extractos Vegetales/farmacología , Extractos Vegetales/toxicidad , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/metabolismo , RatasRESUMEN
We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-ß (Aß) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Aß. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Antioxidantes/farmacología , Inhibidores de la Colinesterasa/farmacología , Quinonas/química , Tacrina/química , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Antioxidantes/química , Barrera Hematoencefálica/efectos de los fármacos , Técnicas de Química Sintética , Inhibidores de la Colinesterasa/química , Cristalografía por Rayos X , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Células Hep G2/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Terapia Molecular Dirigida , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas WistarAsunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Naftiridinas/síntesis química , Naftiridinas/farmacología , Péptidos beta-Amiloides/metabolismo , Sitios de Unión , Línea Celular Tumoral , Colinesterasas/química , Colinesterasas/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Modelos Moleculares , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/metabolismo , Estereoisomerismo , Tacrina/farmacologíaRESUMEN
Focus of this work was the development and characterization of a new immobilized enzyme reactor (IMER) containing human recombinant butyrylcholinesterase (rBChE) for the on-line kinetic characterization of specific, pseudo-irreversible and brain-targeted BChE inhibitors as potential drug candidates for Alzheimer's disease (AD). Specifically, a rBChE-IMER containing 0.99 U of covalently bound target enzyme was purposely developed and inserted into a HPLC system connected to a UV-vis detector. Selected reversible cholinesterase inhibitors, (-)-phenserine and (-)-cymserine analogues, were then kinetically characterized by rBChE-IMER, and by classical in solution assays and their carbamoylation and decarbamoylation constants were determined. The results support the elucidation of the potency, inhibition duration, mode of action and specific structure/activity relations of these agents and allow cross-validation of the two assay techniques.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Butirilcolinesterasa/química , Inhibidores de la Colinesterasa/aislamiento & purificación , Enzimas Inmovilizadas/química , Butirilcolinesterasa/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Enzimas Inmovilizadas/efectos de los fármacos , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacosRESUMEN
Starting from a structure-based drug design, new acetylcholinesterase inhibitors were designed and synthesized as analogues of donepezil. The compounds were composed by an aromatic function and a tertiary amino moiety connected by a suitable spacer. In particular, the benzophenone nucleus and the N,N-benzylmethylamine function were selected. The easily accessible three-step synthesis of these compounds resulted to be significantly less difficult and expensive than that of donepezil. Several compounds possess anti-cholinesterase activity in the order of micro and sub-micromolar. Particularly, compounds 1 and 10 were the most potent inhibitors of the series.
Asunto(s)
Acetilcolinesterasa/efectos de los fármacos , Benzofenonas/química , Benzofenonas/farmacología , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Benzofenonas/síntesis química , Inhibidores de la Colinesterasa/síntesis química , Evaluación Preclínica de Medicamentos , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Espectrometría de Masa por Ionización de ElectrosprayAsunto(s)
Alcanos/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Etilaminas/uso terapéutico , Alcanos/síntesis química , Alcanos/química , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Etilaminas/síntesis química , Etilaminas/química , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Heterodimers 4 and 5 were effective inhibitors of acetylcholinesterase (AChE) activity and AChE-induced amyloid-beta (A beta) aggregation. The peculiar biological profile of 4 can be relevant in studying the molecular basis underlying the nonclassical action of AChE and in addressing the question whether AChE inhibitors can affect the neurotoxic cascade leading to Alzheimer's disease. Compound 4 emerged as the most potent heterodimer so far available to inhibit AChE-induced A beta aggregation.