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1.
RSC Med Chem ; 12(7): 1154-1163, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34355181

RESUMO

As part of our efforts to develop sustainable drugs for Alzheimer's disease (AD), we have been focusing on the inexpensive and largely available cashew nut shell liquid (CNSL) as a starting material for the identification of new acetylcholinesterase (AChE) inhibitors. Herein, we decided to investigate whether cardanol, a phenolic CNSL component, could serve as a scaffold for improved compounds with concomitant anti-amyloid and antioxidant activities. Ten new derivatives, carrying the intact phenolic function and an aminomethyl functionality, were synthesized and first tested for their inhibitory potencies towards AChE and butyrylcholinesterase (BChE). 5 and 11 were found to inhibit human BChE at a single-digit micromolar concentration. Transmission electron microscopy revealed the potential of five derivatives to modulate Aß aggregation, including 5 and 11. In HORAC assays, 5 and 11 performed similarly to standard antioxidant ferulic acid as hydroxyl scavenging agents. Furthermore, in in vitro studies in neuronal cell cultures, 5 and 11 were found to effectively inhibit reactive oxygen species production at a 10 µM concentration. They also showed a favorable initial ADME/Tox profile. Overall, these results suggest that CNSL is a promising raw material for the development of potential disease-modifying treatments for AD.

2.
Eur J Med Chem ; 225: 113779, 2021 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-34418785

RESUMO

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.

3.
Molecules ; 26(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299650

RESUMO

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.


Assuntos
Acetilcolinesterase , Butirilcolinesterase/química , Inibidores da Colinesterase , Simulação de Acoplamento Molecular , Phyllanthus/química , Casca de Planta/química , Extratos Vegetais/química , Acetilcolinesterase/química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/isolamento & purificação , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Humanos , Estrutura Molecular
4.
Molecules ; 26(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071439

RESUMO

In the last years, the connection between the endocannabinoid system (eCS) and neuroprotection has been discovered, and evidence indicates that eCS signaling is involved in the regulation of cognitive processes and in the pathophysiology of Alzheimer's disease (AD). Accordingly, pharmacotherapy targeting eCS could represent a valuable contribution in fighting a multifaceted disease such as AD, opening a new perspective for the development of active agents with multitarget potential. In this paper, a series of coumarin-based carbamic and amide derivatives were designed and synthesized as multipotent compounds acting on cholinergic system and eCS-related targets. Indeed, they were tested with appropriate enzymatic assays on acetyl and butyryl-cholinesterases and on fatty acid amide hydrolase (FAAH), and also evaluated as cannabinoid receptor (CB1 and CB2) ligands. Moreover, their ability to reduce the self-aggregation of beta amyloid protein (Aß42) was assessed. Compounds 2 and 3, bearing a carbamate function, emerged as promising inhibitors of hAChE, hBuChE, FAAH and Aß42 self-aggregation, albeit with moderate potencies, while the amide 6 also appears a promising CB1/CB2 receptors ligand. These data prove for the new compounds an encouraging multitarget profile, deserving further evaluation.


Assuntos
Canabinoides/química , Receptores Colinérgicos/química , Doença de Alzheimer/tratamento farmacológico , Amidoidrolases , Peptídeos beta-Amiloides/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Carbamatos/farmacologia , Química Farmacêutica/métodos , Colinérgicos , Cumarínicos/uso terapêutico , Desenho de Fármacos , Endocanabinoides/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Conformação Proteica , Ratos , Receptores de Canabinoides , Rivastigmina/farmacologia
5.
Biochim Biophys Acta Biomembr ; 1863(9): 183641, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33984320

RESUMO

The ability of styrene-maleic acid (SMAc) co-polymers to spontaneously insert into biological membranes can be exploited to extract G protein-coupled receptors (GPCRs) embedded in styrene-maleic acid lipid particles (SMALPs), preserving the native environment around the protein and thus enhancing the feasibility of functional studies. So far, the SMALP technology has been primarily employed on non-mammalian cells and protocols are not optimized for adherent human cell lines, which cannot be harvested in large amounts. In this work, a fine investigation of key parameters affecting the formation of SMALPs was undertaken with the purpose of maximizing the yield of extraction of a recombinant form of human ß2-adrenergic receptor (rhß2AR) from HEK293T cells. The study highlighted an important influence of ionic strength on the membrane solubilization efficiency and GPCR purification yield of SMAc co-polymers: by lowering the salt concentration of all buffers used in previously published SMALP protocols, the water solubility and extraction efficiency of the selected SMAc co-polymer (commercially supplied as a potassium salt) were enhanced. In-line combination of size-exclusion chromatography (SEC) with immobilized metal affinity chromatography (IMAC) allowed further improvement of the final rhß2AR yield by reducing the loss of SMALP-embedded GPCRs during the fractionation and purification of SMALPs. The overall findings of this study show that the available SMALP protocols can be significantly optimized in several aspects in order to increase the efficiency of GPCR solubilization and isolation from low-yielding expression systems.


Assuntos
Lipídeos/química , Maleatos/química , Poliestirenos/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Células Cultivadas , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/química
6.
J Med Chem ; 64(8): 4972-4990, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33829779

RESUMO

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.


Assuntos
Ligantes , Fármacos Neuroprotetores/química , Extratos Vegetais/química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Anacardium/química , Anacardium/metabolismo , Sítios de Ligação , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Domínio Catalítico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Desenho de Fármacos , Humanos , Lipopolissacarídeos/farmacologia , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Simulação de Dinâmica Molecular , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Nozes/química , Nozes/metabolismo , Relação Estrutura-Atividade , Tacrina/química , Tacrina/metabolismo
7.
ACS Chem Neurosci ; 12(8): 1328-1342, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33797877

RESUMO

Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial nature of Alzheimer's disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3ß and as calcium channel antagonists via the Biginelli multicomponent reaction. Among these MTDLs, (±)-BIGI-3h was identified as a promising new hit compound showing in vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant effects and brain penetration, along with the ability to inhibit the aggregation of both τ protein and ß-amyloid peptide. The in vivo studies have shown that (±)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits.


Assuntos
Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Glicogênio Sintase Quinase 3 beta , Humanos , Ligantes , Monoaminoxidase/metabolismo
8.
ACS Chem Neurosci ; 12(9): 1698-1715, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33852284

RESUMO

Since 2002, no clinical candidate against Alzheimer's disease has reached the market; hence, an effective therapy is urgently needed. We followed the so-called "multitarget directed ligand" approach and designed 36 novel tacrine-phenothiazine heterodimers which were in vitro evaluated for their anticholinesterase properties. The assessment of the structure-activity relationships of such derivatives highlighted compound 1dC as a potent and selective acetylcholinesterase inhibitor with IC50 = 8 nM and 1aA as a potent butyrylcholinesterase inhibitor with IC50 = 15 nM. Selected hybrids, namely, 1aC, 1bC, 1cC, 1dC, and 2dC, showed a significant inhibitory activity toward τ(306-336) peptide aggregation with percent inhibition ranging from 50.5 to 62.1%. Likewise, 1dC and 2dC exerted a remarkable ability to inhibit self-induced Aß1-42 aggregation. Notwithstanding, in vitro studies displayed cytotoxicity toward HepG2 cells and cerebellar granule neurons; no pathophysiological abnormality was observed when 1dC was administered to mice at 14 mg/kg (i.p.). 1dC was also able to permeate to the CNS as shown by in vitro and in vivo models. The maximum brain concentration was close to the IC50 value for acetylcholinesterase inhibition with a relatively slow elimination half-time. 1dC showed an acceptable safety and good pharmacokinetic properties and a multifunctional biological profile.


Assuntos
Doença de Alzheimer , Tacrina , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Animais , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Desenho de Fármacos , Camundongos , Fenotiazinas/farmacologia , Relação Estrutura-Atividade , Tacrina/farmacologia
9.
Molecules ; 26(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669839

RESUMO

Despite Alzheimer's disease (AD) incidence being projected to increase worldwide, the drugs currently on the market can only mitigate symptoms. Considering the failures of the classical paradigm "one target-one drug-one disease" in delivering effective medications for AD, polypharmacology appears to be a most viable therapeutic strategy. Polypharmacology can involve combinations of multiple drugs and/or single chemical entities modulating multiple targets. Taking inspiration from an ongoing clinical trial, this work aims to convert a promising cromolyn-ibuprofen drug combination into single-molecule "codrugs." Such codrugs should be able to similarly modulate neuroinflammatory and amyloid pathways, while showing peculiar pros and cons. By exploiting a linking strategy, we designed and synthesized a small set of cromolyn-ibuprofen conjugates (4-6). Preliminary plasma stability and neurotoxicity assays allowed us to select diamide 5 and ethanolamide 6 as promising compounds for further studies. We investigated their immunomodulatory profile in immortalized microglia cells, in vitro anti-aggregating activity towards Aß42-amyloid self-aggregation, and their cellular neuroprotective effect against Aß42-induced neurotoxicity. The fact that 6 effectively reduced Aß-induced neuronal death, prompted its investigation into an in vivo model. Notably, 6 was demonstrated to significantly increase the longevity of Aß42-expressing Drosophila and to improve fly locomotor performance.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Cromolina Sódica/uso terapêutico , Ibuprofeno/uso terapêutico , Polifarmacologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromolina Sódica/síntese química , Cromolina Sódica/química , Cromolina Sódica/farmacologia , Drosophila/efeitos dos fármacos , Desenho de Fármacos , Endocitose/efeitos dos fármacos , Ibuprofeno/síntese química , Ibuprofeno/química , Ibuprofeno/farmacologia , Imunomodulação/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/toxicidade , Agregados Proteicos/efeitos dos fármacos , Ratos Wistar
10.
Hepatology ; 74(4): 2058-2073, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33710623

RESUMO

BACKGROUND AND AIMS: Circulating albumin in cirrhosis can be dysfunctional because of accumulating structural damages, leading to the concept of effective albumin concentration (eAlb), referring to the albumin portion presenting structural and functional integrity. We aimed to estimate eAlb in patients with decompensated cirrhosis and analyze its relationships with albumin function and clinical outcomes as compared to total albumin concentration (tAlb). APPROACH AND RESULTS: We evaluated 319 patients with cirrhosis hospitalized for acute decompensation (AD) with and without acute-on-chronic liver failure (ACLF) and 18 age- and sex-comparable outpatients with compensated cirrhosis. tAlb was quantified by standard assay, whereas eAlb was estimated combining liquid chromatography/electrospray ionization/mass spectrometry and standard methods. Albumin binding and detoxification efficiency were evaluated by electron paramagnetic resonance analysis. Circulating albumin in patients with decompensated cirrhosis displayed multiple structural abnormalities, with reversible oxidation and glycation being the most frequent. As a result, eAlb progressively declined with the worsening of cirrhosis and was superior to tAlb in stratifying patients between compensated cirrhosis, AD, and ACLF, as well as patients with and without complications. Moreover, eAlb, but not tAlb, was closely associated with binding capacities in ACLF. Finally, eAlb at admission predicted the occurrence of ACLF within 30 days and mortality at 90 days better than tAlb. CONCLUSIONS: This large, observational study provides the evidence in patients with decompensated cirrhosis that eAlb can be quantified and differentiated from tAlb routinely measured in clinical practice. As compared to tAlb, eAlb is more closely associated with disease severity and albumin dysfunction and carries a greater prognostic power. These results prompt future research assessing eAlb as a biomarker for predicting prognosis and treatment response.

11.
Chembiochem ; 22(9): 1597-1608, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400854

RESUMO

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 84 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 =13 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.

12.
ChemMedChem ; 16(1): 187-198, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716144

RESUMO

Thanks to the widespread use and safety profile of donepezil (1) in the treatment of Alzheimer's disease (AD), one of the most widely adopted multi-target-directed ligand (MTDL) design strategies is to modify its molecular structure by linking a second fragment carrying an additional AD-relevant biological property. Herein, supported by a proposed combination therapy of 1 and the quinone drug idebenone, we rationally designed novel 1-based MTDLs targeting Aß and oxidative pathways. By exploiting a bioisosteric replacement of the indanone core of 1 with a 1,4-naphthoquinone, we ended up with a series of highly merged derivatives, in principle devoid of the "physicochemical challenge" typical of large hybrid-based MTDLs. A preliminary investigation of their multi-target profile identified 9, which showed a potent and selective butyrylcholinesterase inhibitory activity, together with antioxidant and antiaggregating properties. In addition, it displayed a promising drug-like profile.


Assuntos
Donepezila/química , Ligantes , Fármacos Neuroprotetores/química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Donepezila/metabolismo , Donepezila/farmacologia , Donepezila/uso terapêutico , Desenho de Fármacos , Humanos , Indanos/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade
13.
J Med Chem ; 64(1): 812-839, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33356266

RESUMO

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.


Assuntos
Acetilcolinesterase/metabolismo , Antioxidantes/metabolismo , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Acetilcolinesterase/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Butirilcolinesterase/química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Estresse Oxidativo/efeitos dos fármacos , Relação Estrutura-Atividade , Distribuição Tecidual
14.
iScience ; 23(10): 101604, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33205017

RESUMO

SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.

15.
Mol Pharm ; 17(9): 3609-3621, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32786955

RESUMO

Protein inactivation either during the production process or along the gastrointestinal tract is the major problem associated with the development of oral delivery systems for biological drugs. This work presents an evaluation of the structural integrity and the biological activity of a model protein, catalase, after its encapsulation in glyceryl trimyristate-based solid lipid microparticles (SLMs) obtained by the spray congealing technology. Circular dichroism and fluorescence spectroscopies were used to assess the integrity of catalase released from SLMs. The results confirmed that no conformational change occurred during the production process and both the secondary and tertiary structures were retained. Catalase is highly sensitive to temperature and undergoes denaturation above 60 °C; nevertheless, spray congealing allowed the retention of most biological activity due to the loading of the drug at the solid state, markedly reducing the risk of denaturation. Catalase activity after exposure to simulated gastric conditions (considering both acidic pH and the presence of gastric digestive hydrolases) ranged from 35 to 95% depending on the carrier: increasing of both the fatty acid chain length and the degree of substitution of the glyceride enhanced residual enzyme activity. SLMs allowed the protein release in a simulated intestinal environment and were not cytotoxic against HT29 cells. In conclusion, the encapsulation of proteins into SLMs by spray congealing might be a promising strategy for the formulation of nontoxic and inexpensive oral biotherapeutic products.


Assuntos
Catalase/administração & dosagem , Catalase/química , Lipídeos/química , Estômago/efeitos dos fármacos , Administração Oral , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Portadores de Fármacos/química , Ácidos Graxos/química , Células HT29 , Humanos , Concentração de Íons de Hidrogênio , Microesferas , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos
16.
Viruses ; 12(9)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854437

RESUMO

Parvovirus B19 (B19V), an ssDNA virus in the family Parvoviridae, is a human pathogenic virus, responsible for a wide range of clinical manifestations, still in need of effective and specific antivirals. DNA structures, including G-quadruplex (G4), have been recognised as relevant functional features in viral genomes, and small-molecule ligands binding to these structures are promising antiviral compounds. Bioinformatic tools predict the presence of potential G4 forming sequences (PQSs) in the genome of B19V, raising interest as targets for antiviral strategies. Predictions locate PQSs in the genomic terminal regions, in proximity to replicative origins. The actual propensity of these PQSs to form G4 structures was investigated by circular dichroism spectroscopic analysis on synthetic oligonucleotides of corresponding sequences. No signature of G4 structures was detected, and the interaction with the G4 ligand BRACO-19 (N,N'-(9-{[4-(dimethylamino)phenyl]amino}acridine-3,6-diyl)bis(3-pyrrolidin-1-ylpropanamide) did not appear consistent with the stabilisation of G4 structures. Any potential role of PQSs in the viral lifecycle was then assessed in an in vitro infection model system, by evaluating any variation in replication or expression of B19V in the presence of the G4 ligands BRACO-19 and pyridostatin. Neither showed a significant inhibitory activity on B19V replication or expression. Experimental challenge did not support bioinformatic predictions. The terminal regions of B19V are characterised by relevant sequence and symmetry constraints, which are functional to viral replication. Our experiments suggest that these impose a stringent requirement prevailing over the propensity of forming actual G4 structures.


Assuntos
DNA Viral/química , Quadruplex G , Parvovirus B19 Humano/genética , Acridinas/farmacologia , Aminoquinolinas/farmacologia , Antivirais/farmacologia , Células Cultivadas , Dicroísmo Circular , Biologia Computacional , DNA Viral/metabolismo , Células Precursoras Eritroides/virologia , Genoma Viral , Humanos , Parvovirus B19 Humano/efeitos dos fármacos , Parvovirus B19 Humano/fisiologia , Ácidos Picolínicos/farmacologia , Replicação Viral/efeitos dos fármacos
17.
J Med Chem ; 63(17): 9360-9390, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32706255

RESUMO

Oxidative stress is a major pathogenic factor in Alzheimer's disease, but it should not be tackled alone rather together with other key targets to derive effective treatments. The combination of the scaffold of the polar antioxidant lead 7-methoxy-2,2-dimethylchroman-6-ol (CR-6) with that of the lipophilic cholinesterase inhibitor 6-chlorotacrine results in compounds with favorable brain permeability and multiple activities in vitro (acetylcholinesterase, butyrylcholinesterase, ß-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE-1), and Aß42 and tau aggregation inhibition). In in vivo studies on wild-type and APP/presenilin 1 (PS1) mice, two selected compounds were well tolerated and led to positive trends, albeit statistically nonsignificant in some cases, on memory performance, amyloid pathology (reduced amyloid burden and potentiated non-amyloidogenic APP processing), and oxidative stress (reduced cortical oxidized proteins and increased antioxidant enzymes superoxide dismutase 2 (SOD2), catalase, glutathione peroxidase 1 (GPX1), and heme oxygenase 1 (Hmox1) and transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2)). These compounds emerge as interesting brain-permeable multitarget compounds, with a potential as anti-Alzheimer agents beyond that of the original lead CR-6.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Antioxidantes/química , Antioxidantes/farmacologia , Benzopiranos/química , Benzopiranos/farmacologia , Terapia de Alvo Molecular , Doença de Alzheimer/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/uso terapêutico , Benzopiranos/metabolismo , Benzopiranos/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Camundongos , Simulação de Dinâmica Molecular , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Conformação Proteica
18.
Biochem Pharmacol ; 177: 114010, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32360492

RESUMO

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.


Assuntos
Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Acetilcolinesterase , Alcinos/química , Doença de Alzheimer/tratamento farmacológico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Butirilcolinesterase/metabolismo , Catálise , Inibidores da Colinesterase/síntese química , Química Click , Cobre , Reação de Cicloadição , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Tacrina/química , Tacrina/farmacologia , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
19.
Anal Bioanal Chem ; 412(18): 4245-4259, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32367292

RESUMO

Serum levels of early-glycated albumin are significantly increased in patients with diabetes mellitus and may play a role in worsening inflammatory status and sustaining diabetes-related complications. To investigate possible pathological recognition involving early-glycated albumin and the receptor for advanced glycation end products (RAGE), an early-glycated human serum albumin (HSAgly), with a glycation pattern representative of the glycated HSA form abundant in diabetic patients, and the recombinant human RAGE ectodomain (VC1) were used. Biorecognition between the two interactants was investigated by combining surface plasmon resonance (SPR) analysis and affinity chromatography coupled with mass spectrometry (affinity-MS) for peptide extraction and identification. SPR analysis proved early-glycated albumin could interact with the RAGE ectodomain with a steady-state affinity constant of 6.05 ± 0.96 × 10-7 M. Such interaction was shown to be specific, as confirmed by a displacement assay with chondroitin sulfate, a known RAGE binder. Affinity-MS studies were performed to map the surface area involved in the recognition. These studies highlighted that a region surrounding Lys525 and part of subdomain IA were involved in VC1 recognition. Finally, an in silico analysis highlighted (i) a key role for glycation at Lys525 (the most commonly glycated residue in HSA in diabetic patients) through a triggering mechanism similar to that previously observed for AGEs or advanced lipoxidation end products and (ii) a stabilizing role for subdomain IA. Albeit a moderate affinity for complex formation, the high plasma levels of early-glycated albumin and high percentage of glycation at Lys525 in diabetic patients make this interaction of possible pathological relevance. Graphical abstract.


Assuntos
Receptor para Produtos Finais de Glicação Avançada/metabolismo , Albumina Sérica Humana/metabolismo , Albumina Sérica/metabolismo , Sítios de Ligação , Cromatografia de Afinidade , Diabetes Mellitus/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Receptor para Produtos Finais de Glicação Avançada/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Albumina Sérica/química , Albumina Sérica Humana/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície
20.
Bioorg Chem ; 98: 103753, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32200328

RESUMO

Our goal was the evaluation of a series of N-1,2,3-triazole-isatin derivatives for multi-target activity which included cholinesterase (ChE) inhibition and ß-amyloid (Aß) peptide anti-aggregation. The compounds have shown considerable promise as butyrylcholinesterase (BuChE) inhibitors. Although the inhibition of eel acetylcholinesterase (eeAChE) was weak, the inhibitions against equine BuChE (eqBuChE) and human BuChE (hBuChE) were more significant with a best inhibition against eqBuChE of 0.46 µM. In some cases, these molecules gave better inhibitions for hBuChE than eqBuChE. For greater insights into their mode of action, molecular docking studies were carried out, followed by STD-NMR validation. In addition, some of these compounds showed weak Aß anti-aggregation activity. Hepatotoxicity studies showed that they were non-hepatoxic and neurotoxicity studies using neurite outgrowth experiments led to the conclusion that these compounds are only weakly neurotoxic.


Assuntos
Acetilcolinesterase/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Isatina/farmacologia , Triazóis/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Electrophorus , Células Hep G2 , Cavalos , Humanos , Isatina/química , Estrutura Molecular , Agregados Proteicos , Relação Estrutura-Atividade , Triazóis/química
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