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1.
iScience ; 27(2): 108968, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38327788

RESUMO

Excessive or aberrant NLRP3 inflammasome activation has been implicated in the progression and initiation of many inflammatory conditions; however, currently no NLRP3 inflammasome inhibitors have been approved for therapeutic use in the clinic. Here we have identified that the natural product brazilin effectively inhibits both priming and activation of the NLRP3 inflammasome in cultured murine macrophages, a human iPSC microglial cell line and in a mouse model of acute peritoneal inflammation. Through computational modeling, we predict that brazilin can adopt a favorable binding pose within a site of the NLRP3 protein which is essential for its conformational activation. Our results not only encourage further evaluation of brazilin as a therapeutic agent for NLRP3-related inflammatory diseases, but also introduce this small-molecule as a promising scaffold structure for the development of derivative NLRP3 inhibitor compounds.

2.
Cell Death Discov ; 9(1): 469, 2023 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-38129373

RESUMO

The NLRP3 inflammasome is a component of the inflammatory response to infection and injury, orchestrating the maturation and release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-18, and triggering pyroptotic cell death. Appropriate levels of NLRP3 activation are needed to avoid excessive tissue damage while ensuring host protection. Here we report a role for symmetrical diarylsquaramides as selective K+ efflux-dependent NLRP3 inflammasome enhancers. Treatment of macrophages with squaramides potentiated IL-1ß secretion and ASC speck formation in response to K+ efflux-dependent NLRP3 inflammasome activators without affecting priming, endosome cargo trafficking, or activation of other inflammasomes. The squaramides lowered intracellular K+ concentration which enabled cells to respond to a below-threshold dose of the inflammasome activator nigericin. Taken together these data further highlight the role of ion flux in inflammasome activation and squaramides as an interesting platform for therapeutic development in conditions where enhanced NLRP3 activity could be beneficial.

3.
Molecules ; 28(18)2023 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-37764248

RESUMO

The emergence of resistance to first-line antimalarial drugs calls for the development of new therapies for drug-resistant malaria. The efficacy of quinoline-based antimalarial drugs has prompted the development of novel quinolines. A panel of 4-aminoquinoline hydrazone analogues were tested on the multidrug-resistant K1 strain of Plasmodium falciparum: IC50 values after a 48 h cycle ranged from 0.60 to 49 µM, while the 72 h cycle ranged from 0.026 to 0.219 µM. Time-course assays were carried out to define the activity of the lead compounds, which inhibited over 50% growth in 24 h and 90% growth in 72 h. Cytotoxicity assays with HepG2 cells showed IC50 values of 0.87-11.1 µM, whereas in MDBK cells, IC50 values ranged from 1.66 to 11.7 µM. High selectivity indices were observed for the lead compounds screened at 72 h on P. falciparum. Analyses of stage specificity revealed that the ring stages of the parasite life cycle were most affected. Based on antimalarial efficacy and in vitro safety profiles, lead compound 4-(2-benzylidenehydrazinyl)-6-methoxy-2-methylquinoline 2 was progressed to drug combination studies for the detection of synergism, with a combinatory index of 0.599 at IC90 for the combination with artemether, indicating a synergistic antimalarial activity. Compound 2 was screened on different strains of P. falciparum (3D7, Dd2), which maintained similar activity to K1, suggesting no cross-resistance between multidrug resistance and sensitive parasite strains. In vivo analysis with 2 showed the suppression of parasitaemia with P. yoelii NL (non-lethal)-treated mice (20 mg/kg and 5 mg/kg).


Assuntos
Antimaláricos , Malária Falciparum , Malária , Animais , Camundongos , Antimaláricos/farmacologia , Malária/tratamento farmacológico , Aminoquinolinas , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum
4.
Molecules ; 28(15)2023 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-37570721

RESUMO

Many transmembrane proteins are modulated by intracellular or extracellular pH. Investigation of pH dependence generally proceeds by mutagenesis of a wide set of amino acids, guided by properties such as amino-acid conservation and structure. Prediction of pKas can streamline this process, allowing rapid and effective identification of amino acids of interest with respect to pH dependence. Commencing with the calcium-activated chloride channel bestrophin 1, the carboxylate ligand structure around calcium sites relaxes in the absence of calcium, consistent with a measured lack of pH dependence. By contrast, less relaxation in the absence of calcium in TMEM16A, and maintenance of elevated carboxylate sidechain pKas, is suggested to give rise to pH-dependent chloride channel activity. This hypothesis, modulation of calcium/proton coupling and pH-dependent activity through the extent of structural relaxation, is shown to apply to the well-characterised cytosolic proteins calmodulin (pH-independent) and calbindin D9k (pH-dependent). Further application of destabilised, ionisable charge sites, or electrostatic frustration, is made to other human chloride channels (that are not calcium-activated), ClC-2, GABAA, and GlyR. Experimentally determined sites of pH modulation are readily identified. Structure-based tools for pKa prediction are freely available, allowing users to focus on mutagenesis studies, construct hypothetical proton pathways, and derive hypotheses such as the model for control of pH-dependent calcium activation through structural flexibility. Predicting altered pH dependence for mutations in ion channel disorders can support experimentation and, ultimately, clinical intervention.

5.
iScience ; 26(5): 106758, 2023 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-37216118

RESUMO

Inflammation driven by DNA sensors is now understood to be important to disease pathogenesis. Here, we describe new inhibitors of DNA sensing, primarily of the inflammasome forming sensor AIM2. Biochemistry and molecular modeling has revealed 4-sulfonic calixarenes as potent inhibitors of AIM2 that likely work by binding competitively to the DNA-binding HIN domain. Although less potent, these AIM2 inhibitors also inhibit DNA sensors cGAS and TLR9 demonstrating a broad utility against DNA-driven inflammatory responses. The 4-sulfonic calixarenes inhibited AIM2-dependent post-stroke T cell death, highlighting a proof of concept that the 4-sulfonic calixarenes could be effective at combating post-stroke immunosuppression. By extension, we propose a broad utility against DNA-driven inflammation in disease. Finally, we reveal that the drug suramin, by virtue of its structural similarities, is an inhibitor of DNA-dependent inflammation and propose that suramin could be rapidly repurposed to meet an increasing clinical need.

6.
Molecules ; 28(8)2023 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-37110551

RESUMO

Bestrophin 1 (Best1) is a chloride channel that localises to the plasma membrane of retinal pigment epithelium (RPE) cells. Mutations in the BEST1 gene are associated with a group of untreatable inherited retinal dystrophies (IRDs) called bestrophinopathies, caused by protein instability and loss-of-function of the Best1 protein. 4PBA and 2-NOAA have been shown to rescue the function, expression, and localisation of Best1 mutants; however, it is of interest to find more potent analogues as the concentration of the drugs required is too high (2.5 mM) to be given therapeutically. A virtual docking model of the COPII Sec24a site, where 4PBA has been shown to bind, was generated and a library of 1416 FDA-approved compounds was screened at the site. The top binding compounds were tested in vitro in whole-cell patch-clamp experiments of HEK293T cells expressing mutant Best1. The application of 25 µM tadalafil resulted in full rescue of Cl- conductance, comparable to wild type Best1 levels, for p.M325T mutant Best1 but not for p.R141H or p.L234V mutants.


Assuntos
Canais de Cloreto , Epitélio Pigmentado da Retina , Humanos , Bestrofinas/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Tadalafila , Células HEK293 , Mutação , Epitélio Pigmentado da Retina/metabolismo , Proteínas de Transporte Vesicular/genética
7.
Molecules ; 27(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36234744

RESUMO

The NLRP3 inflammasome is currently an exciting target for drug discovery due to its role in various inflammatory diseases; however, to date, no NLRP3 inhibitors have reached the clinic. Several studies have used natural products as hit compounds to facilitate the design of novel selective NLRP3 inhibitors. Here, we review selected natural products reported in the literature as NLRP3 inhibitors, with a particular focus on those targeting gout. To complement this survey, we also report a virtual screen of the ZINC20 natural product database, predicting favored chemical features that can aid in the design of novel small molecule NLRP3 inhibitors.


Assuntos
Produtos Biológicos , Gota , Produtos Biológicos/farmacologia , Humanos , Inflamassomos , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR
8.
Dis Model Mech ; 15(10)2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-36073607

RESUMO

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.


Assuntos
Anticonvulsivantes , Pentilenotetrazol , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Benzaldeídos/efeitos adversos , Drosophila , Homeostase , Camundongos , Neurônios , Pentilenotetrazol/efeitos adversos , Pirazóis/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/prevenção & controle
9.
Protein Sci ; 31(10): e4420, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36173167

RESUMO

The NLRP3 inflammasome is a cytoplasmic complex that regulates the activation of inflammatory cytokines and, given its implication in a range of diseases, is an important therapeutic target. The cofactor ATP and the centrosomal kinase NEK7 are important for NLRP3 activation. Here we have constructed and simulated computational models of full-length monomeric NLRP3 to shed light on the importance of NEK7 and cofactor interactions for its conformation and dynamics in aqueous solution. We find that molecular dynamics simulation reproduces well the features of the recently published cryo-EM structure of the ADP-bound NLRP3-NEK7 complex; on the removal of NEK7, the NLRP3 molecule adopts a more compact closed form during simulations. Replacement of ADP by ATP promotes a rearrangement of hydrogen-bonding interactions, domain interfaces, and a degree of opening of the NLRP3 conformation. We also examine the dynamics of an acidic loop of the LRR domain of NLRP3, which samples in a region observed in the NEK7-bound cryo-EM structure but not in an oligomeric form of inactive NLRP3. During the molecular dynamics simulations of NLRP3, we find some plasticity in its topology that suggests access routes for ATP to the cofactor pocket not immediately evident from the existing NEK7-bound cryo-EM structure. These computed dynamical trajectories of NLRP3 provide insight into coordinates of deformation that may be key for cofactor binding and inflammasome activation.


Assuntos
Inflamassomos , Quinases Relacionadas a NIMA , Proteína 3 que Contém Domínio de Pirina da Família NLR , Difosfato de Adenosina , Trifosfato de Adenosina , Simulação por Computador , Citocinas/metabolismo , Hidrogênio , Inflamassomos/química , Inflamassomos/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
10.
J Med Chem ; 65(6): 4783-4797, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35290041

RESUMO

A set of meta-substituted 3-arylisoquinolinones have been identified that show substantial cytotoxicity in breast, liver, lung and colon cancer cell lines; these are up to 700-fold more active than the corresponding para analogues. These compounds were initially proposed as inhibitors of N-ribosyl dihydronicotinamide (NRH): quinone oxidoreductase 2 (NQO2) but were found to be inactive against the enzyme. Instead, COMPARE analysis suggested that 6-fluoro-3-(meta-fluorophenyl)isoquinolin-1(2H)-one (4) could mimic colchicine and interact with microtubules, a recognized target for cancer therapy. Subsequent docking, molecular dynamics simulations, and free energy analysis further suggested that compound 4 bound well into the colchicine-binding pocket of tubulin. Indeed, 4 suppressed tubulin polymerization, caused G2/M cell cycle arrest, and induced apoptosis. Also, 4 inhibited the formation of endothelial cell capillary-like tubes and further disrupted the structure of preestablished tubes; the effects were not observed with para analogue 5. In accordance with this, the computed free energy of binding of 5 to tubulin was lower in magnitude than that for 4 and appeared to arise in part from the inability of the para substituent to occupy a tubulin subpocket, which is possible in the meta orientation. In conclusion, the antiproliferative potential of the novel 3-arylisoquinolinones is markedly influenced by a subtle change in the structure (meta versus para). The meta-substituted isoquinolinone 4 is a microtubule-destabilizing agent with potential tumor-selectivity and antiangiogenic and vascular disrupting features.


Assuntos
Antineoplásicos , Tubulina (Proteína) , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células , Colchicina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Microtúbulos , Estrutura Molecular , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química
11.
Br J Pharmacol ; 179(11): 2713-2732, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34877651

RESUMO

BACKGROUND AND PURPOSE: QT prolongation and intracellular Ca2+ loading with diastolic Ca2+ release via ryanodine receptors (RyR2) are the predominant mechanisms underlying hypokalaemia-induced ventricular arrhythmia. We investigated the antiarrhythmic actions of two RyR2 inhibitors: dantrolene and VK-II-86, a carvedilol analogue lacking antagonist activity at ß-adrenoceptors, in hypokalaemia. EXPERIMENTAL APPROACH: Surface ECG and ventricular action potentials (APs) were recorded from whole-heart murine Langendorff preparations. Ventricular arrhythmia incidence was compared in hearts perfused with low [K+ ], and those pretreated with dantrolene or VK-II-86. Whole-cell patch clamping was used in murine and canine ventricular cardiomyocytes to study effects of dantrolene and VK-II-86 on AP parameters in low [K+ ] and effects of VK-II-86 on the inward rectifier current (IK1 ), late sodium current (INa_L ) and the L-type Ca2+ current (ICa ). Effects of VK-II-86 on IKr were investigated in transfected HEK-293 cells. A fluorogenic probe quantified the effects of VK-II-86 on oxidative stress in hypokalaemia. KEY RESULTS: Dantrolene reduced the incidence of ventricular arrhythmias induced by low [K+ ] in explanted murine hearts by 94%, whereas VK-II-86 prevented all arrhythmias. VK-II-86 prevented hypokalaemia-induced AP prolongation and depolarization but did not alter AP parameters in normokalaemia. Hypokalaemia was associated with decreased IK1 and IKr , and increased INa-L , and ICa . VK-II-86 prevented all hypokalaemia-induced changes in ion channel activity and oxidative stress. CONCLUSIONS AND IMPLICATIONS: VK-II-86 prevents hypokalaemia-induced arrhythmogenesis by normalizing calcium homeostasis and repolarization reserve. VK-II-86 may provide an effective treatment in hypokalaemia and other arrhythmias caused by delayed repolarization or Ca2+ overload.


Assuntos
Hipopotassemia , Canal de Liberação de Cálcio do Receptor de Rianodina , Potenciais de Ação , Animais , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Cálcio/metabolismo , Carvedilol/farmacologia , Dantroleno/efeitos adversos , Cães , Células HEK293 , Humanos , Hipopotassemia/complicações , Hipopotassemia/tratamento farmacológico , Camundongos , Miócitos Cardíacos , Sódio/metabolismo
12.
Materials (Basel) ; 14(22)2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34832380

RESUMO

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their in silico molecular docking into segments of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. This is believed to be the target of the erythromycin A derivative, azithromycin, which has mild anti-malarial activity. The erythromycin B derivatives were evaluated on the multi-drug (chloroquine, pyrimethamine, and sulfadoxine)-resistant strain K1 of P. falciparum for asexual growth inhibition on asynchronous culture. The erythromycin B derivatives were identified as active in vitro inhibitors of asexual growth of P. falciparum with low micro-molar IC50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC50 of 68.6 µM, d-erythromycin B 86.8 µM, and erythromycin B 9-oxime 146.0 µM on the multi-drug-resistant K1 of P. falciparum. Based on the molecular docking, it seems that a small number of favourable interactions or the presence of unfavourable interactions of investigated derivatives of erythromycin B with in silico constructed segment from the exit tunnel from the apicoplast of P. falciparum is the reason for their weak in vitro anti-malarial activities.

13.
Biochem Biophys Rep ; 26: 100943, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33778168

RESUMO

The pathogenesis of Alzheimer's disease (AD) is correlated with the misfolding and aggregation of amyloid-beta protein (Aß). Here we report that the antibiotic benzylpenicillin (BP) can specifically bind to Aß, modulate the process of aggregation and supress its cytotoxic effect, initially via a reversible binding interaction, followed by covalent bonding between specific functional groups (nucleophiles) within the Aß peptide and the beta-lactam ring. Mass spectrometry and computational docking supported covalent modification of Aß by BP. BP was found to inhibit aggregation of Aß as revealed by the Thioflavin T (ThT) fluorescence assay and atomic force microscopy (AFM). In addition, BP treatment was found to have a cytoprotective activity against Aß-induced cell cytotoxicity as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell toxicity assay. The specific interaction of BP with Aß suggests the possibility of structure-based drug design, leading to the identification of new drug candidates against AD. Moreover, good pharmacokinetics of beta-lactam antibiotics and safety on long-time use make them valuable candidates for drug repurposing towards neurological disorders such as AD.

14.
Molecules ; 25(23)2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33255820

RESUMO

Inflammasomes are protein complexes which are important in several inflammatory diseases. Inflammasomes form part of the innate immune system that triggers the activation of inflammatory cytokines interleukin (IL)-1ß and IL-18. The inflammasome most studied in sterile inflammation and non-communicable disease is the NLRP3 inflammasome. Upon activation by diverse pathogen or disease associated signals, NLRP3 nucleates the oligomerization of an adaptor protein ASC forming a platform (the inflammasome) for the recruitment and activation of the protease caspase-1. Active caspase-1 catalyzes the processing and release of IL-1ß and IL-18, and via cleavage of the pore forming protein gasdermin D can drive pyroptotic cell death. This review focuses on the structural basis and mechanism for NLRP3 inflammasome signaling in the context of drug design, providing chemical structures, activities, and clinical potential of direct inflammasome inhibitors. A cryo-EM structure of NLRP3 bound to NEK7 protein provides structural insight and aids in the discovery of novel NLRP3 inhibitors utilizing ligand-based or structure-based approaches.


Assuntos
Descoberta de Drogas , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Ensaios Clínicos como Assunto , Desenho de Fármacos , Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunidade Inata , Inflamação/etiologia , Inflamação/metabolismo , Modelos Moleculares , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Ligação Proteica , Relação Estrutura-Atividade
15.
Elife ; 92020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33216713

RESUMO

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1ß and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered an attractive therapeutic target. However, there is much regarding the mechanism of NLRP3 activation that remains unknown. Chloride efflux is suggested as an important step in NLRP3 activation, but which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of chloride channels in the regulation of NLRP3 in murine macrophages. Specifically, we identify LRRC8A, an essential component of volume-regulated anion channels (VRAC), as a vital regulator of hypotonicity-induced, but not DAMP-induced, NLRP3 inflammasome activation. Although LRRC8A was dispensable for canonical DAMP-dependent NLRP3 activation, this was still sensitive to chloride channel inhibitors, suggesting there are additional and specific chloride sensing and regulating mechanisms controlling NLRP3.


Inflammation is a critical part of a healthy immune system, which protects us against harmful pathogens (such as bacteria or viruses) and works to restore damaged tissues. In the immune cells of our body, the inflammatory process can be activated through a group of inflammatory proteins that together are known as the NLRP3 inflammasome complex. While inflammation is a powerful mechanism that protects the human body, persistent or uncontrolled inflammation can cause serious, long-term damage. The inappropriate activation of the NLRP3 inflammasome has been implicated in several diseases, including Alzheimer's disease, heart disease, and diabetes. The NLRP3 inflammasome can be activated by different stimuli, including changes in cell volume and exposure to either molecules produced by damaged cells or toxins from bacteria. However, the precise mechanism through which the NLRP3 becomes activated in response to these stimuli was not clear. The exit of chloride ions from immune cells is known to activate the NLRP3 inflammasome. Chloride ions exit the cell through proteins called anion channels, including volume-regulated anion channels (VRACs), which respond to changes in cell volume. Green et al. have found that, in immune cells from mice grown in the lab called macrophages, VRACs are the only chloride channels involved in activating the NLRP3 inflammasome when the cell's volume changes. However, when the macrophages are exposed to molecules produced by damaged cells or toxins from bacteria, Green et al. discovered that other previously unidentified chloride channels are involved in activating the NLRP3 inflammasome. These results suggest that it might be possible to develop drugs to prevent the activation of the NLRP3 inflammasome that selectively target specific sets of chloride channels depending on which stimuli are causing the inflammation. Such a selective approach would minimise the side effects associated with drugs that generically suppress all NLRP3 activity by directly binding to NLRP3 itself. Ultimately, this may help guide the development of new, targeted anti-inflammatory drugs that can help treat the symptoms of a variety of diseases in humans.


Assuntos
Alarminas/imunologia , Inflamassomos/imunologia , Inflamação/imunologia , Proteínas de Membrana/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/imunologia , Feminino , Humanos , Inflamassomos/genética , Inflamação/genética , Macrófagos/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Pressão Osmótica
16.
Neurobiol Aging ; 94: 24-33, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32512325

RESUMO

Aggregation of amyloid ß1-42 (Aß1-42) peptide within the brain is considered one of the main causes of the neuropathological changes associated with Alzheimer's disease. Resveratrol is a well-known antioxidant but has also been reported to bind to Aß1-42 peptide, thereby reducing aggregation. However, little is known of the precise mechanism by which resveratrol reduces Aß1-42 peptide aggregation. Using the thioflavin-T assay, the ability of resveratrol to reduce the extent of Aß1-42 peptide aggregation was investigated. The findings of the present study demonstrate that interaction of resveratrol with Aß1-42 peptide resulted in the cleavage of Aß1-42 peptide into smaller fragments, as detected by matrix assisted laser desorption ionization-time of flight mass spectrometry. Atomic force microscopy analyses revealed Aß1-42 peptide, under control conditions, aggregated into oligomers, protofibrils, and fibrils, whereas there was a distinct lack of these structures when Aß1-42 peptide was incubated with resveratrol. Following 10 days incubation of Aß1-42 peptide with resveratrol, particles with a mean z-height of 1.940 nm (range 0.675-3.275 nm) were observed, which are characteristic of shorter peptide species. In cell-based studies, resveratrol significantly reduced the cytotoxicity of Aß1-42 peptide toward SH-SY5Y human neuroblastoma cells, suggesting a protective effect of the polyphenol. We therefore propose a novel mechanism by which resveratrol disrupts Aß1-42 aggregation by mediating fragmentation of Aß1-42 into smaller peptides, which have no propensity to aggregate further.


Assuntos
Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Resveratrol/metabolismo , Resveratrol/farmacologia , Peptídeos beta-Amiloides/toxicidade , Antioxidantes , Humanos , Fragmentos de Peptídeos/toxicidade , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , Ligação Proteica , Células Tumorais Cultivadas
17.
Invest Ophthalmol Vis Sci ; 61(5): 28, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32421148

RESUMO

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy. Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp. Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1. Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.


Assuntos
Antineoplásicos/farmacologia , Bestrofinas/genética , Oftalmopatias Hereditárias/tratamento farmacológico , Regulação da Expressão Gênica/fisiologia , Glicolatos/farmacologia , Fenilbutiratos/farmacologia , Doenças Retinianas/tratamento farmacológico , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting , Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Cicloeximida/farmacologia , Eletroforese em Gel de Poliacrilamida , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Genes Recessivos , Células HEK293/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transfecção
18.
Chem Sci ; 11(43): 11720-11728, 2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34094411

RESUMO

The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18, and contributes to inflammation exacerbating disease. Fenamate non-steroidal anti-inflammatory drugs (NSAIDs) were recently described as NLRP3 inflammasome inhibitors via chloride channel inhibition. Fenamate NSAIDs inhibit cyclooxygenase (COX) enzymes, limiting their potential as therapeutics for NLRP3-associated diseases due to established side effects. The aim here was to develop properties of the fenamates that inhibit NLRP3, and at the same time to reduce COX inhibition. We synthesised a library of analogues, with feedback from in silico COX docking potential, and IL-1ß release inhibitory activity. Through iterative screening and rational chemical design, we established a collection of chloride channel inhibiting active lead molecules with potent activity at the canonical NLRP3 inflammasome and no activity at COX enzymes, but only in response to stimuli that activated NLRP3 by a K+ efflux-dependent mechanism. This study identifies a model for the isolation and removal of unwanted off-target effects, with the enhancement of desired activity, and establishes a new chemical motif for the further development of NLRP3 inflammasome inhibitors.

19.
Eur J Med Chem ; 182: 111649, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31514018

RESUMO

(NRH):quinone oxidoreductase 2 (NQO2) is associated with various processes involved in cancer initiation and progression probably via the production of ROS during quinone metabolism. Thus, there is a need to develop inhibitors of NQO2 that are active in vitro and in vivo. As part of a strategy to achieve this we have used the 4-aminoquinoline backbone as a starting point and synthesized 21 novel analogues. The syntheses utilised p-anisidine with Meldrum's acid and trimethyl orthoacetate or trimethyl orthobenzoate to give the 4-hydrazin-quinoline scaffold, which was derivatised with aldehydes or acid chlorides to give hydrazone or hydrazide analogues, respectively. The hydrazones were the most potent inhibitors of NQO2 in cell free systems, some with low nano-molar IC50 values. Structure-activity analysis highlighted the importance of a small substituent at the 2-position of the 4-aminoquinoline ring, to reduce steric hindrance and improve engagement of the scaffold within the NQO2 active site. Cytotoxicity and NQO2-inhibitory activity in vitro was evaluated using ovarian cancer SKOV-3 and TOV-112 cells (expressing high and low levels of NQO2, respectively). Generally, the hydrazones were more toxic than hydrazide analogues and further, toxicity is unrelated to cellular NQO2 activity. Pharmacological inhibition of NQO2 in cells was measured using the toxicity of CB1954 as a surrogate end-point. Both the hydrazone and hydrazide derivatives are functionally active as inhibitors of NQO2 in the cells, but at different inhibitory potency levels. In particular, 4-((2-(6-methoxy-2-methylquinolin-4-yl)hydrazono)methyl)phenol has the greatest potency of any compound yet evaluated (53 nM), which is 50-fold lower than its toxicity IC50. This compound and some of its analogues could serve as useful pharmacological probes to determine the functional role of NQO2 in cancer development and response to therapy.


Assuntos
Aminoquinolinas/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Hidrazonas/farmacologia , Quinona Redutases/antagonistas & inibidores , Aminoquinolinas/síntese química , Aminoquinolinas/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Modelos Moleculares , Estrutura Molecular , Quinona Redutases/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
20.
J Radioanal Nucl Chem ; 317(2): 977-984, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30100651

RESUMO

N-Methyl carbamoylimidazole is a safe and practical alternative to methyl isocyanate for carbamoylation reactions. We have developed a new chemical route for its synthesis from methyl iodide and applied this to the synthesis of N-[11C]methyl carbamoylimidazole as a new [11C]synthon to radiolabel biomolecules for PET imaging research. N-[11C]methyl carbamoylimidazole was prepared from [11C]methyl iodide in 70-74% radiochemical yield (decay corrected) and can be used in situ for further reaction without purification. The reactivity of N-[11C]methyl carbamoylimidazole was demonstrated in a series of [11C]carbamoylation reactions.

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