RESUMO
Reactive oxygen species (ROS) are produced by every aerobic cell during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Superoxide Dismutases (SOD) are antioxidant proteins that convert superoxide anions (O2â¢-) to hydrogen peroxide (H2O2) and dioxygen. Using the differential in the level of oxidative stress between normal and cancer cells, SOD mimetics can show an antitumoral effect and prevent oxaliplatin-induced peripheral neuropathy. New Pt(IV) conjugate prodrugs (OxPt-x-Mn1C1A (x = 1, 1-OH, 2)), combining oxaliplatin and a Mn SOD mimic (MnSODm Mn1C1A) with a covalent link, were designed. Their stability in buffer and in the presence of sodium ascorbate was studied. In vitro, their antitumoral activity was assessed by the viability and ROS production of tumor cell lines (CT16, HCT 116, KC) and fibroblasts (primary culture and NIH 3T3). In vivo, a murine model of colorectal cancer was created with subcutaneous injection of CT26 cells in Balb/c mice. Tumor size and volume were measured weekly in four groups: vehicle, oxaliplatin, and oxaliplatin associated with MnSODm Mn1C1A and the bis-conjugate OxPt-2-Mn1C1A. Oxaliplatin-induced peripheral neuropathy (OIPN) was assessed using a Von Frey test reflecting chronic hypoalgesia. Tolerance to treatment was assessed with a clinical score including four items: weight loss, weariness, alopecia, and diarrhea. In vitro, Mn1C1A associated with oxaliplatin and Pt(IV) conjugates treatment induced significantly higher production of H2O2 in all cell lines and showed a significant improvement of the antitumoral efficacy compared to oxaliplatin alone. In vivo, the association of Mn1C1A to oxaliplatin did not decrease its antitumoral activity, while OxPt-2-Mn1C1A had lower antitumoral activity than oxaliplatin alone. Mn1C1A associated with oxaliplatin significantly decreased OIPN and also improved global clinical tolerance of oxaliplatin. A neuroprotective effect was observed, associated with a significantly improved tolerance to oxaliplatin without impairing its antitumoral activity.
Assuntos
Antineoplásicos , Doenças do Sistema Nervoso Periférico , Camundongos , Animais , Oxaliplatina/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Superóxidos , Antineoplásicos/uso terapêutico , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/patologia , Superóxido Dismutase , Camundongos Endogâmicos BALB CRESUMO
By using the differential in level of oxidative status between normal and cancer cells, SuperOxide Dismutase (SOD) mimetics can have anti-tumor efficacy and prevent oxaliplatin-induced peripheral neuropathy. Our objective was to evaluate the neuroprotective efficacy of MAG, a new SOD mimic. In vitro, the effects of MAG alone or with oxaliplatin were studied on colon cancer cells (HT29 and CT26) and on normal fibroblast cells (NIH3T3). The cell viability (by crystal violet) as well as the production of reactive forms of oxygen and glutathione (by spectrofluorimetric assay) was measured. In vivo, efficacy on tumor growth was assessed in mice grafted with CT26 colon cancer cells. The effects on induced neurotoxicity were measured by specific behavioral Von Frey nociception, cold-plate tests, specific functional neuromuscular assay and electron microscopy. In vitro, MAG induced a production of hydrogen peroxide in all cells. At 24 h-incubation, MAG exhibits a cytotoxic activity in all cell lines. A cytotoxic additive effect of MAG and oxaliplatin was observed through oxidative burst. In vivo, oxaliplatin-treated mice associated with MAG did not counteract oxaliplatin's antitumoral efficacy. After 4 weeks of treatment with oxaliplatin combined with MAG, behavioral and functional tests showed a decrease in peripheral neuropathy induced by oxaliplatin in vivo. Electron microscopy analyses on sciatic nerves revealed an oxaliplatin-induced demyelination which is prevented by the association of MAG to this chemotherapy. In conclusion, MAG prevents the appearance of sensitive axonal neuropathy and neuromuscular disorders induced by oxaliplatin without affecting its antitumor activity.
RESUMO
The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.
Assuntos
Antivirais/química , Antivirais/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Naproxeno/análogos & derivados , Células A549 , Animais , Sítios de Ligação , Inibidores de Ciclo-Oxigenase 2/química , Cães , Desenho de Fármacos , Reposicionamento de Medicamentos , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Naproxeno/farmacologia , Proteínas do Nucleocapsídeo , Oseltamivir/farmacologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ressonância de Plasmônio de Superfície , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismoRESUMO
Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer's disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aß42 peptide, in correlation with Nrf2 activation. Comparatively, lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aß42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aß oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo.
Assuntos
Doença de Alzheimer/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Geneticamente Modificados , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Cloreto de Lítio/farmacologia , Longevidade/efeitos dos fármacos , Longevidade/genética , Camundongos , Microscopia Confocal , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiadiazóis/farmacologia , Triazóis/farmacologiaRESUMO
Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,ß-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.
Assuntos
Antineoplásicos/química , Indóis/química , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Antioxidantes/metabolismo , Domínio Catalítico , Bovinos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Células HCT116 , Humanos , Concentração Inibidora 50 , Células MCF-7 , Camundongos , Naftoquinonas/química , Estresse Oxidativo , Domínios Proteicos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Selenocisteína/química , Tiorredoxinas/metabolismoRESUMO
An organometallic derivative of praziquantel was studied directly in worms by using inductively coupled plasma-mass spectrometry (ICP-MS) for quantification and synchrotron-based imaging. X-ray fluorescence (XRF) and IR absorption spectromicroscopy were used for the first time in combination to directly locate this organometallic drug candidate in schistosomes. The detection of both CO (IR) and Cr (XRF) signatures proved that the Cr(CO)3 core remained intact in the worms. Images showed a preferential accumulation at the worm's tegument, consistent with a possible targeting of the calcium channel but not excluding other biological targets inside the worm.
Assuntos
Praziquantel/química , Schistosoma mansoni/química , Animais , Cromo/química , Espectrometria de Massas , Microscopia , Imagem Óptica , Praziquantel/síntese química , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/metabolismo , Espectrofotometria Infravermelho , Estereoisomerismo , Espectroscopia por Absorção de Raios XRESUMO
A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 µM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements.
Assuntos
Histidina/química , Manganês/química , Proteínas/química , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica , Simulação de Dinâmica Molecular , Peptídeos/química , Estrutura Terciária de ProteínaRESUMO
The transcription factor Nrf2 regulates the expression of a large network of cytoprotective and metabolic enzymes and proteins. Compounds that directly and reversibly inhibit the interaction between Nrf2 and its main negative regulator Keap1 are potential pharmacological agents for a range of disease types including neurodegenerative conditions and cancer. We describe the development of a series of 1,4-diphenyl-1,2,3-triazole compounds that inhibit the Nrf2-Keap1 protein-protein interaction (PPI) in vitro and in live cells and up-regulate the expression of Nrf2-dependent gene products.
Assuntos
Heme Oxigenase-1/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/metabolismo , Triazóis/química , Linhagem Celular Tumoral , Química Click , Simulação por Computador , Bases de Dados de Compostos Químicos , Relação Dose-Resposta a Droga , Polarização de Fluorescência , Células HEK293 , Heme Oxigenase-1/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Isotiocianatos/química , Isotiocianatos/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch , Simulação de Acoplamento Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/química , Ligação Proteica , Relação Estrutura-Atividade , Sulfóxidos , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
Pulse electron-electron double resonance (PELDOR) is a versatile technique for probing the structures and functions of complex biological systems. Despite the recent interest in high-spin metal-ions for high field/frequency applications, PELDOR measurements of Mn(ii) remain relatively underexplored. Here we present Mn(ii)-Mn(ii) PELDOR distance measurements at 94 GHz on polyproline II (PPII) helices doubly spin-labeled with Mn(ii)DOTA, which are distinguished by their small zero-field interaction. The measured Mn-Mn distances and distribution profiles were in good agreement with the expected values from molecular models. Additional features in the frequency-domain spectra became apparent at certain combinations of detect and pump frequencies. Spin-Hamiltonian calculations showed that they likely arose from contributions from the pseudo-secular component of the dipolar interaction that were found to be non-negligible for Mn(ii)DOTA. However, the influence of the pseudo-secular component on the distance distribution profiles apparently was limited. The results show the potential of Mn(ii)DOTA spin labels for high-field PELDOR distance measurements in proteins and other biological systems.
Assuntos
Complexos de Coordenação/química , Compostos Heterocíclicos com 1 Anel/química , Manganês , Peptídeos/química , Marcadores de Spin , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Maleimidas/química , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
Octahedral d(6) low-spin Re(I) tricarbonyl complexes are of considerable interest as noninvasive imaging probes and have been deeply studied owing to their biological stability, low toxicity, large Stokes shifts, and long luminescence lifetimes. We reported recently the bimodal IR and luminescence imaging of a Re(I) tricarbonyl complex with a Pyta ligand (4-(2-pyridyl)-1,2,3-triazole) in cells and labeled such metal-carbonyl complexes SCoMPIs for single-core multimodal probes for imaging. Re(I) tricarbonyl complexes have unique photophysical properties allowing for their unequivocal detection in cells but also present some weaknesses such as a very low luminescence quantum yield in aqueous medium. Further optimizations would thus be desirable. We therefore developed new Re(I) tricarbonyl complexes prepared from different ancillary ligands. Complexes with benzothiadiazole-triazole ligands show interesting luminescent quantum yields in acetonitrile and may constitute valuable luminescent metal complexes in organic media. A series of complexes with bidentate 1-(2-quinolinyl)-1,2,3-triazole (Taquin) and 1-(2-pyridyl)-1,2,3-triazole (Tapy) ligands bearing various 4-substituted alkyl side chains has been designed and synthesized with efficient procedures. Their photophysical properties have been characterized in acetonitrile and in a H2O/DMSO (98/2) mixture and compared with those of the parent Quinta- and Pyta-based complexes. Tapy complexes bearing long alkyl chains show impressive enhancement of their luminescent properties relative to the parent Pyta complex. Theoretical calculations have been performed to further characterize this new class of rhenium tricarbonyl complexes. Preliminary cellular imaging studies in MDA-MB231 breast cancer cells reveal a strong increase in the luminescence signal in cells incubated with the Tapy complex substituted with a C12 alkyl chain. This study points out the interesting potential of the Tapy ligand in coordination chemistry, which has been so far underexploited.
Assuntos
Complexos de Coordenação/química , Substâncias Luminescentes/química , Rênio/química , Triazóis/química , Alquilação , Neoplasias da Mama/diagnóstico , Linhagem Celular Tumoral , Feminino , Humanos , Luminescência , Medições Luminescentes , Imagem ÓpticaRESUMO
Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the cap'n'collar basic-region leucine-zipper transcription factor Nrf2 is a potential strategy to enhance expression of antioxidant and free radical detoxification gene products regulated by Nrf2. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer-chemopreventive agents. We describe the structure-activity relationships for a series of peptides based upon regions of the Nrf2 Neh2 domain, of varying length and sequence, that interact with the Keap1 Kelch domain and disrupt the interaction with Nrf2. We have also investigated sequestosome-1 (p62) and prothymosin-α sequences that have been reported to interact with Keap1. To achieve this we have developed a high-throughput fluorescence polarization (FP) assay to screen inhibitors. In addition to screening synthetic peptides, we have used a phage display library approach to identify putative peptide ligands with non-native sequence motifs. Candidate peptides from the phage display library screening protocol were evaluated in the FP assay to quantify their binding activity. Hybrid peptides based upon the Nrf2 "ETGE" motif and the sequestosome-1 "Keap1-interaction region" have superior binding activity compared to either native peptide alone.