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1.
J Enzyme Inhib Med Chem ; 36(1): 2128-2138, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34583607

RESUMO

Influenza viruses represent a major threat to human health and are responsible for seasonal epidemics, along with pandemics. Currently, few therapeutic options are available, with most drugs being at risk of the insurgence of resistant strains. Hence, novel approaches targeting less explored pathways are urgently needed. In this work, we assayed a library of nitrobenzoxadiazole derivatives against the influenza virus A/Puerto Rico/8/34 H1N1 (PR8) strain. We identified three promising 4-thioether substituted nitrobenzoxadiazoles (12, 17, and 25) that were able to inhibit viral replication at low micromolar concentrations in two different infected cell lines using a haemagglutination assay. We further assessed these molecules using an In-Cell Western assay, which confirmed their potency in the low micromolar range. Among the three molecules, 12 and 25 displayed the most favourable profile of activity and selectivity and were selected as hit compounds for future optimisation studies.


Assuntos
4-Cloro-7-nitrobenzofurazano/farmacologia , Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , Animais , Antivirais/síntese química , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
2.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070875

RESUMO

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.


Assuntos
Subunidades Proteicas/química , Fator 2 Associado a Receptor de TNF/química , Tirosina/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Termodinâmica , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
J Cell Physiol ; 234(9): 15885-15897, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30741416

RESUMO

Glutathione transferases (GSTs) play an important role in retinal pathophysiology. Within this family, the GSTP isoform is known as an endogenous regulator of cell survival and proliferation pathways and of cellular responses to oxidative stress. In the present study we silenced GSTP in R28 cells, a retinal precursor cell line with markers of both glial and neuronal origin, and obtained stable clones which were viable and, unexpectedly, characterized by a more neuronal phenotype. The degree of neuronal differentiation was inversely correlated with GSTP residual expression levels. The clone with the lowest expression of GSTP showed metabolic reprogramming, a more favorable redox status and, despite its neuronal phenotype, a sensitivity to glutamate and 4-hydroxynonenal toxicity comparable to that of control cells. Altogether, our evidence shows that near full depletion of GSTP in retinal precursor cells, triggers neuronal differentiation and prosurvival metabolic changes.

4.
Chemotherapy ; 64(3): 138-145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31639786

RESUMO

BACKGROUND: PD-L1 is a membrane protein with inhibitory effects on immune responses, whose expression has been correlated with high aggressiveness and the propensity of melanoma to metastasize. The nitrobenzoxadiazole (NBD) NBDHEX and its analog MC3181 are endowed with strong antitumor activity towards melanoma and a significant ability to reduce its adhesion and invasiveness. Therefore, we investigated whether PD-L1 status could affect cell sensitivity to the cytotoxic effects of NBDs. We then evaluated the effects of NBDHEX on PD-L1 expression and autophagy in melanoma cells. We used the BRAF-mutated A375 melanoma cell line and an A375 variant population enriched for PD-L1+ cells as a model. The cytotoxic effects of NBDs were evaluated in comparison to those of the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. METHODS: The effect of NBDHEX on autophagy was determined by measuring LC3-II and p62 protein levels by Western blot. The cytotoxic activity of the compounds was evaluated by sulforhodamine B assay. PD-L1 expression and plasma membrane localization were analyzed by FACS and Western blot analysis. RESULTS: NBDHEX behaves as a late-autophagy inhibitor in A375 melanoma cells, as previously found in other tumor cell lines. NBDHEX and MC3181 showed strong and comparable cytotoxic activity in both parental and PD-L1+ A375 cells, with IC50 values in the sub-micromolar range. Conversely, cells sorted for high PD-L1 expression had lower sensitivity to both the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. NBDHEX treatment did not change the total expression and cell surface localization of PD-L1 in both parental and PD-L1+ A375 cells. CONCLUSIONS: Our data suggest that NBDs may represent a promising treatment strategy for melanoma with elevated PD-L1 expression.


Assuntos
Autofagia/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Glutationa Transferase/antagonistas & inibidores , Nitrobenzenos/farmacologia , Oxidiazóis/farmacologia , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Cloroquina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/metabolismo , Humanos , Melanoma , Nitrobenzenos/química , Oxidiazóis/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Vemurafenib/farmacologia
5.
J Enzyme Inhib Med Chem ; 34(1): 1131-1139, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31169043

RESUMO

The antitumor agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (1) is a potent inhibitor of GSTP1-1, a glutathione S-transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of 1 (compound 3) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, 3 is susceptible to rapid metabolic hydrolysis. In an effort to improve the metabolic stability of 3, its ester group has been replaced by an amide, leading to N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexyl)benzamide (4). Unlike 3, compound 4 was stable to human liver microsomal carboxylesterases, but retained the ability to disrupt the interaction between GSTP1-1 and TRAF2 regardless of GSH levels. Moreover, 4 exhibited both a higher stability in the presence of GSH and a greater cytotoxicity towards cultured A375 melanoma cells, in comparison with 1 and its analog 2. These findings suggest that 4 deserves further preclinical testing.


Assuntos
4-Cloro-7-nitrobenzofurazano/farmacologia , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glutationa S-Transferase pi/antagonistas & inibidores , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , Antineoplásicos/síntese química , Antineoplásicos/química , Benzamidas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Glutationa S-Transferase pi/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
6.
Biotechnol Appl Biochem ; 65(1): 38-45, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28960521

RESUMO

The ability of a C-terminal truncated form of TRAF2 to bind synthetic vesicles has been quantitatively studied by steady-state fluorescence energy transfer from the protein to large unilamellar vesicles (LUVs) prepared with different lipid mixtures. The dissociation constants, the free energy of binding, and the average number of phospholipids interacting with truncated TRAF2 have been evaluated from the corresponding binding curves. The results indicate that the protein strongly interacts with the lipid bilayer, preferentially in the monomeric state. These findings have been discussed in terms of their possible role in the activity of TRAF2 in vivo.


Assuntos
Bicamadas Lipídicas/química , Fator 2 Associado a Receptor de TNF/química , Sítios de Ligação , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares
7.
J Biol Chem ; 291(52): 26739-26749, 2016 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-27815499

RESUMO

Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (although smaller values of nH are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role, safeguarding DNA from DNDGIC, display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs. Beside the well known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity.


Assuntos
Evolução Molecular , Compostos Ferrosos/farmacologia , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Glutationa/análogos & derivados , Óxido Nítrico/metabolismo , Cristalografia por Raios X , Glutationa/farmacologia , Humanos , Cinética
8.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(9): 813-822, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28499815

RESUMO

In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state. Generalized Polarization measurements and spectral phasor analysis revealed that truncated-TRAF2 affects the membrane fluidity, especially when vesicles are heated up at physiological temperature. The addition of nanomolar concentration of TRAF2 in GUVs also seems to exert a mechanical action, as demonstrated by the formation of intraluminal vesicles, a process in which ganglioside GM1 plays a crucial role.


Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Fator 2 Associado a Receptor de TNF/química , Fator 2 Associado a Receptor de TNF/metabolismo , Difusão , Fluorescência , Gangliosídeo G(M1)/metabolismo , Humanos , Fluidez de Membrana/fisiologia , Ligação Proteica/fisiologia , Domínios Proteicos , Lipossomas Unilamelares/metabolismo
9.
J Transl Med ; 14: 37, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26847645

RESUMO

BACKGROUND: Nitrobenzoxadiazole derivatives (NBDs), including NBDHEX and the recently developed MC3181, are promising anticancer agents able to target glutathione transferase and inhibit both its catalytic activity and ability to sequester TNF-receptor associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK). NBDs have been shown to impair the growth and survival of a broad-spectrum of tumor types, in vitro and in vivo. Herein, we evaluated the effects of the new compound MC3181 on U-2OS osteosarcoma cells and investigated the impact of both NBDHEX and MC3181 on autophagy. METHODS: Cell viability was evaluated by sulforhodamine B assay. The dissociation of the TRAF2-GSTP1-1 complex was detected by proximity ligation assay, while the phospho-activation of JNK was assessed by western blotting. The effects of NBDs on autophagy were evaluated by GFP-LC3 puncta formation, western blotting for LC3-II and p62, and LC3 turnover assay in the presence of bafilomycin A1. The role of JNK in the reduction of autophagic flux caused by NBDs was investigated using JNK1 shRNA-transfected cells. Fluorogenic caspase activity assay and flow cytometric analysis of DNA content were used to determine the cytotoxic effects of NBDs on JNK1-silenced cells. RESULTS: Similar to NBDHEX, MC3181 reduced viability and activated TRAF2/JNK signaling in U-2OS cells. Moreover, NBDs induced the accumulation of autophagic vesicles and LC3-II while reducing both basal and nutritional stress-induced autophagic flux. Furthermore, increased levels of both LC3-II and the autophagy selective substrate p62 were observed in different tumor cell lines treated with NBDs, the concurrent increase of these markers being consistent with an impairment of autophagosome clearance. Autophagy inhibition by NBDs required JNK activity: NBDs caused autophagy inhibition and caspase-3 activation in JNK-positive U-2OS, but no autophagic flux inhibition or caspase-3 activation in JNK-silenced cells. CONCLUSIONS: Our demonstration that NBDs can act as late-phase autophagy inhibitors opens new opportunities to fully exploit their therapeutic potential. This may not rely solely on their effectiveness in inducing cell cycle arrest and apoptosis, but also on their ability to weaken the capacity of tumor cells to endure stress conditions via autophagy. In addition, this study provides evidence that JNK can participate in impairing autophagy.


Assuntos
Autofagia/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Oxidiazóis/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Proteínas Associadas aos Microtúbulos/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/metabolismo
10.
J Enzyme Inhib Med Chem ; 31(6): 924-30, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26329912

RESUMO

CONTEXT: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to overcome drug resistance in oncology practice. OBJECTIVE: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding γ-oxa-glutamyl isostere (compounds 1 and 5, respectively) have been disclosed as GST inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD thioethers. MATERIALS AND METHODS: Synthesis of derivatives 1 and 5 and in vitro evaluation on human GSTP1-1 and M2-2 are reported. RESULTS: Conjugates 1 and 5 were found to be low micromolar inhibitors of both isoforms. Furthermore, they display a threefold reduction in selectivity for GSTM2-2 over the P1-1 isozyme in comparison with the potent non-peptide inhibitor nitrobenzoxadiazolyl-thiohexanol (NBDHEX). DISCUSSION AND CONCLUSIONS: Spectroscopic data are congruent with the formation of a stable sigma-complex between GSH and the inhibitors in the protein active site. Conjugate 5 is suitable for in vivo modulation of GST activity in cancer treatment.


Assuntos
4-Cloro-7-nitrobenzofurazano/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa Transferase/antagonistas & inibidores , Glutationa/farmacologia , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Glutationa/química , Glutationa S-Transferase pi/metabolismo , Glutationa Transferase/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
11.
Biochemistry ; 54(40): 6153-61, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26390021

RESUMO

TNF receptor-associated factors (TRAFs) are characterized by an oligomeric structure that plays a fundamental role in the binding process with membrane receptors. In this work, we studied the trimer-to-monomer (T ↔ 3M) equilibrium transition of the TRAF2 C-terminal domain using both chemical (dilution/guanidinium hydrochloride) and mechanical stress (high pressure) to induce the dissociation of the native protein into subunits. The experimental results and computer simulations indicate that stable monomers exist and that their population accounts for 15% of the total TRAF2 molecules already at a physiological intracellular concentration (≈1 µM), being instead the predominant species in the nanomolar concentration range. Because the total amount of TRAF2 changes during a cell cycle, the monomer-trimer equilibrium can be crucial for regulating the activities of TRAF2 in vivo.


Assuntos
Multimerização Proteica , Fator 2 Associado a Receptor de TNF/química , Linhagem Celular , Humanos , Simulação de Dinâmica Molecular , Estabilidade Proteica , Fator 2 Associado a Receptor de TNF/análise
12.
Int J Biochem Cell Biol ; 167: 106508, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38142771

RESUMO

TNF receptor-associated factor 2 (TRAF2) is involved in different cellular processes including signal transduction and transcription regulation. We here provide evidence of a direct interaction between the TRAF domain of TRAF2 and the monosialotetrahexosylganglioside (GM1). Previously, we showed that the TRAF domain occurs mainly in a trimeric form in solution, but it can also exist as a stable monomer when in the nanomolar concentration range. Here, we report that the quaternary structure of the TRAF domain is also affected by pH changes, since a weakly acidic pH (5.5) favors the dissociation of the trimeric TRAF domain into stable monomers, as previously observed at neutral pH (7.6) with the diluted protein. The TRAF domain-GM1 binding was similar at pH 5.5 and 7.6, suggesting that GM1 interacts with both the trimeric and monomeric forms of the protein. However, only the monomeric protein appeared to cause membrane deformation and inward vesiculation in GM1-containing giant unilamellar vesicles (GUVs). The formation of complexes between GM1 and TRAF2, or its TRAF domain, was also observed in cultured human leukemic HAP1 cells expressing either the truncated TRAF domain or the endogenous full length TRAF2. The GM1-protein complexes were observed after treatment with tunicamycin and were more concentrated in cells undergoing apoptosis, a condition which is known to cause cytoplasm acidification. These findings open the avenue for future studies aimed at deciphering the physiopathological relevance of the TRAF domain-GM1 interaction.


Assuntos
Gangliosídeo G(M1) , Transdução de Sinais , Humanos , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação da Expressão Gênica , NF-kappa B/metabolismo
13.
Biochemistry ; 51(37): 7304-12, 2012 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-22920299

RESUMO

The role played by glutathione transferase P1-1 (GSTP1-1) in modulating the c-Jun N-terminal kinase (JNK) pathway has been extensively investigated using JNK isoforms known to exert opposite effects in the cells. We have expressed isoform JNK1α2, which has been reported to transmit a pro-apoptotic signal, and we have analyzed both the phosphorylation level and the activity of this kinase in the presence of GSTP1-1. Contrary to what previous studies suggest, we found that GSTP1-1 is able to form a complex with the unphosphorylated and inactive JNK1α2 isoform, even in the absence of the substrate. We also analyzed the consequences of this interaction on the activity of both enzymes. The complex strongly reduced the extent of activation of JNK1α2 and preserved GSTP1-1 from inactivation. Unexpectedly, glutathione (GSH) exerted a negative effect on the affinity of GSTP1-1 for JNK1α2, suggesting that the intracellular levels of this thiol may allow a fine-tuning of the MAPK signaling pathway. Moreover, we found that the adduct formed by GSH and the strong GSTP1-1 inhibitor NBDHEX abolishes the interaction between GSTP1-1 and JNK1α2. These data confirm and extend at the molecular level previous evidence obtained in tumor cell lines.


Assuntos
Glutationa S-Transferase pi/metabolismo , Glutationa/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Linhagem Celular Tumoral , Glutationa/genética , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa S-Transferase pi/genética , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Oxidiazóis/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia
14.
Int J Biochem Cell Biol ; 145: 106193, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35257890

RESUMO

The scaffold protein Tumor Necrosis Factor Receptor-Associated Factor 2 (TRAF2) has been reported to play a key role in the endoplasmic reticulum (ER) stress-induced activation of c-Jun N-terminal Kinase (JNK) and hence autophagy. Autophagy is a highly conserved catabolic process, whose dysregulation is involved in the pathogenesis of various human diseases, including cancer. We investigated the involvement of TRAF2 in autophagy regulation in the human leukemic HAP1 cell line, under both basal and ER stress conditions. In TRAF2-knockout HAP1 cell line (KO), the basal autophagic flux was higher than in the parental cell line (WT). Moreover, tunicamycin-induced ER stress stimulated JNK activation and autophagy both in WT and KO HAP1. On the other hand, re-expression of a TRAF2 C-terminal fragment (residues ,310-501), in a TRAF2-KO cellular background, rendered HAP1 cells unable to activate both JNK and autophagy upon ER stress induction. Of note, this apparent dominant negative effect of the C-terminal fragment was observed even in the absence of the endogenous, full-length TRAF2 molecule. Furthermore, the expression of the C-terminal fragment resulted in both protein kinase B (AKT) pathway activation and increased resistance to the toxic effects induced by prolonged ER stress conditions. These findings indicate that TRAF2 is dispensable for the activation of both JNK and autophagy in HAP1 cells, while the TRAF2 C-terminal domain may play an autonomous role in regulating the cellular response to ER stress.


Assuntos
Estresse do Retículo Endoplasmático , Leucemia , Fator 2 Associado a Receptor de TNF/metabolismo , Apoptose , Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/farmacologia , Ubiquitina-Proteína Ligases/metabolismo
15.
Pharmaceuticals (Basel) ; 15(2)2022 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-35215282

RESUMO

This work describes the activity of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and of its newly identified carboxylic acid metabolite on the human malaria parasite Plasmodium falciparum. NBDHEX has been previously identified as a potent cytotoxic agent against murine and human cancer cells as well as towards the protozoan parasite Giardia duodenalis. We show here that NBDHEX is active in vitro against all blood stages of P. falciparum, with the rare feature of killing the parasite stages transmissible to mosquitoes, the gametocytes, with a 4-fold higher potency than that on the pathogenic asexual stages. This activity importantly translates into blocking parasite transmission through the Anopheles vector in mosquito experimental infections. A mass spectrometry analysis identified covalent NBDHEX modifications in specific cysteine residues of five gametocyte proteins, possibly associated with its antiparasitic effect. The carboxylic acid metabolite of NBDHEX retains the gametocyte preferential inhibitory activity of the parent compound, making this novel P. falciparum transmission-blocking chemotype at least as a new tool to uncover biological processes targetable by gametocyte selective drugs. Both NBDHEX and its carboxylic acid metabolite show very limited in vitro cytotoxicity on VERO cells. This result and previous evidence that NBDHEX shows an excellent in vivo safety profile in mice and is orally active against human cancer xenografts make these molecules potential starting points to develop new P. falciparum transmission-blocking agents, enriching the repertoire of drugs needed to eliminate malaria.

16.
Cell Biosci ; 12(1): 108, 2022 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842713

RESUMO

BACKGROUND: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. RESULTS: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1ß and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit. CONCLUSIONS: This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

17.
J Biomol Struct Dyn ; 39(1): 319-329, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31980009

RESUMO

The oligomeric state of TRAF2 (tumor necrosis factor-receptor associated factor 2), a TNF (tumor necrosis factor) receptor-associated factor, is crucial for membrane binding and probably plays a fundamental role in regulating the protein function in vivo. In this study we have combined molecular dynamics with the protein contact network approach to characterize the interaction of the three identical subunits of TRAF2. The average structure obtained after a 225 ns simulation reveals that two clusters of different size are formed, one of which includes almost completely two subunits, while the third monomer appears to be more independent. This picture is also confirmed by the estimated average number of inter-subunit contacts and by the comparison of side chains mobility in each monomer. The analysis of equilibrium pressure-induced dissociation measurements supports such findings, indicating that the dimeric-monomeric (2 + 1) might be prevalent with respect to the trimeric configuration, especially in the case of more diluted samples. These findings suggest that the formation of monomeric species, which is crucial for the formation of intra-luminal vesicles, might depend on preferential asymmetric interactions among the three subunits.Communicated by Ramaswamy H. Sarma.


Assuntos
Simulação de Dinâmica Molecular , Receptores do Fator de Necrose Tumoral , Substâncias Macromoleculares , NF-kappa B/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fatores de Necrose Tumoral
18.
Biochemistry ; 49(1): 226-35, 2010 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-19950984

RESUMO

Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-pi or GST-mu. Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here indicate that brostallicin binds reversibly to both isoenzymes with K(d) values in the micromolar range (the affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is slowly converted into the final products. This intermediate, identified as the alpha-chloroamido derivative of the GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1, has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the treatment of tumor overexpressing the pi or mu class GST.


Assuntos
Antineoplásicos/farmacologia , Glutationa S-Transferase pi/metabolismo , Glutationa Transferase/metabolismo , Guanidinas/farmacologia , Pirróis/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Catálise , Linhagem Celular Tumoral , DNA/metabolismo , Feminino , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa Transferase/antagonistas & inibidores , Humanos , Cinética
19.
Arch Biochem Biophys ; 500(2): 116-22, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20494652

RESUMO

Chemoresistance is a multifactorial phenomenon and many studies clearly show that a coordinated expression of efflux transporter proteins and phase II conjugating enzymes in tumor cells is linked to the development of the multidrug resistance phenotype. In particular, the overexpression of glutathione S-transferases and efflux pumps in tumors may reduce the reactivity of various anticancer drugs. In recent years it has become evident that glutathione S-transferases are also involved in the control of apoptosis through the inhibition of the JNK signaling pathway. As such, the glutathione S-transferase superfamily has become the focus of extensive pharmaceutical research in attempt to generate more efficient anticancer agents. Here we present an overview of the GST inhibitors and the GST-activated pro-drugs utilized to date to overcome drug resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Glutationa Transferase/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Transporte Biológico Ativo , Resistência a Múltiplos Medicamentos/fisiologia , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Humanos , Neoplasias/patologia , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia
20.
Biochem Pharmacol ; 178: 114060, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32473836

RESUMO

The 7-nitrobenzo[c][1,2,5]oxadiazole (NBD) derivative NBDHEX (compound 1) and its analogue MC3181 (compound 2) have been found to be potent inhibitors of tumor cell growth in vitro and therapeutically active and safe in mice bearing human melanoma xenografts. To enhance the aqueous solubility of these compounds, we synthesized the hemisuccinate of 1 (compound 3) and the phosphate monoesters of 1 and 2 (compound 4 and 5, respectively). These novel NBD derivatives displayed a solubility in the conventional phosphate-buffered saline up to 150-fold higher than that of 1, and up to 4-fold higher than that of 2. Notably, solubility of phosphates 4 and 5 in a potassium phosphate buffer at pH 7.4, was up to 500-fold higher than that of 1, and ~10-fold higher than that of 2. Compounds 3-5 retained high cytotoxicity towards cultured human melanoma and osteosarcoma cells and were cleaved in vitro by both human and murine hydrolases, thus releasing the corresponding parent compound (i.e., 1 or 2). Interestingly, esters 3-5 displayed high inhibitory activity towards the glutathione transferase (GST) isoform GSTP1-1 and showed a reactivity towards reduced glutathione comparable to that of the respective parent compound. Finally, both 4 and 5 were safe and effective when administered intravenously or orally as an aqueous solution to mice xenografted with A375 human melanoma tumors. Collectively, these results and the previously observed synergistic interaction between 1 and 2 and various approved anticancer drugs, suggest the possible utility of phosphates 4 and 5 as single agents and in combination regimens in cancers with unmet medical need, including melanoma.


Assuntos
4-Cloro-7-nitrobenzofurazano/metabolismo , Antineoplásicos/metabolismo , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa S-Transferase pi/metabolismo , Neoplasias/metabolismo , Água/metabolismo , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ésteres/química , Ésteres/metabolismo , Feminino , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Solubilidade , Água/química , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
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