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PLoS Negl Trop Dis ; 14(5): e0008339, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32437349


Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.

Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Tolueno/análogos & derivados , Trypanosoma brucei brucei/efeitos dos fármacos , Antiprotozoários/isolamento & purificação , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Ensaios de Triagem em Larga Escala , NADH NADPH Oxirredutases/química , Ligação Proteica , Conformação Proteica , Tolueno/isolamento & purificação , Tolueno/farmacologia , Trypanosoma brucei brucei/enzimologia
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702


Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.

Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
PLoS Negl Trop Dis ; 12(11): e0006969, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30475811


Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts.

Antiprotozoários/química , Inibidores Enzimáticos/química , Leishmania/enzimologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmaniose/parasitologia , Modelos Moleculares , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADP/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
SLAS Discov ; 22(7): 897-905, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28346095


Pseudomonas infections are common among hospitalized, immunocompromised, and chronic lung disease patients. These infections are recalcitrant to common antibacterial therapies due to inherent antibiotic resistance. To meet the need of new anti- Pseudomonas drugs, a sensitive, homogenous, and robust assay was developed with the aim of identifying inhibitors of acyl-coenzyme A synthetases (ACSs) from Pseudomonas. Given the importance of fatty acids for in vivo nutrition of Pseudomonas, such inhibitors might have the potential to reduce the bacterial fitness during infection. The assay, based on a coupled reaction between the Pseudomonas spp. ACS and the firefly luciferase, allowed the identification of three classes of inhibitors by screening of a diverse compound collection. These compounds were confirmed to reversibly bind ACS with potencies in the micromolar range. Two classes were found to compete with acyl-coenzyme A, while the third one was competitive with fatty acid binding. Although these compounds inhibit the bacterial ACS in cell-free assays, they show modest or no effect on Pseudomonas growth in vitro.

Acil Coenzima A/antagonistas & inibidores , Acil Coenzima A/metabolismo , Inibidores Enzimáticos/farmacologia , Pseudomonas/metabolismo , Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Luciferases de Vaga-Lume/metabolismo , Infecções por Pseudomonas/microbiologia
Eur J Pharmacol ; 762: 283-92, 2015 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-26048310


Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy.

Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Fatores Imunológicos/farmacologia , Melanoma/patologia , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Imidazóis/farmacologia , Melanoma/genética , Invasividade Neoplásica , Metástase Neoplásica , Oximas/farmacologia
Amino Acids ; 44(1): 293-300, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22139409


Aloin, a natural anthracycline from aloe plant, is a hydroxyanthraquinone derivative shown to have antitumor properties. This study demonstrated that aloin exerted inhibition of cell proliferation, adhesion and invasion abilities of B16-F10 melanoma cells under non-cytotoxic concentrations. Furthermore, aloin induced melanoma cell differentiation through the enhancement of melanogenesis and transglutaminase activity. To improve the growth-inhibiting effect of anticancer agents, we found that the combined treatment of cells with aloin and low doses of cisplatin increases the antiproliferative activity of aloin. The results suggest that aloin possesses antineoplastic and antimetastatic properties, exerted likely through the induction of melanoma cell differentiation.

Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Cisplatino/farmacologia , Emodina/análogos & derivados , Transglutaminases/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Emodina/farmacologia , Melaninas/biossíntese , Melanoma Experimental , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Espermina/metabolismo