RESUMEN
Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.
Asunto(s)
Ácidos Grasos/farmacología , Nitrocompuestos/farmacología , Sirtuinas/metabolismo , Acetilación , Humanos , Estrés Oxidativo , Conformación Proteica , Sirtuinas/química , Sirtuinas/genéticaRESUMEN
There is a growing interest to study and address neglected tropical diseases (NTD). To this end, in silico methods can serve as the bridge that connects academy and industry, encouraging the development of future treatments against these diseases. This chapter discusses current challenges in the development of new therapies, available computational methods and successful cases in computer-aided design with particular focus on human trypanosomiasis. Novel targets are also discussed. As a case study, we identify amentoflavone as a potential inhibitor of TcSir2rp3 (sirtuine) from Trypanosoma cruzi (20.03 µM) with a workflow that integrates chemoinformatic approaches, molecular modeling, and theoretical affinity calculations, as well as in vitro assays.
Asunto(s)
Biflavonoides/química , Enfermedad de Chagas , Simulación por Computador , Inhibidores Enzimáticos/química , Proteínas Protozoarias , Sirtuinas , Tripanocidas/química , Trypanosoma cruzi/enzimología , Biflavonoides/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/enzimología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Sirtuinas/antagonistas & inhibidores , Sirtuinas/química , Tripanocidas/uso terapéuticoRESUMEN
Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi, affecting more than 7 million people in the world. Benznidazole and nifurtimox are the only drugs available for treatment and in addition to causing several side effects, are only satisfactory in the acute phase of the disease. Sirtuins are NAD+-dependent deacetylases involved in several biological processes, which have become drug target candidates in various disease settings. T. cruzi presents two sirtuins, one cytosolic (TcSir2rp1) and the latter mitochondrial (TcSir2rp3). Here, we characterized the effects of human sirtuin inhibitors against T. cruzi sirtuins as an initial approach to develop specific parasite inhibitors. We found that, of 33 compounds tested, two inhibited TcSir2rp1 (15 and 17), while other five inhibited TcSir2rp3 (8, 12, 13, 30, and 32), indicating that specific inhibitors can be devised for each one of the enzymes. Furthermore, all inhibiting compounds prevented parasite proliferation in cultured mammalian cells. When combining the most effective inhibitors with benznidazole at least two compounds, 17 and 32, demonstrated synergistic effects. Altogether, these results support the importance of exploring T. cruzi sirtuins as drug targets and provide key elements to develop specific inhibitors for these enzymes as potential targets for Chagas disease treatment.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Nitroimidazoles/farmacología , Sirtuinas/antagonistas & inhibidores , Sirtuinas/metabolismo , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Línea Celular , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/parasitología , Histona Desacetilasas del Grupo III/antagonistas & inhibidores , Concentración 50 Inhibidora , Macaca mulatta , Simulación del Acoplamiento Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sirtuinas/química , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidadRESUMEN
Giardia duodenalis is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. Treatment of giardiasis is limited to nitroheterocyclic compounds as metronidazole and benzimidazoles as albendazole, where remarkably treatment failure is relatively common. Consequently, the need for new options to treat this disease is underscored. We predicted by a bioinformatic approach that nicotinamide inhibits Giardia sirtuins by the nicotinamide exchange pathway, and since sirtuins are involved in cell cycle control, they could be related with arrest and decrease of viability. When trophozoites were treated with nicotinamide (NAM), a strong arrest of Giardia trophozoites in G2 phase was observed and at the same time changes in transcriptional expression of sirtuins were produced. Interestingly, the G2 arrest is not related to double-strand breaks, which strengthens the role of sirtuins in the control of the Giardia cell cycle. Results with NAM-treated trophozoites as predicted demonstrate antigiardial effects and thus open new options for the treatment of giardiasis, either with the combination of nicotinamide with another antigiardial drug, or with the design of specific inhibitors for Giardia sirtuins.
Asunto(s)
Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Giardia lamblia/efectos de los fármacos , Niacinamida/farmacología , Sirtuinas/metabolismo , Complejo Vitamínico B/farmacología , Secuencia de Aminoácidos , Giardia lamblia/citología , Giardia lamblia/genética , Giardia lamblia/metabolismo , Humanos , Alineación de Secuencia , Sirtuinas/antagonistas & inhibidores , Sirtuinas/química , Sirtuinas/genéticaRESUMEN
Sirtuins are a conserved family of NAD-dependent protein deacylases. Initially proposed as histone deacetylases, it is now known that they act on a variety of proteins including transcription factors and metabolic enzymes, having a key role in the regulation of cellular homeostasis. Seven isoforms are identified in mammals (SIRT1-7), all of them sharing a conserved catalytic core and showing differential subcellular localization and activities. Oxidative stress can affect the activity of sirtuins at different levels: expression, posttranslational modifications, protein-protein interactions, and NAD levels. Mild oxidative stress induces the expression of sirtuins as a compensatory mechanism, while harsh or prolonged oxidant conditions result in dysfunctional modified sirtuins more prone to degradation by the proteasome. Oxidative posttranslational modifications have been identified in vitro and in vivo, in particular cysteine oxidation and tyrosine nitration. In addition, oxidative stress can alter the interaction with other proteins, like SIRT1 with its protein inhibitor DBC1 resulting in a net increase of deacetylase activity. In the same way, manipulation of cellular NAD levels by pharmacological inhibition of other NAD-consuming enzymes results in activation of SIRT1 and protection against obesity-related pathologies. Nevertheless, further research is needed to establish the molecular mechanisms of redox regulation of sirtuins to further design adequate pharmacological interventions.
Asunto(s)
Estrés Oxidativo , Sirtuinas/metabolismo , Envejecimiento/patología , Animales , Enfermedad , Humanos , Terapia Molecular Dirigida , Unión Proteica , Sirtuinas/químicaRESUMEN
Sirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue from Leishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectinaffinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylated in vivo.