RESUMO
Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.
Assuntos
Amida Sintases/antagonistas & inibidores , Antineoplásicos/farmacologia , Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Amida Sintases/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Antiprotozoários/síntese química , Antiprotozoários/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leishmania infantum/enzimologia , Macrófagos/efeitos dos fármacos , Estrutura Molecular , Relação Estrutura-Atividade , Trypanosoma cruzi/enzimologiaRESUMO
Trypanosomatid-caused diseases are among the neglected infectious diseases with the highest disease burden, affecting about 27 million people worldwide and, in particular, socio-economically vulnerable populations. Trypanothione synthetase (TryS) is considered one of the most attractive drug targets within the thiol-polyamine metabolism of typanosomatids, being unique, essential and druggable. Here, we have compiled a dataset of 401 T. brucei TryS inhibitors that includes compounds with inhibitory data reported in the literature, but also in-house acquired data. QSAR classifiers were derived and validated from such dataset, using publicly available and open-source software, thus assuring the portability of the obtained models. The performance and robustness of the resulting models were substantially improved through ensemble learning. The performance of the individual models and the model ensembles was further assessed through retrospective virtual screening campaigns. At last, as an application example, the chosen model-ensemble has been applied in a prospective virtual screening campaign on DrugBank 5.1.6 compound library. All the in-house scripts used in this study are available on request, whereas the dataset has been included as supplementary material.
Assuntos
Amida Sintases/química , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Aprendizado de Máquina , Algoritmos , Amida Sintases/antagonistas & inibidores , Amida Sintases/metabolismo , Antiprotozoários/química , Antiprotozoários/farmacologia , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Inibidores Enzimáticos/farmacologia , Humanos , Redes e Vias Metabólicas , Modelos Teóricos , Curva ROC , Relação Estrutura-AtividadeRESUMO
Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.
Assuntos
Benzazepinas/química , Glutationa/análogos & derivados , Leishmania/metabolismo , Espermidina/análogos & derivados , Animais , Glutationa/biossíntese , Espermidina/biossínteseRESUMO
To validate therapeutic targets in metabolic pathways of trypanosomatids, the criterion of enzyme essentiality determined by gene knockout or knockdown is usually being applied. Since, it is often found that most of the enzymes/proteins analyzed are essential, additional criteria have to be implemented for drug target prioritization. Metabolic control analysis (MCA), often in conjunction with kinetic pathway modeling, offers such possibility for prioritization. MCA is a theoretical and experimental approach to analyze how metabolic pathways are controlled. It involves strategies to perform quantitative analyses to determine the degree in which an enzyme controls a pathway flux, a value called flux control coefficient ([Formula: see text]). By determining the [Formula: see text] of individual steps in a metabolic pathway, the distribution of control of the pathway is established, that is, the identification of the main flux-controlling steps. Therefore, MCA can help in ranking pathway enzymes as drug targets from a metabolic perspective. In this chapter, three approaches to determine [Formula: see text] are reviewed: (1) In vitro pathway reconstitution, (2) manipulation of enzyme activities within parasites, and (3) in silico kinetic modeling of the metabolic pathway. To perform these methods, accurate experimental data of enzyme activities, metabolite concentrations and pathway fluxes are necessary. The methodology is illustrated with the example of trypanothione metabolism of Trypanosoma cruzi and protocols to determine such experimental data for this metabolic process are also described. However, the MCA strategy can be applied to any metabolic pathway in the parasite and general directions to perform it are provided in this chapter.
Assuntos
Desenvolvimento de Medicamentos/métodos , Metabolômica/métodos , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Extratos Celulares/isolamento & purificação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Simulação por Computador , Glutationa/análogos & derivados , Glutationa/metabolismo , Humanos , Cinética , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espermidina/análogos & derivados , Espermidina/metabolismo , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.
Assuntos
Antioxidantes/metabolismo , Glutamato-Cisteína Ligase/genética , Glutationa/análogos & derivados , Proteínas de Protozoários/genética , Espermidina/análogos & derivados , Tiorredoxinas/genética , Trypanosoma cruzi/efeitos dos fármacos , Amida Sintases/genética , Amida Sintases/metabolismo , Butionina Sulfoximina/farmacologia , Linhagem Celular , Combinação de Medicamentos , Resistência a Medicamentos/genética , Fibroblastos/parasitologia , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/metabolismo , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Humanos , Peróxido de Hidrogênio/farmacologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Nitroimidazóis/farmacologia , Oxirredução , Estresse Oxidativo , Peroxidases/genética , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Espermidina/antagonistas & inibidores , Espermidina/biossíntese , Tiorredoxinas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids. METHODS AND RESULTS: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000⯵M) and heavy metal (1-500⯵M) toxicity. Treatment with TryS inhibitors (5-30⯵M) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. CONCLUSIONS: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti-trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.