RESUMO
Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/ultraestrutura , Esquistossomose mansoni/tratamento farmacológico , Animais , Anti-Helmínticos/farmacologia , Resistência a Medicamentos , Microscopia Eletrônica de Varredura , Oviposição/efeitos dos fármacos , Praziquantel/farmacologia , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/patologiaRESUMO
The only drug currently available for treatment of the neglected disease Schistosomiasis is Praziquantel, and the possible emergence of resistance makes research on novel therapeutic agents necessary and urgent. To this end, the targeting of Schistosoma mansoni epigenetic enzymes, which regulate the parasitic life cycle, emerged as a promising approach. Due to the strong effects of human sirtuin inhibitors on parasite survival and reproduction, Schistosoma sirtuins were postulated as potential therapeutic targets. In vitro testing of synthetic substrates of S. mansoni sirtuin 2 (SmSirt2) and kinetic experiments on a myristoylated peptide demonstrated lysine long-chain deacylation as an intrinsic SmSirt2 activity in addition to its known deacetylase activity for the first time. Focused in vitro screening of the GSK Kinetobox library and structure-activity relationships of identified hits led to the first SmSirt2 inhibitors with activity in the low micromolar range. Several SmSirt2 inhibitors showed potency against both larval schistosomes (viability) and adult worms (pairing, egg laying) in culture without general toxicity to human cancer cells.
Assuntos
Proteínas de Helminto/antagonistas & inibidores , Schistosoma mansoni/metabolismo , Sirtuína 2/antagonistas & inibidores , Animais , Proteínas de Helminto/metabolismo , Humanos , Cinética , Larva/efeitos dos fármacos , Larva/metabolismo , Lisina/química , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Oxidiazóis/química , Oxidiazóis/metabolismo , Oxidiazóis/farmacologia , Oxidiazóis/uso terapêutico , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose/tratamento farmacológico , Sirtuína 2/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9-370 nM), with belinostat, panobinostat and vorinostat having 8-45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 µM) or S. mansoni schistosomula (IC50 > 10 µM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 â¼10 µM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4-7 and 4-10 after infection (P < 0.05), respectively.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Leishmania/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Acetilação , Administração Oral , Animais , Depsipeptídeos/farmacologia , Células HEK293 , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/administração & dosagem , Indóis/farmacologia , Indóis/uso terapêutico , Concentração Inibidora 50 , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/tratamento farmacológico , Malária/parasitologia , Camundongos , Panobinostat , Parasitemia/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium knowlesi/crescimento & desenvolvimento , Schistosoma mansoni/crescimento & desenvolvimento , Sulfonamidas/farmacologia , VorinostatRESUMO
Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae.
RESUMO
The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/ß HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.
Assuntos
Epigênese Genética , Proteínas de Helminto/química , Histona Desacetilases/química , Dobramento de Proteína , Schistosoma mansoni/enzimologia , Animais , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Inibidores de Histona Desacetilases/química , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Estrutura Secundária de Proteína , Schistosoma mansoni/genéticaRESUMO
BACKGROUND: The chemotherapy of schistosomiasis currently depends on the use of a single drug, praziquantel. In order to develop novel chemotherapeutic agents we are investigating enzymes involved in the epigenetic modification of chromatin. Sirtuins are NAD+ dependent lysine deacetylases that are involved in a wide variety of cellular processes including histone deacetylation, and have been demonstrated to be therapeutic targets in various pathologies, including cancer. METHODOLOGY PRINCIPAL FINDINGS: In order to determine whether Schistosoma mansoni sirtuins are potential therapeutic targets we first identified and characterized their protein sequences. Five sirtuins (SmSirt) are encoded in the S. mansoni genome and phylogenetic analysis showed that they are orthologues of mammalian Sirt1, Sirt2, Sirt5, Sirt6 and Sirt7. Both SmSirt1 and SmSirt7 have large insertion in the catalytic domain compared to their mammalian orthologues. SmSirt5 is the only mitochondrial sirtuin encoded in the parasite genome (orthologues of Sirt3 and Sirt4 are absent) and transcripts corresponding to at least five splicing isoforms were identified. All five sirtuins are expressed throughout the parasite life-cycle, but with distinct patterns of expression. Sirtuin inhibitors were used to treat both schistosomula and adult worms maintained in culture. Three inhibitors in particular, Sirtinol, Salermide and MS3 induced apoptosis and death of schistosomula, the separation of adult worm pairs, and a reduction in egg laying. Moreover, Salermide treatment led to a marked disruption of the morphology of ovaries and testes. Transcriptional knockdown of SmSirt1 by RNA interference in adult worms led to morphological changes in the ovaries characterized by a marked increase in mature oocytes, reiterating the effects of sirtuin inhibitors and suggesting that SmSirt1 is their principal target. CONCLUSION SIGNIFICANCE: Our data demonstrate the potential of schistosome sirtuins as therapeutic targets and validate screening for selective sirtuin inhibitors as a strategy for developing new drugs against schistosomiasis.
Assuntos
Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Sirtuínas/genética , Sirtuínas/metabolismo , Animais , Anti-Helmínticos/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas de Helminto/antagonistas & inibidores , Humanos , Masculino , Dados de Sequência Molecular , Testes de Sensibilidade Parasitária , Filogenia , Schistosoma mansoni/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sirtuínas/antagonistas & inibidoresRESUMO
The histone modifying enzymes (HME) represent particularly promising targets for the development of alternatives to praziquantel, the only currently available drug to combat schistosomiasis. The inhibition of these enzymes frequently arrests the cell cycle or induces apoptosis in cancer cells, but not in normal cells and numerous HME inhibitors are under investigation as potential anticancer agents. The recent resolution of the genome sequences of Schistosoma mansoni and Schistosoma japonicum has allowed us to identify all the schistosome genes encoding histone acetyltransferases, deacetylases, methyltransferases and demethylases. We have chosen a strategy using phylogenetic screening with inhibitors of HME classes, screening of individual HME targets by both high-throughput and reasoned (in silico docking using resolved crystal structures) approaches in a project funded by the European Community, named SEtTReND (Schistosome Epigenetics: Targets, Regulation, New Drugs). The initial focus is on the class I histone deacetylase (HDAC) 8 since the comparison of the catalytic site of the schistosome and human enzymes shows crucial differences, rendering possible the development of inhibitors specific for SmHDAC8. However, phenotypic screening shows that inhibitors of all HME classes tested were able to induce apoptosis and death in parasites in vitro, indicating that other enzymes may prove to be viable targets.
Assuntos
Desenho de Fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Schistosoma/efeitos dos fármacos , Schistosoma/metabolismo , Esquistossomose/tratamento farmacológico , Esquistossomicidas/uso terapêutico , Animais , Humanos , Schistosoma/crescimento & desenvolvimentoRESUMO
Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.
Assuntos
Cromatina/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Histona Desacetilases/metabolismo , Schistosoma/efeitos dos fármacos , Animais , Cromatina/metabolismo , Desenho de Fármacos , Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Schistosoma/enzimologiaRESUMO
Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.