RESUMO
The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactone's effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3Galt6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.
RESUMO
The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.
Assuntos
Antineoplásicos , Glicoconjugados , Antineoplásicos/química , Glicoconjugados/química , Glicosídeos/farmacologia , Canais de Translocação SEC/metabolismoRESUMO
Buruli ulcer (BU) is a neglected tropical disease caused by subcutaneous infection with Mycobacterium ulcerans and its exotoxin mycolactone. BU displays coagulative necrosis and widespread fibrin deposition in affected skin tissues. Despite this, the role of the vasculature in BU pathogenesis remains almost completely unexplored. We hypothesise that fibrin-driven ischemia can be an 'indirect' route to mycolactone-dependent tissue necrosis by a mechanism involving vascular dysfunction. Here, we tracked >900 vessels within contiguous tissue sections from eight BU patient biopsies. Our aim was to evaluate their vascular and coagulation biomarker phenotype and explore potential links to fibrin deposition. We also integrated this with our understanding of mycolactone's mechanism of action at Sec61 and its impact on proteins involved in maintaining normal vascular function. Our findings showed that endothelial cell dysfunction is common in skin tissue adjacent to necrotic regions. There was little evidence of primary haemostasis, perhaps due to mycolactone-dependent depletion of endothelial von Willebrand factor. Instead, fibrin staining appeared to be linked to the extrinsic pathway activator, tissue factor (TF). There was significantly greater than expected fibrin staining around vessels that had TF staining within the stroma, and this correlated with the distance it extended from the vessel basement membrane. TF-induced fibrin deposition in these locations would require plasma proteins outside of vessels, therefore we investigated whether mycolactone could increase vascular permeability in vitro. This was indeed the case, and leakage was further exacerbated by IL-1ß. Mycolactone caused the loss of endothelial adherens and tight junctions by the depletion of VE-cadherin, TIE-1, TIE-2 and JAM-C; all Sec61-dependent proteins. Taken together, our findings suggest that both vascular and lymphatic vessels in BU lesions become "leaky" during infection, due to the unique action of mycolactone, allowing TF-containing structures and plasma proteins into skin tissue, ultimately leading to local coagulopathy and tissue ischemia.
Assuntos
Úlcera de Buruli/metabolismo , Fibrina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucina-1beta/metabolismo , Macrolídeos/metabolismo , Mycobacterium ulcerans/metabolismo , Pele , Tromboplastina/metabolismo , Adolescente , Adulto , Idoso , Úlcera de Buruli/microbiologia , Úlcera de Buruli/patologia , Criança , Feminino , Células Endoteliais da Veia Umbilical Humana/microbiologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pele/irrigação sanguínea , Pele/metabolismo , Pele/microbiologiaRESUMO
The Mycobacterium ulcerans exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of Sqstm1 reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in eif2ak3-/-/perk-/- cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease.Abbreviations: ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A1; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.
Assuntos
Autofagia , Úlcera de Buruli , Fator de Iniciação 2 em Eucariotos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrolídeos , Camundongos , Fator de Iniciação 2 em Procariotos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Canais de Translocação SEC/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitina/metabolismoRESUMO
The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon 'breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Macrolídeos/farmacologia , Canais de Translocação SEC/metabolismo , Animais , Células HCT116 , Células HEK293 , Humanos , Camundongos , Células RAW 264.7RESUMO
The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of â¼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more 'metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.
Assuntos
Proteínas de Protozoários/metabolismo , Proteínas Repressoras/metabolismo , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , Interferência de RNA , Proteínas Repressoras/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a devastating necrotizing skin disease. Key to its pathogenesis is mycolactone, the exotoxin virulence factor that is both immunosuppressive and cytotoxic. The discovery that the essential Sec61 translocon is the major cellular target of mycolactone explains much of the disease pathology, including the immune blockade. Sec61 inhibition leads to a loss in production of nearly all cytokines from monocytes, macrophages, dendritic cells and T cells, as well as antigen presentation pathway proteins and costimulatory molecules. However, there has long been evidence that the immune system is not completely incapable of responding to M. ulcerans infection. In particular, IL-1ß was recently shown to be present in BU lesions, and to be induced from M. ulcerans-exposed macrophages in a mycolactone-dependent manner. This has important implications for our understanding of BU, showing that mycolactone can act as the "second signal" for IL-1ß production without inhibiting the pathways of unconventional secretion it uses for cellular release. In this Perspective article, we validate and discuss this recent advance, which is entirely in-line with our understanding of mycolactone's inhibition of the Sec61 translocon. However, we also show that the IL-1 receptor, which uses the conventional secretory pathway, is sensitive to mycolactone blockade at Sec61. Hence, a more complete understanding of the mechanisms regulating IL-1ß function in skin tissue, including the transient intra-macrophage stage of M. ulcerans infection, is urgently needed to uncover the double-edged sword of IL-1ß in BU pathogenesis, treatment and wound healing.
Assuntos
Úlcera de Buruli/imunologia , Interleucina-1beta/imunologia , Macrolídeos/metabolismo , Macrófagos/imunologia , Canais de Translocação SEC/metabolismo , Humanos , Mycobacterium ulcerans/patogenicidadeRESUMO
Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
Assuntos
Macrolídeos/farmacologia , Microssomos/química , Ribossomos/química , Canais de Translocação SEC/química , Animais , Sítios de Ligação , Sistema Livre de Células/metabolismo , Cães , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidade , Pâncreas/química , Pâncreas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Ribossomos/metabolismo , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Monoallelic exclusion ensures that the African trypanosome Trypanosoma brucei exclusively expresses only 1 of thousands of different variant surface glycoprotein (VSG) coat genes. The active VSG is transcribed from 1 of 15 polycistronic bloodstream-form VSG expression sites (ESs), which are controlled in a mutually exclusive fashion. Unusually, T. brucei uses RNA polymerase I (Pol I) to transcribe the active ES, which is unprecedented among eukaryotes. This active ES is located within a unique extranucleolar Pol I body called the expression-site body (ESB). A stringent restriction mechanism prevents T. brucei from expressing multiple ESs at the same time, although how this is mediated is unclear. By using drug-selection pressure, we generated VSG double-expresser T. brucei lines, which have disrupted monoallelic exclusion, and simultaneously express 2 ESs in a dynamic fashion. The 2 unstably active ESs appear epigenetically similar to fully active ESs as determined by using chromatin immunoprecipitation for multiple epigenetic marks (histones H3 and H1, TDP1, and DNA base J). We find that the double-expresser cells, similar to wild-type single-expresser cells, predominantly contain 1 subnuclear ESB, as determined using Pol I or the ESB marker VEX1. Strikingly, simultaneous transcription of the 2 dynamically transcribed ESs is normally observed only when the 2 ESs are both located within this single ESB. This colocalization is reversible in the absence of drug selection. This discovery that simultaneously active ESs dynamically share a single ESB demonstrates the importance of this unique subnuclear body in restricting the monoallelic expression of VSG.
Assuntos
Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Linhagem Celular , Epigênese Genética , Transporte Proteico , Transcrição Gênica , Trypanosoma brucei brucei/genéticaRESUMO
Mycolactone is the exotoxin virulence factor of Mycobacterium ulcerans that causes the neglected tropical disease Buruli ulcer. We recently showed it to be a broad spectrum inhibitor of Sec61-dependent co-translational translocation of proteins into the endoplasmic reticulum (ER). An outstanding question is the molecular pathway linking this to its known cytotoxicity. We have now used translational profiling to better understand the reprogramming that occurs in cells exposed to mycolactone. Gene ontology identified enrichment in genes involved in cellular response to stress, and apoptosis signalling among those showing enhanced translation. Validation of these results supports a mechanism by which mycolactone activates an integrated stress response meditated by phosphorylation of eIF2α via multiple kinases (PERK, GCN, PKR) without activation of the ER stress sensors IRE1 or ATF6. The response therefore uncouples the integrated stress response from ER stress, and features translational and transcriptional modes of genes expression that feature the key regulatory transcription factor ATF4. Emphasising the importance of this uncoupled response in cytotoxicity, downstream activation of this pathway is abolished in cells expressing mycolactone-resistant Sec61α variants. Using multiple genetic and biochemical approaches, we demonstrate that eIF2α phosphorylation is responsible for mycolactone-dependent translation attenuation, which initially protects cells from cell death. However, chronic activation without stress remediation enhances autophagy and apoptosis of cells by a pathway facilitated by ATF4 and CHOP. Our findings demonstrate that priming events at the ER can result in the sensing of stress within different cellular compartments.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Toxinas Bacterianas/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Macrolídeos/toxicidade , Canais de Translocação SEC/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Humanos , Camundongos , Transporte Proteico/efeitos dos fármacos , Canais de Translocação SEC/genéticaRESUMO
A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2 ng/ml and as early as 8 hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.
Assuntos
Antibacterianos/uso terapêutico , Úlcera de Buruli/tratamento farmacológico , Fibrina/metabolismo , Macrolídeos/uso terapêutico , Mycobacterium ulcerans/fisiologia , Trombomodulina/metabolismo , Úlcera de Buruli/diagnóstico , Úlcera de Buruli/metabolismo , Úlcera de Buruli/microbiologia , Células Endoteliais/metabolismo , Humanos , Macrolídeos/metabolismo , Necrose/microbiologia , Pele/microbiologia , Pele/patologiaRESUMO
Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.
Assuntos
Retículo Endoplasmático/metabolismo , Mediadores da Inflamação/metabolismo , Macrolídeos/metabolismo , Mycobacterium ulcerans/patogenicidade , Animais , Úlcera de Buruli/metabolismo , Úlcera de Buruli/microbiologia , Úlcera de Buruli/patologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Retículo Endoplasmático/patologia , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Mycobacterium ulcerans/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nitroaromatic prodrugs are used to treat a range of microbial infections with selectivity achieved by specific activation reactions. For trypanosomatid parasites, this is mediated by type I nitroreductases. Here, we demonstrate that the causative agent of leishmaniasis, Leishmania major, expresses an FMN-containing nitroreductase (LmNTR) that metabolizes a wide range of substrates, and based on electron donor and acceptor preferences, it may function as an NADH:quinone oxidoreductase. Using gene deletion approaches, we demonstrate that this activity is essential to L. major promastigotes, the parasite forms found in the insect vector. Intriguingly, LmNTR(+/-) heterozygote promastigote parasites could readily differentiate into infectious metacyclic cells but these were unable to establish infections in cultured mammalian cells and caused delayed pathology in mice. Furthermore, we exploit the LmNTR activity evaluating a library of nitrobenzylphosphoramide mustards using biochemical and phenotypic screens. We identify a subset of compounds that display significant growth inhibitory properties against the intracellular parasite form found in the mammalian hosts. The leishmanicidal activity was shown to be LmNTR-specific as the LmNTR(+/-) heterozygote promastigotes displayed resistance to the most potent mustards. We conclude that LmNTR can be targeted for drug development by exploiting its prodrug activating property or by designing specific inhibitors to block its endogenous function.
Assuntos
Leishmania major/enzimologia , Nitrorredutases/metabolismo , Pró-Fármacos/farmacologia , Tripanossomicidas/farmacologia , Alelos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Mononucleotídeo de Flavina/metabolismo , Heterozigoto , Humanos , Cinética , Leishmania major/efeitos dos fármacos , Leishmania major/crescimento & desenvolvimento , Leishmania major/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Mostarda/química , Compostos de Mostarda/farmacologia , Nitroimidazóis/química , Nitroimidazóis/farmacologia , Nitrorredutases/antagonistas & inibidores , Pró-Fármacos/química , Especificidade por Substrato/efeitos dos fármacos , Tripanossomicidas/químicaRESUMO
The nitroheterocycle nifurtimox, as part of a nifurtimox-eflornithine combination therapy, represents one of a limited number of treatments targeting Trypanosoma brucei, the causative agent of human African trypanosomiasis. The mode of action of this prodrug involves an initial activation reaction catalyzed by a type I nitroreductase (NTR), an enzyme found predominantly in prokaryotes, leading to the formation of a cytotoxic unsaturated open-chain nitrile metabolite. Here, we evaluate the trypanocidal activities of a library of other 5-nitrofurans against the bloodstream form of T. brucei as a preliminary step in the identification of additional nitroaromatic compounds that can potentially partner with eflornithine. Biochemical screening against the purified enzyme revealed that all 5-nitrofurans were effective substrates for T. brucei NTR (TbNTR), with the preferred compounds having apparent kcat/Km values approximately 50-fold greater than those of nifurtimox. For several compounds, in vitro reduction by this nitroreductase yielded products characterized by mass spectrometry as either unsaturated or saturated open-chain nitriles. When tested against the bloodstream form of T. brucei, many of the derivatives displayed significant growth-inhibitory properties, with the most potent compounds generating 50% inhibitory concentrations (IC50s) around 200 nM. The antiparasitic activities of the most potent agents were demonstrated to be NTR dependent, as parasites having reduced levels of the enzyme displayed resistance to the compounds, while parasites overexpressing TbNTR showed hypersensitivity. We conclude that other members of the 5-nitrofuran class of nitroheterocycles have the potential to treat human African trypanosomiasis, perhaps as an alternative partner prodrug to nifurtimox, in the next generation of eflornithine-based combinational therapies.
Assuntos
Nitrofuranos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Nitrorredutases/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
Nitroheterocyclic prodrugs are used to treat infections caused by Trypanosoma cruzi and Trypanosoma brucei. A key component in selectivity involves a specific activation step mediated by a protein homologous with type I nitroreductases, enzymes found predominantly in prokaryotes. Using data from determinations based on flavin cofactor, oxygen-insensitive activity, substrate range, and inhibition profiles, we demonstrate that NTRs from T. cruzi and T. brucei display many characteristics of their bacterial counterparts. Intriguingly, both enzymes preferentially use NADH and quinones as the electron donor and acceptor, respectively, suggesting that they may function as NADH:ubiquinone oxidoreductases in the parasite mitochondrion. We exploited this preference to determine the trypanocidal activity of a library of aziridinyl benzoquinones against bloodstream-form T. brucei. Biochemical screens using recombinant NTR demonstrated that several quinones were effective substrates for the parasite enzyme, having K(cat)/K(m) values 2 orders of magnitude greater than those of nifurtimox and benznidazole. In tests against T. brucei, antiparasitic activity mirrored the biochemical data, with the most potent compounds generally being preferred enzyme substrates. Trypanocidal activity was shown to be NTR dependent, as parasites with elevated levels of this enzyme were hypersensitive to the aziridinyl agent. By unraveling the biochemical characteristics exhibited by the trypanosomal NTRs, we have shown that quinone-based compounds represent a class of trypanocidal compound.
Assuntos
Benzoquinonas/farmacologia , Nitrorredutases/antagonistas & inibidores , Pró-Fármacos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Benzoquinonas/química , Escherichia coli/genética , Cinética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Terapia de Alvo Molecular , NAD/química , NAD/metabolismo , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Nitrorredutases/química , Nitrorredutases/metabolismo , Pró-Fármacos/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Especificidade por Substrato , Tripanossomicidas/química , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologiaRESUMO
Benznidazole is the frontline drug used against Trypanosoma cruzi, the causative agent of Chagas disease. However, treatment failures are often reported. Here, we demonstrate that independently acquired mutations in the gene encoding a mitochondrial nitroreductase (TcNTR) can give rise to distinct drug-resistant clones within a single population. Following selection of benznidazole-resistant parasites, all clones examined had lost one of the chromosomes containing the TcNTR gene. Sequence analysis of the remaining TcNTR allele revealed 3 distinct mutant genes in different resistant clones. Expression studies showed that these mutant proteins were unable to activate benznidazole. This correlated with loss of flavin mononucleotide binding. The drug-resistant phenotype could be reversed by transfection with wild-type TcNTR. These results identify TcNTR as a central player in acquired resistance to benznidazole. They also demonstrate that T. cruzi has a propensity to undergo genetic changes that can lead to drug resistance, a finding that has implications for future therapeutic strategies.
Assuntos
Resistência a Medicamentos/genética , Nitroimidazóis/farmacologia , Nitrorredutases/genética , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Alelos , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Clonagem Molecular , Regulação da Expressão Gênica , Variação Genética , Dados de Sequência Molecular , Mutação , Nitrorredutases/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Ratos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Células VeroRESUMO
Benznidazole, a 2-nitroimidazole, is the front-line treatment used against American trypanosomiasis, a parasitic infection caused by Trypanosoma cruzi. Despite nearly 40 years of use, the trypanocidal activity of this prodrug is not fully understood. It has been proposed that benznidazole activation leads to the formation of reductive metabolites that can cause a series of deleterious effects, including DNA damage and thiol depletion. Here, we show that the key step in benznidazole activation involves an NADH-dependent trypanosomal type I nitroreductase. This catalyzes an oxygen-insensitive reaction with the interaction of enzyme, reductant, and prodrug occurring through a ping-pong mechanism. Liquid chromatography/mass spectrometry (LC/MS) analysis of the resultant metabolites identified 4,5-dihydro-4,5-dihydroxyimidazole as the major product of a reductive pathway proceeding through hydroxylamine and hydroxy intermediates. The breakdown of this product released the reactive dialdehyde glyoxal, which, in the presence of guanosine, generated guanosine-glyoxal adducts. These experiments indicate that the reduction of benznidazole by type I nitroreductase activity leads to the formation of highly reactive metabolites and that the expression of this enzyme is key to the trypanocidal properties displayed by the prodrug.
Assuntos
Doença de Chagas/tratamento farmacológico , Nitroimidazóis/metabolismo , Nitrorredutases/metabolismo , Pró-Fármacos/metabolismo , Tripanossomicidas/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Biotransformação , Doença de Chagas/parasitologia , Cromatografia Líquida , Dano ao DNA/efeitos dos fármacos , Glioxal/metabolismo , Guanosina/metabolismo , Humanos , Hidroxilamina/metabolismo , Imidazóis/metabolismo , Cinética , Espectrometria de Massas , NAD/metabolismo , Nitroimidazóis/farmacologia , Pró-Fármacos/farmacologia , Proteínas de Protozoários/metabolismo , Espectrofotometria , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologiaRESUMO
A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.
Assuntos
Leishmania/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/síntese química , Nitrorredutases/metabolismo , Compostos Organofosforados/síntese química , Pró-Fármacos/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Escherichia coli/enzimologia , Humanos , Concentração Inibidora 50 , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/farmacologia , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Chagas disease and African sleeping sickness are trypanosomal infections that represent important public health problems in Latin America and Africa, respectively. The restriction of these diseases to the poorer parts of the world has meant that they have been largely neglected and limited progress has been made in their treatment. The nitroheterocyclic prodrugs nifurtimox and benznidazole, in use against Chagas disease for >40 years, remain the only agents available for this infection. In the case of African sleeping sickness, nifurtimox has recently been added to the arsenal of medicines, with the nitroheterocycle fexinidazole currently under evaluation. For a long time, the cytotoxic mechanism of these drugs was poorly understood: nifurtimox was thought to act via production of superoxide anions and nitro radicals, while the mode of benznidazole action was more obscure. The trypanocidal activity of nitroheterocyclic drugs is now known to depend on a parasite type I nitroreductase (NTR). This enzyme is absent from mammalian cells, a difference that forms the basis for the drug selectivity. The role of this enzyme in drug activation has been genetically and biochemically validated. It catalyses the 2-electron reduction of nitroheterocyclic compounds within the parasite, producing toxic metabolites without significant generation of superoxide. Recognition that this enzyme is responsible for activation of nitroheterocyclic prodrugs has allowed screening for compounds that preferentially target the parasite. This approach has led to the identification of two new classes of anti-trypanosomal agents, nitrobenzylphosphoramide mustards and aziridinyl nitrobenzamides, and promises to yield new, safer, more effective drugs.
Assuntos
Doença de Chagas/tratamento farmacológico , Nitrocompostos/farmacologia , Nitrorredutases/metabolismo , Pró-Fármacos/farmacologia , Proteínas de Protozoários/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , África , Animais , Benzamidas/síntese química , Benzamidas/farmacologia , Doença de Chagas/parasitologia , Doença de Chagas/transmissão , Humanos , Insetos Vetores/parasitologia , América Latina , Nifurtimox/síntese química , Nifurtimox/farmacologia , Nitrocompostos/síntese química , Nitroimidazóis/síntese química , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/síntese química , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/síntese química , Tripanossomicidas/síntese química , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologia , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/transmissãoRESUMO
The prodrug nifurtimox has been used for more than 40 years to treat Chagas disease and forms part of a recently approved combinational therapy that targets West African trypanosomiasis. Despite this, its mode of action is poorly understood. Detection of reactive oxygen and nitrogen intermediates in nifurtimox-treated extracts led to the proposal that this drug induces oxidative stress in the target cell. Here, we outline an alternative mechanism involving reductive activation by a eukaryotic type I nitroreductase. Several enzymes proposed to metabolize nifurtimox, including prostaglandin F2α synthase and cytochrome P450 reductase, were overexpressed in bloodstream-form Trypanosoma brucei. Only cells with elevated levels of the nitroreductase displayed altered susceptibility to this nitrofuran, implying a key role in drug action. Reduction of nifurtimox by this enzyme was shown to be insensitive to oxygen and yields a product characterized by LC/MS as an unsaturated open-chain nitrile. This metabolite was shown to inhibit both parasite and mammalian cell growth at equivalent concentrations, in marked contrast to the parental prodrug. These experiments indicate that the basis for the selectivity of nifurtimox against T. brucei lies in the expression of a parasite-encoded type I nitroreductase.