RESUMO
Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Endossomos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Semicarbazonas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Aminas , Animais , Transporte Biológico/fisiologia , Caspase 1/metabolismo , Cromatografia Líquida , Endossomos/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , Macrófagos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Estrutura Molecular , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Semicarbazonas/química , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
There is a high unmet need for early detection approaches for diffuse gastric cancer (DGC). We examined whether the stool proteome of mouse models of gastric cancer (GC) and individuals with hereditary diffuse gastric cancer (HDGC) have utility as biomarkers for early detection. Proteomic mass spectrometry of the stool of a genetically engineered mouse model driven by oncogenic KrasG12D and loss of p53 and Cdh1 in gastric parietal cells [known as Triple Conditional (TCON) mice] identified differentially abundant proteins compared with littermate controls. Immunoblot assays validated a panel of proteins, including actinin alpha 4 (ACTN4), N-acylsphingosine amidohydrolase 2 (ASAH2), dipeptidyl peptidase 4 (DPP4), and valosin-containing protein (VCP), as enriched in TCON stool compared with littermate control stool. Immunofluorescence analysis of these proteins in TCON stomach sections revealed increased protein expression compared with littermate controls. Proteomic mass spectrometry of stool obtained from patients with HDGC with CDH1 mutations identified increased expression of ASAH2, DPP4, VCP, lactotransferrin (LTF), and tropomyosin-2 relative to stool from healthy sex- and age-matched donors. Chemical inhibition of ASAH2 using C6 urea ceramide was toxic to GC cell lines and GC patient-derived organoids. This toxicity was reversed by adding downstream products of the S1P synthesis pathway, which suggested a dependency on ASAH2 activity in GC. An exploratory analysis of the HDGC stool microbiome identified features that correlated with patient tumors. Herein, we provide evidence supporting the potential of analyzing stool biomarkers for the early detection of DGC. Prevention Relevance: This study highlights a novel panel of stool protein biomarkers that correlate with the presence of DGC and has potential use as early detection to improve clinical outcomes.
Assuntos
Biomarcadores Tumorais , Detecção Precoce de Câncer , Fezes , Proteômica , Neoplasias Gástricas , Fezes/química , Fezes/microbiologia , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Animais , Humanos , Camundongos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/genética , Detecção Precoce de Câncer/métodos , Feminino , Proteômica/métodos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas/métodos , Modelos Animais de DoençasRESUMO
Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT), as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6) background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36-74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT.
Assuntos
Alelos , Antraz/genética , Bacillus anthracis , Cromossomos de Mamíferos/genética , Variação Genética , Imunidade Inata/genética , Animais , Antraz/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Cromossomos de Mamíferos/imunologia , Inflamação/genética , Inflamação/imunologia , Camundongos , Locos de Características Quantitativas/imunologiaRESUMO
Anthrax is caused by Bacillus anthracis and can result in nearly 100% mortality due in part to anthrax toxin. Antimalarial amodiaquine (AQ) acts as a host-oriented inhibitor of anthrax toxin endocytosis. Here, we determined the pharmacokinetics and safety of AQ in mice, rabbits, and humans as well as the efficacy in the fly, mouse, and rabbit models of anthrax infection. In the therapeutic-intervention studies, AQ nearly doubled the survival of mice infected subcutaneously with a B. anthracis dose lethal to 60% of the animals (LD60). In rabbits challenged with 200 LD50 of aerosolized B. anthracis, AQ as a monotherapy delayed death, doubled the survival rate of infected animals that received a suboptimal amount of antibacterial levofloxacin, and reduced bacteremia and toxemia in tissues. Surprisingly, the anthrax efficacy of AQ relies on an additional host macrophage-directed antibacterial mechanism, which was validated in the toxin-independent Drosophila model of Bacillus infection. Lastly, a systematic literature review of the safety and pharmacokinetics of AQ in humans from over 2â¯000 published articles revealed that AQ is likely safe when taken as prescribed, and its pharmacokinetics predicts anthrax efficacy in humans. Our results support the future examination of AQ as adjunctive therapy for the prophylactic anthrax treatment.
Assuntos
Antraz , Bacillus anthracis , Amodiaquina , Animais , Antraz/tratamento farmacológico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Levofloxacino , Camundongos , Coelhos , Revisões Sistemáticas como AssuntoRESUMO
Inflammasomes activate caspase-1 in response to molecular signals from pathogens and other dangerous stimuli as a part of the innate immune response. A previous study discovered a small-molecule, 4-fluoro- N'-[1-(2-pyridinyl)ethylidene]benzohydrazide, which we named DN1, that reduces the cytotoxicity of anthrax lethal toxin (LT). We determined that DN1 protected cells irrespectively of LT concentration and reduced the pathogenicity of an additional bacterial exotoxin and several viruses. Using the LT cytotoxicity pathway, we show that DN1 does not prevent LT internalization and catalytic activity or caspase-1 activation. Moreover, DN1 does not affect the proteolytic activity of host cathepsin B, which facilitates the cytoplasmic entry of toxins. PubChem Bioactivities lists two G protein-coupled receptors (GPCR), type-1 angiotensin II receptor and apelin receptor, as targets of DN1. The inhibition of phosphatidylinositol 3-kinase, phospholipase C, and protein kinase B, which are downstream of GPCR signaling, synergized with DN1 in protecting cells from LT. We hypothesize that DN1-mediated antagonism of GPCRs modulates signal transduction pathways to induce a cellular state that reduces LT-induced pyroptosis downstream of caspase-1 activation. DN1 also reduced the susceptibility of Drosophila melanogaster to toxin-associated bacterial infections. Future experiments will aim to further characterize how DN1 modulates signal transduction pathways to inhibit pyroptotic cell death in LT-sensitive macrophages. DN1 represents a novel chemical probe to investigate host cellular mechanisms that mediate cell death in response to pathogenic agents.
Assuntos
Antraz/fisiopatologia , Antibacterianos/farmacologia , Antígenos de Bactérias/toxicidade , Bacillus anthracis/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Morte Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Antraz/tratamento farmacológico , Antraz/metabolismo , Antraz/microbiologia , Antibacterianos/química , Bacillus anthracis/genética , Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Caspase 1/genética , Caspase 1/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Drosophila melanogaster , Feminino , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Exploiting the host endocytic trafficking pathway is a common mechanism by which bacterial exotoxins gain entry to exert virulent effects upon the host cells. A previous study identified a small-molecule, 1-(2,6-dimethyl-1-piperidinyl)-3-[(2-isopropyl-5-methylcyclohexyl)oxy]-2-propanol, that blocks the process of anthrax lethal toxin (LT) cytotoxicity. Here, we report the characterization of the bioactivity of this compound, which we named RC1. We found that RC1 protected host cells independently of LT concentration and also blocked intoxication by other bacterial exotoxins, suggesting that the target of the compound is a host factor. Using the anthrax LT intoxication pathway as a reference, we show that while anthrax toxin is able to bind to cells and establish an endosomal pore in the presence of the drug, the toxin is unable to translocate into the cytosol. We demonstrate that RC1 does not inhibit the toxin directly but rather reduces the enzymatic activity of host cathepsin B that mediates the escape of toxins into the cytoplasm from late endosomes. We demonstrate that the pathogenicity of Human cytomegalovirus and Herpes simplex virus 1, which relies on cathepsin B protease activity, is reduced by RC1. This study reveals the potential of RC1 as a broad-spectrum host-oriented therapy against several aggressive and deadly pathogens.
Assuntos
Antídotos/farmacologia , Antivirais/farmacologia , Catepsina B/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Piperidinas/farmacologia , Animais , Toxinas Bacterianas/antagonistas & inibidores , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/crescimento & desenvolvimento , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , CamundongosRESUMO
The current anthrax vaccine requires improvements for rapidly invoking longer-lasting neutralizing antibody responses with fewer doses from a well-defined formulation. Designing antigens that target neutralizing antibody epitopes of anthrax protective antigen, a component of anthrax toxin, may offer a solution for achieving a vaccine that can induce strong and long lasting antibody responses with fewer boosters. Here we report implementation of a strategy for developing epitope focused virus nanoparticle vaccines against anthrax by using immunogenic virus particles to present peptides derived from anthrax toxin previously identified in (1) neutralizing antibody epitope mapping studies, (2) toxin crystal structure analyses to identify functional regions, and (3) toxin mutational analyses. We successfully expressed two of three peptide epitopes from anthrax toxin that, in previous reports, bound antibodies that were partially neutralizing against toxin activity, discovered cross-reactivity between vaccine constructs and toxin specific antibodies raised in goats against native toxin and showed that antibodies induced by our vaccine constructs also cross-react with native toxin. While protection against intoxication in cellular and animal studies were not as effective as in previous studies, partial toxin neutralization was observed in animals, demonstrating the feasibility of using plant-virus nanoparticles as a platform for epitope defined anthrax vaccines.
Assuntos
Vacinas contra Antraz/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Portadores de Fármacos , Epitopos/imunologia , Vírus do Mosaico do Tabaco/genética , Animais , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Reações Cruzadas , Epitopos/genética , Feminino , Vetores Genéticos , Cabras , Camundongos Endogâmicos C57BL , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
A longstanding and still-increasing threat to the effective treatment of infectious diseases is resistance to antimicrobial countermeasures. Potentially, the targeting of host proteins and pathways essential for the detrimental effects of pathogens offers an approach that may discover broad-spectrum anti-pathogen countermeasures and circumvent the effects of pathogen mutations leading to resistance. Here we report implementation of a strategy for discovering broad-spectrum host-oriented therapies against multiple pathogenic agents by multiplex screening of drugs for protection against the detrimental effects of multiple pathogens, identification of host cell pathways inhibited by the drug, and screening for effects of the agent on other pathogens exploiting the same pathway. We show that a clinically used antimalarial drug, Amodiaquine, discovered by this strategy, protects host cells against infection by multiple toxins and viruses by inhibiting host cathepsin B. Our results reveal the practicality of discovering broadly acting anti-pathogen countermeasures that target host proteins exploited by pathogens.
Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Vírus/efeitos dos fármacos , Amodiaquina/química , Amodiaquina/farmacologia , Animais , Catepsina B/metabolismo , Morte Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Aprovação de Drogas , Ebolavirus/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células HeLa , Humanos , Metaboloma/efeitos dos fármacos , Camundongos , Modelos Biológicos , Células RAW 264.7 , Estados Unidos , United States Food and Drug AdministrationRESUMO
EGA, 1, prevents the entry of multiple viruses and bacterial toxins into mammalian cells by inhibiting vesicular trafficking. The cellular target of 1 is unknown, and a structure-activity relationship study was conducted in order to develop a strategy for target identification. A compound with midnanomolar potency was identified (2), and three photoaffinity labels were synthesized (3-5). For this series, the expected photochemistry of the phenyl azide moiety is a more important factor than the IC50 of the photoprobe in obtaining a successful photolabeling event. While 3 was the most effective reversible inhibitor of the series, it provided no protection to cells against anthrax lethal toxin (LT) following UV irradiation. Conversely, 5, which possessed weak bioactivity in the standard assay, conferred robust irreversible protection vs LT to cells upon UV photolysis.