RESUMEN
Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in â¼P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.
Asunto(s)
Cisteína Endopeptidasas/fisiología , Evasión Inmune/fisiología , Macrófagos/parasitología , Proteínas Protozoarias/fisiología , Animales , Arginasa/biosíntesis , Cisteína Endopeptidasas/deficiencia , Dipéptidos/farmacología , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-12/biosíntesis , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Fenilalanina/análogos & derivados , Piperazinas , Compuestos de Tosilo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Compuestos de Vinilo/farmacologíaRESUMEN
The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Citocromo P-450 CYP3A/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Hepatocitos/metabolismo , Proteínas Nucleares/fisiología , Adenosina Trifosfatasas/genética , Animales , Células Cultivadas , Citocromo P-450 CYP3A/análisis , Hepatocitos/enzimología , Masculino , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal , Transporte de Proteínas , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drug-drug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Receptores de Citocinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Células Cultivadas , Citocromo P-450 CYP3A/genética , Retículo Endoplásmico/metabolismo , Estabilidad de Enzimas , Células HEK293 , Células Hep G2 , Hepatocitos/citología , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Immunoblotting , Hígado/citología , Hígado/enzimología , Masculino , Microscopía Fluorescente , Complejo de la Endopetidasa Proteasomal/metabolismo , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Chagas' disease, caused by infection with the parasite Trypanosoma cruzi, is the major cause of heart failure in Latin America. Classic clinical manifestations result from the infection of heart muscle cells leading to progressive cardiomyopathy. To ameliorate disease, chemotherapy must eradicate the parasite. Current drugs are ineffective and toxic, and new therapy is a critical need. To expedite drug screening for this neglected disease, we have developed and validated a cell-based, high-throughput assay that can be used with a variety of untransfected T. cruzi isolates and host cells and that simultaneously measures efficacy against the intracellular amastigote stage and toxicity to host cells. T. cruzi-infected muscle cells were incubated in 96-well plates with test compounds. Assay plates were automatically imaged and analyzed based on size differences between the DAPI (4',6-diamidino-2-phenylindole)-stained host cell nuclei and parasite kinetoplasts. A reduction in the ratio of T. cruzi per host cell provided a quantitative measure of parasite growth inhibition, while a decrease in count of the host nuclei indicated compound toxicity. The assay was used to screen a library of clinically approved drugs and identified 55 compounds with activity against T. cruzi. The flexible assay design allows the use of various parasite strains, including clinical isolates with different biological characteristics (e.g., tissue tropism and drug sensitivity), and a broad range of host cells and may even be adapted to screen for inhibitors against other intracellular pathogens. This high-throughput assay will have an important impact in antiparasitic drug discovery.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Hepatocitos/parasitología , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Músculo Esquelético/parasitología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Bovinos , Línea Celular , Línea Celular Tumoral , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Hepatocitos/citología , Hepatocitos/ultraestructura , Humanos , Músculo Esquelético/citología , Músculo Esquelético/ultraestructura , Pruebas de Sensibilidad Parasitaria , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Chagas' disease, the leading cause of heart failure in Latin America, is caused by the kinetoplastid protozoan Trypanosoma cruzi. The sterols of T. cruzi resemble those of fungi, both in composition and in biosynthesis. Azole inhibitors of sterol 14alpha-demethylase (CYP51) successfully treat fungal infections in humans, and efforts to adapt the success of antifungal azoles posaconazole and ravuconazole as second-use agents for Chagas' disease are under way. However, to address concerns about the use of azoles for Chagas' disease, including drug resistance and cost, the rational design of nonazole CYP51 inhibitors can provide promising alternative drug chemotypes. We report the curative effect of the nonazole CYP51 inhibitor LP10 in an acute mouse model of T. cruzi infection. Mice treated with an oral dose of 40 mg LP10/kg of body weight twice a day (BID) for 30 days, initiated 24 h postinfection, showed no signs of acute disease and had histologically normal tissues after 6 months. A very stringent test of cure showed that 4/5 mice had negative PCR results for T. cruzi, and parasites were amplified by hemoculture in only two treated mice. These results compare favorably with those reported for posaconazole. Electron microscopy and gas chromatography-mass spectrometry (GC-MS) analysis of sterol composition confirmed that treatment with LP10 blocked the 14alpha-demethylation step and induced breakdown of parasite cell membranes, culminating in severe ultrastructural and morphological alterations and death of the clinically relevant amastigote stage of the parasite.
Asunto(s)
Aminopiridinas/farmacología , Antiprotozoarios/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Aminopiridinas/administración & dosificación , Aminopiridinas/química , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/química , Dominio Catalítico , Enfermedad de Chagas/parasitología , Sistema Enzimático del Citocromo P-450/química , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Femenino , Humanos , Indoles/administración & dosificación , Indoles/química , Ratones , Ratones Endogámicos C3H , Microscopía Electrónica de Transmisión , Modelos Moleculares , Proteínas Protozoarias/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Esteroles/biosíntesis , Trypanosoma cruzi/ultraestructuraRESUMEN
The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.
Asunto(s)
Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Giardia lamblia/metabolismo , Lisosomas/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/ultraestructura , Endosomas/genética , Endosomas/ultraestructura , Giardia lamblia/genética , Giardia lamblia/ultraestructura , Lisosomas/genética , Lisosomas/ultraestructura , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)(2) (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-kappaB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 microM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 muM with marked suppression at >100 microM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Indeed, we found a marked (approximately 4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2alpha-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2alpha kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2alpha kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes.
Asunto(s)
Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores del Citocromo P-450 CYP3A , Hígado/enzimología , Inhibidores de Proteasoma , Proteínas Serina-Treonina Quinasas/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Células Cultivadas , Citocromo P-450 CYP3A/biosíntesis , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Activación Enzimática , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Leupeptinas/farmacología , Hígado/efectos de los fármacos , Masculino , Biosíntesis de Proteínas/efectos de los fármacos , ARN/metabolismo , Ratas , Ratas Sprague-Dawley , UbiquitinaciónRESUMEN
Selective inhibition is needed for drugs targeting the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi, etiologic agent of Chagas' disease. 6-(2,2-Dichloroacetamido)chrysene, was shown herein to be a selective inhibitor of the trypanosomal enzyme. SAR analysis revealed that the 6-amido moiety was essential, but the dichloroaceto moiety was not essential for achieving the low K(i) for this inhibitor. Understanding the molecular basis for these interactions could facilitate the design of selective inhibitors without a chrysene moiety.
Asunto(s)
Crisenos/química , Inhibidores Enzimáticos/química , Hipoxantina Fosforribosiltransferasa/química , Tripanocidas/química , Trypanosoma cruzi/química , Animales , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Unión ProteicaRESUMEN
A phenotypic high-throughput screen using â¼100,000 compounds prepared using Diversity-Oriented Synthesis yielded stereoisomeric compounds with nanomolar growth-inhibition activity against the parasite Trypanosoma cruzi, the etiological agent of Chagas disease. After evaluating stereochemical dependence on solubility, plasma protein binding and microsomal stability, the SSS analogue (5) was chosen for structure-activity relationship studies. The p-phenoxy benzyl group appended to the secondary amine could be replaced with halobenzyl groups without loss in potency. The exocyclic primary alcohol is not needed for activity but the isonicotinamide substructure is required for activity. Most importantly, these compounds are trypanocidal and hence are attractive as drug leads for both acute and chronic stages of Chagas disease. Analogue (5) was nominated as the molecular libraries probe ML341 and is available through the Molecular Libraries Probe Production Centers Network.
RESUMEN
Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas' disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite's replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection.
Asunto(s)
Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/parasitología , Interacciones Huésped-Parásitos , Trypanosoma cruzi/crecimiento & desarrollo , Animales , Enfermedad de Chagas/enzimología , Enfermedad de Chagas/genética , Metabolismo Energético , Células HeLa , Humanos , Ratones , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , Trypanosoma cruzi/fisiologíaRESUMEN
BACKGROUND: Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority. METHODOLOGY/PRINCIPAL FINDINGS: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of â¼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM. CONCLUSIONS/SIGNIFICANCE: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/aislamiento & purificación , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450 , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Simulación de Dinámica Molecular , Pruebas de Sensibilidad ParasitariaRESUMEN
Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (â¼10(11) viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January.
Asunto(s)
Abejas/microbiología , Abejas/virología , Crithidia/genética , Metagenoma , Nosema/genética , Estaciones del Año , Agricultura , Migración Animal , Animales , Crithidia/fisiología , Datos de Secuencia Molecular , Nosema/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia , Factores de TiempoRESUMEN
The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania/efectos de los fármacos , Leishmaniasis/parasitología , Animales , Antiprotozoarios/química , Línea Celular Tumoral , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Leishmania/crecimiento & desarrollo , Estadios del Ciclo de Vida/efectos de los fármacos , Macrófagos/parasitología , Naloxona/análogos & derivados , Naloxona/química , Naloxona/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique â¼ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.
Asunto(s)
Antiparasitarios/aislamiento & purificación , Antiparasitarios/metabolismo , Proteasas de Cisteína/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Proteasas/metabolismo , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Biolistics of the flatworm parasite Schistosoma mansoni facilitates the accurate spatial expression of transgenes under the control of gene-specific promoter elements. To improve transgene expression, either in the number of positive worms and/or an increased transgene signal per worm, we tested plasmid constructs incorporating 5' and 3' gene-specific genomic fragments, and parts of the open reading frame for two S. mansoni proteases, cathepsins F and D (SmCF and SmCD). GFP-expression was gut-localized, a novel finding for SmCD and consistent with previous data for SmCF. The mCherry fluorescent protein can also operate as a reporter. Though certain constructs imparted stronger and better distributed signals per positive worm, the low yields throughout (1-5% positive per experiment) precluded further quantifications of improvement. Electroporation of the same constructs was also weakly efficient (1-10% positives per experiment). However, reporter signals were found in tissues other than the gut, which may represent dysregulated transcription.
Asunto(s)
Genes Reporteros , Proteínas del Helminto/genética , Péptido Hidrolasas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Schistosoma mansoni/genética , Transformación Genética , Animales , Biolística , Catepsina D/genética , Catepsina D/metabolismo , Catepsina F/genética , Catepsina F/metabolismo , Regulación de la Expresión Génica , Proteínas del Helminto/metabolismo , Péptido Hidrolasas/metabolismo , Schistosoma mansoni/metabolismoRESUMEN
A century after discovering that the Trypanosoma cruzi parasite is the etiological agent of Chagas disease, treatment is still plagued by limited efficacy, toxicity, and the emergence of drug resistance. The development of inhibitors of the major T. cruzi cysteine protease, cruzain, has been demonstrated to be a promising drug discovery avenue for this neglected disease. Here we establish that a nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitor substantially ameliorates symptoms of acute Chagas disease in a mouse model with no apparent toxicity. A high-resolution crystal structure confirmed the mode of inhibition and revealed key binding interactions of this novel inhibitor class. Subsequent structure-guided optimization then resulted in inhibitor analogues with improvements in potency despite minimal or no additions in molecular weight. Evaluation of the analogues in cell culture showed enhanced activity. These results suggest that nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors have the potential to fulfill the urgent need for improved Chagas disease chemotherapy.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Cetonas/síntesis química , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/síntesis química , Animales , Bovinos , Células Cultivadas , Cisteína Endopeptidasas , Femenino , Cetonas/química , Cetonas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Ratones Endogámicos C3H , Modelos Moleculares , Pruebas de Sensibilidad Parasitaria , Quinolinas/síntesis química , Quinolinas/química , Quinolinas/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Triazoles/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Lysosomes were first described as vacuolar structures containing various hydrolytic enzymes at acidic pH. Subsequent studies revealed that the lysosome/vacuolar system is complex and composed of distinct membrane-enclosed vesicles including endosomes, primary and mature lysosomes, autophagic vesicles, residual bodies, multivesicular bodies, and digestive lysosomes. Lysosomes express a battery of hydrolytic enzymes including proteases, acid phosphatases, glycosidases, and lipases. Parasitic protozoa also possess complex intracellular lysosomes/endosomes/vesicles involved in digestion, transport and recycling of molecules similar to those of mammalian cells. Unique characteristics are ascribed to lysosomes of different parasites and may even differ between parasite stages. Transport of hydrolases and proteins to parasite lysosomes is directed either from the Golgi complex via endosomal vesicles or from endocytic vesicles originated in the cell surface. Inhibition of lysosomal proteases demonstrated that different proteolytic machineries catabolize distinct classes of proteins, and this selectivity may be exploited for the development of effective antiparasitic drugs. This review describes lysosomal molecules that are either validated or potential drug targets for Chagas' disease, sleeping sickness, leishmaniasis, toxoplasmosis, malaria, amebiasis, and giardiasis.
Asunto(s)
Eucariontes/efectos de los fármacos , Lisosomas/efectos de los fármacos , Infecciones por Protozoos/tratamiento farmacológico , Animales , Antiprotozoarios/farmacología , Sistemas de Liberación de Medicamentos , Eucariontes/metabolismo , Humanos , Lisosomas/enzimología , Lisosomas/metabolismoRESUMEN
Chagas' disease, caused by the parasite Trypanosoma cruzi, remains the leading cause of cardiopathy in Latin America with about 12 million people infected. Classic clinical manifestations derive from infection of muscle cells leading to progressive cardiomyopathy, while some patients develop megacolon or megaesophagus. A very aggressive clinical course including fulminant meningoencephalitis has been reported in patients who contract Chagas' disease in the background of immunodeficiency. This includes patients with human immunodeficiency virus infection as well as patients receiving immunosuppressive therapy for organ transplant. Currently, only two drugs are approved for the treatment of Chagas' disease, nifurtimox and benznidazole. Both have significant limitations due to common and serious side effects as well as limited availability. A promising group of new drug leads for Chagas' disease is cysteine protease inhibitors targeting cruzain, the major protease of T. cruzi. The inhibitor N-methyl-Pip-F-homoF-vinyl sulfonyl phenyl (N-methyl-Pip-F-hF-VS phi) is in late-stage preclinical development. Therefore, the question arose as to whether protease inhibitors targeting cruzain would have efficacy in Chagas' disease occurring in the background of immunodeficiency. To address this question, we studied the course of infection in recombinase-deficient (Rag1(-/-)) and normal mice infected with T. cruzi. Infections localized to heart and skeletal muscle in untreated normal animals, while untreated Rag1(-/-) mice showed severe infection in all organs and predominantly in liver and spleen. Treatment with the dipeptide N-methyl-Pip-F-hF-VS phi rescued immunodeficient animals from lethal Chagas' infection. The majority (60 to 100%) of inhibitor-treated Rag1(-/-) mice had increased survival, negative PCR, and normal tissues by histopathological examination.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Homeodominio/genética , Trypanosoma cruzi/efectos de los fármacos , Animales , Bovinos , Células Cultivadas , Enfermedad de Chagas/genética , Enfermedad de Chagas/patología , Cisteína Endopeptidasas , Modelos Animales de Enfermedad , Femenino , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Nifurtimox/farmacología , Nitroimidazoles/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Análisis de Supervivencia , Tripanocidas/farmacologíaRESUMEN
Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli beta-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.