RESUMEN
Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.
Asunto(s)
Haemonchus/genética , MicroARNs/biosíntesis , ARN de Helminto/biosíntesis , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Colestenos/farmacología , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Haemonchus/efectos de los fármacos , Haemonchus/crecimiento & desarrollo , Larva , Masculino , MicroARNs/genética , ARN de Helminto/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Insulina/genética , Especificidad de la EspecieRESUMEN
Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3' UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3' UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species.
Asunto(s)
Antihelmínticos/farmacología , Resistencia a Medicamentos/genética , Expresión Génica , MicroARNs/genética , Nematodos/genética , Animales , Biomarcadores , Resistencia a Medicamentos/fisiología , Femenino , Células HEK293 , Haemonchus/genética , Haemonchus/metabolismo , Humanos , Ivermectina/farmacología , MicroARNs/metabolismo , Nematodos/metabolismo , ARN Mensajero/metabolismoRESUMEN
microRNAs are small non-coding RNAs that are important regulators of gene expression in a range of animals, including nematodes. We have analysed a cluster of four miRNAs from the pathogenic nematode species Haemonchus contortus that are closely linked in the genome. We find that the cluster is conserved only in clade V parasitic nematodes and in some ascarids, but not in other clade III species nor in clade V free-living nematodes. Members of the cluster are present in parasite excretory-secretory products and can be detected in the abomasum and draining lymph nodes of infected sheep, indicating their release in vitro and in vivo. As observed for other parasitic nematodes, H. contortus adult worms release extracellular vesicles (EV). Small RNA libraries were prepared from vesicle-enriched and vesicle-depleted supernatants from both adult worms and L4 stage larvae. Comparison of the miRNA species in the different fractions indicated that specific miRNAs are packaged within vesicles, while others are more abundant in vesicle-depleted supernatant. Hierarchical clustering analysis indicated that the gut is the likely source of vesicle-associated miRNAs in the L4 stage, but not in the adult worm. These findings add to the growing body of work demonstrating that miRNAs released from parasitic helminths may play an important role in host-parasite interactions.
Asunto(s)
Micropartículas Derivadas de Células/genética , Haemonchus/genética , Interacciones Huésped-Parásitos , MicroARNs/genética , MicroARNs/metabolismo , Animales , Medios de Cultivo , Genoma de los Helmintos , Haemonchus/patogenicidad , Estadios del Ciclo de Vida , Linfa/parasitología , Ovinos/parasitologíaRESUMEN
Over the last decade microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important regulators of post-transcriptional gene expression. miRNAs are short, non-coding RNAs that regulate a variety of processes including cancer, organ development and immune function. This class of small RNAs bind with partial complementarity to their target mRNA sequences, most often in the 3'UTR, to negatively regulate gene expression. In parasitic helminths, miRNAs are being increasingly studied for their potential roles in development and host-parasite interactions. The availability of genome data, combined with small RNA sequencing, has paved the way to profile miRNAs expressed at particular developmental stages for many parasitic helminths. While some miRNAs are conserved across species, others appear to be unique to specific parasites, suggesting important roles in adaptation and survival in the host environment. Some miRNAs are released from parasites, in exosomes or in protein complexes, and the potential effects of these on host immune function are being increasingly studied. In addition, release of miRNAs from schistosome and filarial parasites into host plasma can be exploited for the development of specific and sensitive diagnostic biomarkers of infection. Interfering with miRNA function, as well as silencing key components of the pathways they regulate, will progress our understanding of parasite development and provide a novel approach to therapeutic control. RNA interference (RNAi) by siRNAs has proven to be inconsistent in parasitic nematodes. However, the recent successes reported for schistosome and liver fluke RNAi, encourage further efforts to enhance delivery of RNA and improve in vitro culture systems and assays to monitor phenotypic effects in nematodes. These improvements are important for the establishment of reliable functional genomic platforms for novel drug and vaccine development. In this review we focus on the important roles of miRNAs and siRNAs in post-transcriptional gene regulation in veterinary parasitic helminths and the potential value of these in parasite diagnosis and control.
Asunto(s)
Regulación de la Expresión Génica/genética , Helmintiasis Animal/prevención & control , Helmintos/genética , Interacciones Huésped-Parásitos/genética , MicroARNs/inmunología , ARN Interferente Pequeño/inmunología , Animales , Regulación de la Expresión Génica/inmunología , Helmintiasis Animal/diagnóstico , Helmintiasis Animal/genética , Helmintiasis Animal/inmunología , Helmintos/inmunologíaRESUMEN
Inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-5'-monophosphate to xanthosine-5'-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of mycophenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH. The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus, development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery and SAR leading to a novel and potent acridone-based IMPDH inhibitor 4m and its efficacy and GI tolerability when administered orally in a rat adjuvant arthritis model.
Asunto(s)
Acridinas/síntesis química , IMP Deshidrogenasa/antagonistas & inhibidores , Piperazinas/síntesis química , Acridinas/farmacología , Acridinas/toxicidad , Administración Oral , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Disponibilidad Biológica , Línea Celular , Proliferación Celular , Tracto Gastrointestinal/efectos de los fármacos , Semivida , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Macaca fascicularis , Masculino , Piperazinas/farmacología , Piperazinas/toxicidad , Ratas , Ratas Endogámicas Lew , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A series of novel anilino 5-azaimidazoquinoxaline analogues possessing potent in vitro activity against p56Lck and T cell proliferation have been discovered. Subsequent SAR studies led to the identification of compound 4 (BMS-279700) as an orally active lead candidate that blocks the production of proinflammatory cytokines (IL-2 and TNFalpha) in vivo. In addition, an expanded set of imidazoquinoxalines provided several descriptive QSAR models highlighting the influence of significant steric and electronic features. The H-bonding (Met319) contribution to observed binding affinities within a tightly congeneric series was found to be significant.
Asunto(s)
Antiinflamatorios/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Relación Estructura-Actividad Cuantitativa , Quinoxalinas/química , Quinoxalinas/farmacocinética , Animales , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Disponibilidad Biológica , Citocinas/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Femenino , Enlace de Hidrógeno , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Pirazinas/química , Pirazinas/farmacología , Quinoxalinas/farmacología , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.
Asunto(s)
Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Indoles/farmacología , Catálisis , Cinética , Relación Estructura-ActividadRESUMEN
The first reported structure-activity relationships (SARs) about the N-[3-methoxy-4-(5-oxazolyl)phenyl moiety for a series of recently disclosed inosine monophosphate dehydrogenase (IMPDH) inhibitors are described. The syntheses and in vitro inhibitory values for IMPDH II, and T-cell proliferation (for select analogues) are given.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Oxazoles/química , Oxazoles/farmacología , Animales , Carbamatos/química , Carbamatos/farmacología , Línea Celular , Cricetinae , Cricetulus , Concentración 50 Inhibidora , Activación de Linfocitos/efectos de los fármacos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacología , Oxazoles/síntesis química , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiologíaRESUMEN
A series of novel quinolone-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Quinolonas/síntesis química , Quinolonas/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Fenómenos Químicos , Química Física , Cristalografía por Rayos X , Humanos , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimologíaRESUMEN
The synthesis and the structure-activity relationships (SAR) of analogues derived from the introduction of basic residues on ring D of quinolone-based inhibitors of IMPDH are described. This led to the identification of compound 27 as a potent inhibitor of IMPDH with significantly improved aqueous solubility over the lead compound 1.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Quinolonas/síntesis química , Quinolonas/farmacología , Humanos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Ligandos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Modelos Moleculares , Conformación Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A series of anilino(imidazoquinoxaline) analogues bearing solubilizing side chains at the 6- and 7-positions of the fused phenyl ring has been prepared and evaluated for inhibition against Lck enzyme and of T-cell proliferation. Significant improvement of the cellular activity was achieved over the initial lead, compound 2.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Quinoxalinas/síntesis química , Quinoxalinas/farmacología , División Celular/efectos de los fármacos , Humanos , Indicadores y Reactivos , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacosRESUMEN
A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.
Asunto(s)
Amidas/síntesis química , Amidas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/farmacología , Antivirales/toxicidad , Cristalografía por Rayos X , Inhibidores Enzimáticos/toxicidad , Enfermedades Gastrointestinales/inducido químicamente , Enfermedades Gastrointestinales/patología , Inmunosupresores/síntesis química , Inmunosupresores/farmacología , Inmunosupresores/toxicidad , Indicadores y Reactivos , Relación Estructura-ActividadRESUMEN
A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Guanidina/análogos & derivados , Guanidina/síntesis química , IMP Deshidrogenasa/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Guanidina/farmacología , Humanos , Indicadores y Reactivos , Relación Estructura-ActividadRESUMEN
A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.
Asunto(s)
IMP Deshidrogenasa/antagonistas & inhibidores , Triazinas/síntesis química , Triazinas/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Concentración 50 Inhibidora , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Triazinas/químicaRESUMEN
[structure: see text] A modified approach to the synthesis of 2-(N-aryl)-1,3-oxazoles, employing an optimized iminophosphorane/heterocumulene-mediated methodology, and its application to the synthesis of BMS-337197, a potent inhibitor of IMPDH, are described.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , IMP Deshidrogenasa/antagonistas & inhibidores , Oxazoles/síntesis química , Oxazoles/químicaRESUMEN
Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , IMP Deshidrogenasa/antagonistas & inhibidores , Morfolinas/síntesis química , Ácido Micofenólico/análogos & derivados , Oxazoles/síntesis química , Animales , Formación de Anticuerpos/efectos de los fármacos , Artritis Experimental/tratamiento farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratones , Ratones Endogámicos BALB C , Morfolinas/química , Morfolinas/farmacología , Ácido Micofenólico/farmacología , Oxazoles/química , Oxazoles/farmacología , Ratas , Ratas Endogámicas Lew , Relación Estructura-ActividadRESUMEN
We have identified a novel series of 1,5-imidazoquinoxalines as inhibitors of Lck with excellent potency (IC50s<5 nM) as well as good cellular activity against T-cell proliferation (IC50s<1 microM). Structure-activity studies demonstrate the requirement for the core heterocycle in addition to an optimal 2,6-disubstituted aniline group.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Imidazoles/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Quinoxalinas/síntesis química , Familia-src Quinasas/antagonistas & inhibidores , Animales , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Cinética , Activación de Linfocitos/efectos de los fármacos , Modelos Moleculares , Conformación Molecular , Quinoxalinas/farmacología , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T/inmunologíaRESUMEN
A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is described. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are presented.