RESUMO
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.
Assuntos
Haemonchus/genética , MicroRNAs/biossíntese , RNA de Helmintos/biossíntese , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Colestenos/farmacologia , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ontologia Genética , Haemonchus/efeitos dos fármacos , Haemonchus/crescimento & desenvolvimento , Larva , Masculino , MicroRNAs/genética , RNA de Helmintos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Insulina/genética , Especificidade da EspécieRESUMO
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.
Assuntos
Anti-Helmínticos/farmacologia , Resistência a Medicamentos/genética , Expressão Gênica , MicroRNAs/genética , Nematoides/genética , Animais , Biomarcadores , Resistência a Medicamentos/fisiologia , Feminino , Células HEK293 , Haemonchus/genética , Haemonchus/metabolismo , Humanos , Ivermectina/farmacologia , MicroRNAs/metabolismo , Nematoides/metabolismo , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Micropartículas Derivadas de Células/genética , Haemonchus/genética , Interações Hospedeiro-Parasita , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Meios de Cultura , Genoma Helmíntico , Haemonchus/patogenicidade , Estágios do Ciclo de Vida , Linfa/parasitologia , Ovinos/parasitologiaRESUMO
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.
Assuntos
Regulação da Expressão Gênica/genética , Helmintíase Animal/prevenção & controle , Helmintos/genética , Interações Hospedeiro-Parasita/genética , MicroRNAs/imunologia , RNA Interferente Pequeno/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Helmintíase Animal/diagnóstico , Helmintíase Animal/genética , Helmintíase Animal/imunologia , Helmintos/imunologiaRESUMO
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.
Assuntos
Acridinas/síntese química , IMP Desidrogenase/antagonistas & inibidores , Piperazinas/síntese química , Acridinas/farmacologia , Acridinas/toxicidade , Administração Oral , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Disponibilidade Biológica , Linhagem Celular , Proliferação de Células , Trato Gastrointestinal/efeitos dos fármacos , Meia-Vida , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Macaca fascicularis , Masculino , Piperazinas/farmacologia , Piperazinas/toxicidade , Ratos , Ratos Endogâmicos Lew , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Anti-Inflamatórios/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Quinoxalinas/química , Quinoxalinas/farmacocinética , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Citocinas/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Ligação de Hidrogênio , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Pirazinas/química , Pirazinas/farmacologia , Quinoxalinas/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
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.
Assuntos
Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Indóis/farmacologia , Catálise , Cinética , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Oxazóis/química , Oxazóis/farmacologia , Animais , Carbamatos/química , Carbamatos/farmacologia , Linhagem Celular , Cricetinae , Cricetulus , Concentração Inibidora 50 , Ativação Linfocitária/efeitos dos fármacos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , Oxazóis/síntese química , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologiaRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Quinolonas/síntese química , Quinolonas/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Humanos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologiaRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Quinolonas/síntese química , Quinolonas/farmacologia , Humanos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Ligantes , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Quinoxalinas/síntese química , Quinoxalinas/farmacologia , Divisão Celular/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacosRESUMO
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.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Antivirais/síntese química , Antivirais/farmacologia , Antivirais/toxicidade , Cristalografia por Raios X , Inibidores Enzimáticos/toxicidade , Gastroenteropatias/induzido quimicamente , Gastroenteropatias/patologia , Imunossupressores/síntese química , Imunossupressores/farmacologia , Imunossupressores/toxicidade , Indicadores e Reagentes , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Guanidina/análogos & derivados , Guanidina/síntese química , IMP Desidrogenase/antagonistas & inibidores , Antivirais/síntese química , Antivirais/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Guanidina/farmacologia , Humanos , Indicadores e Reagentes , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
IMP Desidrogenase/antagonistas & inibidores , Triazinas/síntese química , Triazinas/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Triazinas/químicaRESUMO
[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.
Assuntos
Inibidores Enzimáticos/síntese química , IMP Desidrogenase/antagonistas & inibidores , Oxazóis/síntese química , Oxazóis/químicaRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , IMP Desidrogenase/antagonistas & inibidores , Morfolinas/síntese química , Ácido Micofenólico/análogos & derivados , Oxazóis/síntese química , Animais , Formação de Anticorpos/efeitos dos fármacos , Artrite Experimental/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Morfolinas/química , Morfolinas/farmacologia , Ácido Micofenólico/farmacologia , Oxazóis/química , Oxazóis/farmacologia , Ratos , Ratos Endogâmicos Lew , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Quinoxalinas/síntese química , Quinases da Família src/antagonistas & inibidores , Animais , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Cinética , Ativação Linfocitária/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Quinoxalinas/farmacologia , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
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.