Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections

Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites

Phenotypic and Molecular Analysis of the Effect of 20-hydroxyecdysone on the Human Filarial Parasite Brugia malayi

A homologue of the ecdysone receptor has been identified and shown to be responsive to 20- hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B. malayi cultured in the presence of 20-hydroxyecdysone produced significantly more microfilariae and abortive immature progeny than control worms, implicating the ecdysone receptor in regulation of embryogenesis and microfilarial development. Transcriptome analyses identified 30 genes whose expression was significantly up-regulated in 20-hydroxyecdysone-treated parasites compared with untreated controls. Of these, 18% were identified to be regulating transcription. A comparative proteomic analysis revealed 932 proteins to be present in greater amounts in extracts of 20- hydroxyecdysone-treated adult females than in extracts prepared from worms cultured in the absence of the hormone. Of the proteins exhibiting a greater than two-fold difference in the 20- hydroxyecdysone-treated versus untreated parasite extracts, 16% were involved in transcriptional regulation. RNA interference (RNAi) phenotype analysis of Caenorhabditis elegans orthologs revealed that phenotypes involved in developmental processes associated with embryogenesis were significantly over-represented in the transcripts and proteins that were up-regulated by exposure to 20-hydroxyecdysone. Taken together, the transcriptomic, proteomic and phenotypic data suggest that the filarial ecdysone receptor may play a role analogous to that in insects, where it serves as a regulator of egg development

The genome of Onchocerca volvulus, agent of river blindness

Human onchocerciasis is a serious neglected tropical disease caused by the filarial nematode Onchocerca volvulus that can lead to blindness and chronic disability. Control of the disease relies largely on mass administration of a single drug, and the development of new drugs and vaccines depends on a better knowledge of parasite biology. Here, we describe the chromosomes of O. volvulus and its Wolbachia endosymbiont. We provide the highest-quality sequence assembly for any parasitic nematode to date, giving a glimpse into the evolution of filarial parasite chromosomes and proteomes. This resource was used to investigate gene families with key functions that could be potentially exploited as targets for future drugs. Using metabolic reconstruction of the nematode and its endosymbiont, we identified enzymes that are likely to be essential for O. volvulus viability. In addition, we have generated a list of proteins that could be targeted by Federal-Drug-Agency-approved but repurposed drugs, providing starting points for anti-onchocerciasis drug development

Molecular and Phenotypic Studies Validating the Role of the Ecdysone Receptor in the Human Parasite \u3ci\u3eBrugia malayi\u3c/i\u3e

Filariasis and onchocerciasis are debilitating diseases affecting 120 million people globally. The massive socio-economic impact of these diseases energized the international community to declare a goal of eliminating filariasis 2020. This resulted in a dramatic increase in the efforts to eliminate filariasis and onchocerciasis, employing a strategy of mass drug administration (MDA). However, these programs rely upon the small arsenal of drugs. This leaves these programs vulnerable to failure in the face of developing resistance and local intolerance to the current drug regimens. Thus, new drugs against these infections are critically needed. A homologue of the ecdysone receptor (EcR), a master regulator of development in insects, has been identified in B. malayi. The potential of the EcR as a drug target has been underscored by work in the agricultural industry, where insecticides targeting the ecdysone developmental pathway are effective and non-toxic to non-target species. As the EcR is absent in humans, it represents an attractive potential chemotherapeutic target. The first study investigates the hypothesis that the ecdysone receptor controls the embryogenesis and molting in the filarial parasite. In-vitro embryogram and in-vivo phenotypic studies were conducted to delineate the effect of 20-hydroxyecdysone on the Brugia malayi parasites. The results suggest that the hormone accelerates embryogenesis and causes precocious molts, resulting in the death of the parasite. Further, transcriptomic and proteomic analysis of the ecdysone treated worms provided evidence that the up-regulated genes participate in embryogenesis. Based upon the validation of the ecdysone receptor as a potential drug target, subsequent studies focused on the development of a drug discovery model to screen for agonists and antagonists of the B. malayi ecdysone receptor. A stable cell line was created to aid the high throughput screening to rapidly identity agonist and antagonist compounds. A total of 7 agonists and 2 antagonists were identified. A homology model of the BmEcR ligand-binding domain was created as an alternate method for virtual screening of small molecules as well as to study the ligand-receptor interactions. The hits identified with the assay were docked in the active site of the BmEcR homology model providing an excellent correspondence of data between the molecular assay and the virtual screening method

Development of a toolkit for <i>piggyBac</i>-mediated integrative transfection of the human filarial parasite <i>Brugia malayi</i>

<div><p>Background</p><p>The human filarial parasites cause diseases that are among the most important causes of morbidity in the developing world. The elimination programs targeting these infections rely on a limited number of drugs, making the identification of new chemotherapeutic agents a high priority. The study of these parasites has lagged due to the lack of reverse genetic methods.</p><p>Methodology/Principal findings</p><p>We report a novel co-culture method that results in developmentally competent infective larvae of one of the human filarial parasites (<i>Brugia malayi</i>) and describe a method to efficiently transfect the larval stages of this parasite. We describe the production of constructs that result in integrative transfection using the <i>piggyBac</i> transposon system, and a selectable marker that can be used to identify transgenic parasites. We describe the production and use of dual reporter plasmids containing both a secreted luciferase selectable marker and fluorescent protein reporters that will be useful to study temporal and spatial patterns of gene expression.</p><p>Conclusions/Significance</p><p>The methods and constructs reported here will permit the efficient production of integrated transgenic filarial parasite lines, allowing reverse genetic technologies to be applied to all life cycle stages of the parasite.</p></div

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections

BACKGROUND: A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. METHODOLOGY/ PRINCIPAL FINDINGS: Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. CONCLUSIONS: The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites

Maps of plasmid constructs used in this study.

<p>ITR = inverted terminal repeat; HPRO = <i>BmHSP70</i> promoter; GX1 = <i>Gaussia</i> luciferase ORF exon 1; HINT = <i>BmHSP70</i> intron 1; GX2 = <i>Gaussia</i> luciferase ORF exon 2; HUTR = <i>BmHSP70 3' UTR;</i> MCS = multiple cloning site (<i>Bcl</i> 1—<i>Bgl</i> II—<i>Mlu</i> 1—<i>Cla</i> 1—<i>Sma</i> 1—<i>Aat</i> II—<i>Nde</i> I—<i>Bcl</i> II); TRASNP = <i>piggyBac</i> transposase ORF; RPRO = <i>Bmrps12</i> promoter; RUTR = <i>Bmrps12</i> 3' UTR; GFP = Green Fluorescent Protein ORF; YFP = Yellow Fluorescent Protein ORF; CHR = Cherry Red ORF.</p

Location of <i>piggyBac</i> insertions in genomes of individual microfilaria.

<p>Location of <i>piggyBac</i> insertions in genomes of individual microfilaria.</p

Secreted GLuc activity in cultures of individual microfilaria derived from adult parasites transfected in the L3-L4 stage.

<p>Bars represent the number of individual microfilariae producing secreted GLuc activity in the range shown. Note that the y-axis is logarithmic.</p

Secreted luciferase activity and fluorescent signal on L3 transfected with dual reporter plasmids.

<p>L3 were transfected with pBACII-BmGluc-MCS, pBACII-BmGluc-GFP, pBACII-BmGluc-YFP, or pBACII-BmGluc-CHR as described in the text. (A) Secreted luciferase activity in cultures of transfected larvae. The arrow indicates the day upon which molting was induced. Control = L3 transfected with pBACII-BmGluc-MCS, GFP = L3 transfected with pBACII-BmGluc-GFP, YFP = L3 transfected with pBACII-BmGluc-YFP and CHR = L3 transfected with pBACII-BmGluc-CHR. (B) Brightfield image of transfected parasites on day 8. (C) GFP channel florescence in the L3 transfected with pBACII-BmGluc-MCS. (D) GFP channel florescence in the L3 transfected with pBACII-BmGluc-GFP. (E) YFP channel florescence in the L3 transfected with pBACII-BmGluc-MCS. (F) YFP channel florescence in the L3 transfected with pBACII-BmGluc-YFP. (G) CHR channel florescence in the L3 transfected with pBACII-BmGluc-MCS. (H) CHR channel florescence in the L3 transfected with pBACII-BmGluc-CHR. In panels B-H bar = 300nm.</p