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

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

Compounds identified in the screen of compounds active against the <i>BmaEcR</i>.

<p>Cells were treated with 10μM of each drug for 48 hrs. The blue bars represent all other compounds tested; 20E is represented as green bar and the red bar indicates the activity present in untreated cells. All columns represent the mean and the error bars the standard deviation in triplicate wells. The horizontal line indicates the activity expected for compounds with no activity.</p

Compounds identified in the screen of compounds active against the <i>BmaEcR</i>.

<p>Cells were treated with 10μM of each drug for 48 hrs. The blue bars represent all other compounds tested; 20E is represented as green bar and the red bar indicates the activity present in untreated cells. All columns represent the mean and the error bars the standard deviation in triplicate wells. The horizontal line indicates the activity expected for compounds with no activity.</p

EC₅₀ values and structures of compounds identified in the preliminary screen of compounds interacting with the <i>Bma</i>EcR using the cell-based luciferase assay.

<p>EC₅₀ values and structures of compounds identified in the preliminary screen of compounds interacting with the <i>Bma</i>EcR using the cell-based luciferase assay.</p

Structure and EC₅₀ values of the compounds predicted to be <i>Bma</i>EcR ligands using the <i>in-silico</i> virtual screening method.

<p>Structure and EC₅₀ values of the compounds predicted to be <i>Bma</i>EcR ligands using the <i>in-silico</i> virtual screening method.</p

XP descriptor analysis given from XP docking of hormones and agonists in <i>B</i>. <i>malayi</i> EcR-LBD binding site.

<p>XP descriptor analysis given from XP docking of hormones and agonists in <i>B</i>. <i>malayi</i> EcR-LBD binding site.</p

<i>In-vitro</i> phenotypic study demonstrating effect of Ponasterone A, Muristerone A and 20E on adult female worms.

<p>The bars depict counts of progeny expelled per ml of media. 1 and 2 represent two biological replicates of the treated and control for the expulsed progeny. The error bars denote the standard deviation of the technical replicates. The colored bars represent each consecutive day of the experiment.</p

Performance of the mammalian cell assay.

<p>Results of a typical assay carried out in a 96 well format are shown. Columns indicate the mean and error bars the standard deviation of six control (uninduced) and six experimental (induced) wells. <b>(A)</b> Assay conducted with the transiently transfected cells with 150ng per well of each construct (<i>Bma</i>EcR- GAL4, RXR-VP16, GLuc reporter) using Lipofectamine, and treated with 10μM 20-hydroxyecdysone for 48 hrs. <b>(B)</b> Assay conducted with the stably transfected cells.</p

Cartoon depiction of the <i>B</i>. <i>malayi</i> EcR LBD homology model.

<p>The secondary structural element are colored in purple. The red dotted circle denotes the active site for the docking studies.</p