Transcription‐inducing activity of natural and synthetic juvenile hormone agonists through the Drosophila Methoprene‐tolerant protein

Abstract

BACKGROUND: Juvenile hormones (JHs) are a class of sesquiterpenoids that play a pivotal role in insect growth and reproduction. Synthetic JH agonists (JHAs), including pyriproxyfen, have been widely used as insecticides to control agricultural pests and disease vectors. The antimetamorphic action of JHAs is mediated by their intracellular receptor, the heterodimer of Methoprene-tolerant (Met) and Taiman (Tai) proteins. Although a range of bioassay systems has been developed to detect the activity of JHAs, each of these systems has its own drawback(s), such as poor reproducibility, the use of radioactive ligands or the effect of endogenous JH-signaling factors.RESULTS: To address these issues, we constructed a new luciferase reporter assay for JHAs in mammalian HEK293T cells transiently transfected with the Drosophila Met and Tai genes. This reporter system gave highly reproducible results and showed nanomolar sensitivity to natural JHs. We then applied this reporter system to a structure-activity relationship (SAR) analysis of 14 natural and synthetic JHAs, leading to identification of the ligand structural factors important for the transcriptioninducing activity.CONCLUSION: Because this reporter system is not affected by the signaling cascade downstream of the JH receptors, it is suitable for evaluating the intrinsic activity of JHAs. The SAR results obtained in this study therefore provide invaluable information on the rational design of novel JHA insecticides.

Reporter gene assays for screening and identification of novel molting hormone- and juvenile hormone-like chemicals

“…The strategy of constructing reporter plasmids is similar to that of constructing RGAs for EcR-USP: DNA fragments containing JHRE identified in genes such as Kr-h1, juvenile hormone esterase (jhe), and early trypsin (ET) were integrated upstream of basal promoters (Table 2). 27,28,[105][106][107][114][115][116][117] Ligand-bound Met heterodimerizes with SRC and binds to JHRE in order to transactivate reporter gene expression ( Fig. 5a).…”

“…5b), insect Kr-h1 JHRE-regulated transactivation was successfully reconstituted using mammalian cells in three studies (Table 2). 105,117,118) Some of these RGAs indicated doesdependent responses against various juvenoids, such as JH III, methyl farnesoate, methoprene, pyriproxyfen, and fenoxy- Abbreviations JHR: juvenile hormone receptor, Aa: Aedes aegypti; Bm: Bombyx mori; Cf: Choristoneura fumiferana; Dm: Drosophila melanogaster; Tc: Tribolium castaneum; Dapma: Daphnia magna; Dappu: Daphnia pulex; jhe: juvenile hormone esterase; ET: early trypsin; MFBS: Met-FISC binding site * Reporter gene assays in insect cells expressing endogenous JHRs and a transcriptional coactivator. Only the reporter plasmid was introduced.…”

“…carb. 27,105,109,117,119,120) RGAs for Met from Daphnia species also have been established. 109,119,120) D. pulex Met exhibited dose-dependent responses against several juvenoids via T. castaneum Kr-h1 JHRE, 118) while the Kr-h1 homolog of D. pulex was not regulated by JH-Met signaling.…”

“…28) Yokoi et al investigated the SAR of 14 natural and synthetic JHAs using their newly established RGA in HEK293T cells. 117) Unlike synthetic EcR ligands, JHAs with potent insect selectivities have not yet been developed. 121) However, a mutational analysis of the PAS-B domain identified two amino acid residues crucial for the ligand selectivity of Met proteins between insects and daphnids.…”

New ways and new hopes for IGR development

“…Importantly, selected compounds were validated in a JHRE-based reporter assay in a human cell line expressing the Drosophila JH receptor proteins Met and Tai. 28) One hit proved to activate the reporter with a sub-micromolar potency, providing a lead compound for further development. Interestingly, this piperazin-based compound is predicted to form a hydrogen bond with a conserved tyrosine residue, previously implicated in forming a hydrogen bond with the epoxide moiety of JH III within the ligand-binding pocket of the other Drosophila JH receptor, Gce.…”