Identification of novel juvenile-hormone signaling activators via high-throughput screening with a chemical library

Abstract

Juvenile hormone (JH) is an insect-specific hormone that regulates molting and metamorphosis. Hence, JH signaling inhibitors (JHSIs) and activators (JHSAs) can be used as effective insect growth regulators (IGRs) for pest management. In our previous study, we established a high-throughput screening (HTS) system for exploration of novel JHSIs and JHSAs using a Bombyx mori cell line (BmN_JF&AR cells) and succeeded in identifying novel JHSIs from a chemical library. Here, we searched for novel JHSAs using this system. The four-step HTS yielded 10 compounds as candidate JHSAs; some of these compounds showed novel basic structures, whereas the others were composed of a 4-phenoxyphenoxymethyl skeleton, the basic structure of several existing JH analogs (pyriproxyfen and fenoxycarb). Topical application of seven compounds to B. mori larvae significantly prolonged the larval period, suggesting that the identified JHSAs may be promising IGRs targeting the JH signaling pathway.

Keywords: high-throughput screening; insect growth regulator; juvenile hormone; juvenile hormone signaling activator; pest management.

Fig. 1. JHSA screening system and scheme of high-throughput screening (HTS). (A) Schematic JHSA assay using BmN_JF&AR cells. Increased reporter activity observed by adding a test compound indicated that the compound had JHSA activity.¹⁸⁾ Met, JH receptor (methoprene tolerant); SRC, steroid receptor coactivator; JHREP, JH response element (kJHRE) and the basal promoter region of α isoform of B. mori Krüppel homolog 1 (BmKr-h1α); Luc2P, luciferase reporter gene containing the first degradation sequence (PEST); Lumi, luminescence. The median effective concentration (EC₅₀) of JH I was 3.7×10⁻¹⁰ M.¹⁸⁾ (B) Flow chart of HTS to identify JHSAs from the DDI chemical library using BmN_JF&AR cells. The DDI chemical library possessed 218,000 compounds in total, and the core library (9600 compounds) was composed of structurally diverse compounds for random screening. The core library was used from the first through third screenings, and analogs of the fourth screening were selected from another approximately 210,000 compounds based on the chemical structure of compounds screened in the third screening. The hit compounds selected by the four-step HTS using BmN_JF&AR cells were evaluated by topical application in B. mori larvae using in vivo assays. Adapted from Kayukawa et al.¹⁸⁾

Fig. 2. Scatter plots of HTS. JHSA activities were examined using the Luciferase reporter assay system in BmN_JF&AR cells. The JHSA activities of compounds in BmN_JF&AR cells were determined using the activation rate (AR [%]), where AR 0% and 100% indicated no activity and maximum JHSA activity, respectively. Gray dots are wells containing a test compound. The red and blue dots indicate positive (JH I, 10 nM) and negative (only DMSO) controls, respectively. Plate layouts are described in Supplemental Fig. S1. (A) The core library (9600 compounds, 5 µM) was screened in the first screening (n=1). The green shadow indicates a plate consisting of 320 compounds, with 16 positive and 16 negative control wells, and the lower plot shows an enlarged image of a plate as an example. (B) The activities of the compounds obtained by the first screening were confirmed in the second screening (confirmation test, n=4). CT, compounds in the confirmation test. (C) The dose–dependent responses of compounds selected by the second screening were examined in the third screening (n=4). The green shadow focuses on a compound, and the dose-dependent response is indicated below. (D) Analogs of the selected compounds in the third screening were evaluated in the fourth screening (analog and dose-response tests, n=3).

Fig. 3. Hit compounds from HTS using BmN_JF&AR cells. (A) The heat map represents the results of the fourth screening. Compounds in the fourth screening were assigned numbers (analog and dose-response test [ADT] no., ADTX-Y). X numbers (large and left of the heat map column) showed the compound groups, and Y numbers (small and left of the heat map column) show each analog in the compound group; the top line for each group (ADTX-1) shows the compound hits identified during the third screening, and the subsequent lines (ADTX-2, -3, -4, etc.) show analogs of ADTX-1. Heat map columns indicate means of AR (%) in fourth screening, and red (100%) and black (0%) areas indicate JHSA activities of 10 nM JH I (positive control) and DMSO alone (negative control), respectively. (B) Summary of JHSA hitting using the 4-step cell-based HTS. Heat maps of the JHSAs, which met the criteria in fourth screening (≥15% at 0.5 µM), were extracted from (A). The JHSAs were renamed JHSA, followed by a number, and the chemical structures are shown. 4-Phenoxyphenoxymethyl (light blue shading) is the basic structure of pyriproxyfen and fenoxycarb, two known JH agonists. Light blue and purple shading indicate 4-(morpholin-4-ylsulfonyl)phenoxymethyl (JHSA4–6) and 5-[(tetrahydro-1,4-oxazepin-4(5H)-yl)sulfonyl]benzimidazol-2-yl, respectively.