Phenylethylamides derived from bacterial secondary metabolites specifically inhibit an insect serotonin receptor

Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune responses and modulates nerve signal in insects. Se-5HTR , a specific receptor of serotonin, has been identified in the beet armyworm, Spodoptera exigua . It is classified into subtype 7 among known 5HTRs. Se-5HTR was expressed in all developmental stages of S . exigua . It was expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited significant immunosuppression by preventing cellular immune responses such as phagocytosis and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in significant immunosuppression. Furthermore, knockout mutant of Se-5HTR by CRISPR-Cas9 led to significant reduction of phagocytotic activity of S . exigua hemocytes. Such immunosuppression was also induced by bacterial secondary metabolites derived from Xenorhabdus and Photorhabdus . To determine specific bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites with respect to cellular immune responses associated with Se-5HTR and selected 10 potent inhibitors. These 10 selected compounds competitively inhibited cellular immune responses against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA derivatives were screened and resulting potent chemicals were used to design a compound to be highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It showed high immunosuppressive activities along with specific and competitive inhibition activity for Se-5HTR. This study reports the first 5HT receptor from S . exigua and provides its specific inhibitor designed from bacterial metabolites and their derivatives.