Identification of host defense peptide-inducing compounds as alternatives to antibiotics using a cell-based high throughput screening assay

Abstract

In-feed antibiotics has been banned from food animals for production purposes. Effective alternatives to antibiotics are needed to maintain animal health and production performance. Modulating of the synthesis of endogenous host defense peptides (HDPs) shows potential as a novel antibiotic alternative strategy to disease control and prevention. To identify small-molecule compounds with the ability to induce HDP synthesis, a high throughput screening (HTS) assay based on a stable HTC/AvBD9-luc cell line expressing a 2-kb avian β-defensin (AvBD9) gene promoter-driven luciferase reporter gene was developed. Libraries of natural and synthetic compounds totaling 5,586 were screened. Using a minimum Z-score of 2.0, 131 hits were identified. After further validation in HTC/AvBD9-luc cells and parental HTC macrophage cells, most compounds showed a strong capacity to induce AvBD9 gene expression in a dose-dependent manner. For example, wortmannin, a phosphoinositide-3-kinase inhibitor, was confirmed to stimulate AvBD9 expression in multiple chicken cell types and the duodenum of chickens. Wortmannin was also shown to synergize with butyrate in inducing AvBD9 gene expression and enhancing the antibacterial activity of chicken monocytes. Additionally, mocetinostat, a histone deacetylase inhibitor, was highly potent in AvBD9 induction in HTC/AvBD9-luc cells and parental HTC cells. Mocetinostat was more efficacious in AvBD9 induction and enhancing the antibacterial activity of chicken cells than its structural analogs, Chidamide and MS-275, two compounds that are highly effective in HDP induction in pigs and humans, respectively. In addition to AvBD9, most other HDP genes were found to be simultaneously induced by wortmannin and mocetinostat in chicken cells. Collectively, these newly-identified HDP inducers have the potential to be developed as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.