Large-Scale Identification of Multiple Classes of Host Defense Peptide-Inducing Compounds for Antimicrobial Therapy

Abstract

The rapid emergence of antibiotic resistance demands new antimicrobial strategies that are less likely to develop resistance. Augmenting the synthesis of endogenous host defense peptides (HDPs) has been proven to be an effective host-directed therapeutic approach. This study aimed to identify small-molecule compounds with a strong ability to induce endogenous HDP synthesis for further development as novel antimicrobial agents. By employing a stable HDP promoter-driven luciferase reporter cell line known as HTC/AvBD9-luc, we performed high-throughput screening of 5002 natural and synthetic compounds and identified 110 hits with a minimum Z-score of 2.0. Although they were structurally and functionally diverse, half of these hits were inhibitors of class I histone deacetylases, the phosphoinositide 3-kinase pathway, ion channels, and dopamine and serotonin receptors. Further validations revealed mocetinostat, a benzamide histone deacetylase inhibitor, to be highly potent in enhancing the expression of multiple HDP genes in chicken macrophage cell lines and jejunal explants. Importantly, mocetinostat was more efficient than entinostat and tucidinostat, two structural analogs, in promoting HDP gene expression and the antibacterial activity of chicken macrophages. Taken together, mocetinostat, with its ability to enhance HDP synthesis and the antibacterial activity of host cells, could be potentially developed as a novel antimicrobial for disease control and prevention.

Keywords: antimicrobial resistance; high-throughput screening; host defense peptide inducers; host defense peptides; mocetinostat.

Figure 1

High-throughput screening to identify host defense peptide-inducing compounds. (A) Identities of small-molecule compound libraries used in the screening. The number of compounds in each library is shown in the parentheses. (B) Z-scores of the 5002 compounds in different libraries. HTC/AvBD9-luc luciferase reporter cell line was stimulated in 384-well plates with 20 μM of each compound for 24 h, followed by luciferase and cell viability assays. The Z-scores for each compound were calculated from luciferase activity normalized to cell viability.

Figure 2

Dose-dependent induction of AvBD9 mRNA expression in chicken HTC cells in response to ten leading HDP inducers. HTC cells were stimulated in duplicate using different concentrations of each compound for 24 h, followed by RT-qPCR analysis of AvBD9 mRNA expression. Sodium butyrate (4 mM) was used as a positive control and an equal volume of solvent as a negative control. The results are means ± SEM of three independent experiments. One-way ANOVA was performed, followed by Dunnett’s test. *** p < 0.001 (relative to the unstimulated control).

Figure 3

Dose- and time-dependent induction of AvBD9 mRNA expression by mocetinostat in two different chicken macrophage cell lines. (A) Chemical structures of mocetinostat and its two structural analogs, entinostat and tucidinostat. (B) Dose-dependent changes in AvBD9 mRNA expression in chicken HTC and HD11 cell lines in response to different concentrations of mocetinostat for 24 h. (C) Time-dependent induction of AvBD9 mRNA in HTC cells in response to 2 μM mocetinostat for various lengths of time. AvBD9 mRNA expression levels were evaluated using RT-qPCR. The results are means ± SEM of three independent experiments. One-way ANOVA was performed, followed by Dunnett’s test. * p < 0.05, and *** p < 0.001 (relative to the unstimulated control).

Figure 4

Induction of multiple HDP genes in chicken HTC macrophages in response to mocetinostat. HTC cells were stimulated in duplicate with 5, 10, and 20 μM mocetinostat for 24 h. HDP gene-expression levels were evaluated using RT-qPCR. The results are means ± SEM of three independent experiments. One-way ANOVA was performed, followed by Dunnett’s test. * p < 0.05, ** p < 0.01, and *** p < 0.001 (relative to the unstimulated control).

Figure 5

Induction of AvBD9 mRNA expression by mocetinostat, entinostat, and tucidinostat in HTC cells and chicken jejunal explants. HTC cells (A) or chicken jejunal explants (B) were exposed to 4 mM butyrate or three different concentrations of mocetinostat, entinostat, and tucidinostat for 24 h, followed by analysis of AvBD9 gene expression using RT-qPCR. The results are means ± SEM of 2–3 independent experiments. One-way ANOVA was performed, followed by Dunnett’s test. * p < 0.05, ** p < 0.01, and *** p < 0.001 (relative to the unstimulated control).

Figure 6

Augmentation of the antibacterial activity of chicken HTC cells by mocetinostat, entinostat, and tucidinostat. Chicken HTCs were stimulated with 10 μM mocetinostat, entinostat, tucidinostat, or 4 mM butyrate for 24 h, followed by cell lysis and incubation of the cell lysate with Escherichia coli (ATCC 25,922) or Salmonella Enteritidis (ATCC 13,076) for different durations. Bacterial turbidity was measured at OD₆₀₀ as an indication of bacterial growth. The results are means ± SEM of two independent experiments. One-way ANOVA was performed, followed by Dunnett’s test. * p < 0.05, ** p < 0.01, and *** p < 0.001 (relative to the unstimulated control at each time point).