Unbiased screening of marine sponge extracts for anti-inflammatory agents combined with chemical genomics identifies girolline as an inhibitor of protein synthesis

Abstract

Toll-like receptors (TLRs) play a critical role in innate immunity, but activation of TLR signaling pathways is also associated with many harmful inflammatory diseases. Identification of novel anti-inflammatory molecules targeting TLR signaling pathways is central to the development of new treatment approaches for acute and chronic inflammation. We performed high-throughput screening from crude marine sponge extracts on TLR5 signaling and identified girolline. We demonstrated that girolline inhibits signaling through both MyD88-dependent and -independent TLRs (i.e., TLR2, 3, 4, 5, and 7) and reduces cytokine (IL-6 and IL-8) production in human peripheral blood mononuclear cells and macrophages. Using a chemical genomics approach, we identified Elongation Factor 2 as the molecular target of girolline, which inhibits protein synthesis at the elongation step. Together these data identify the sponge natural product girolline as a potential anti-inflammatory agent acting through inhibition of protein synthesis.

Figure 1

Screening marine sponge extracts for TLR inhibitors.

Figure 2

Discovery of girolline as a potential inhibitor for TLR5 signalling from marine sponge extracts. (a) The first round of screening was performed on the crude extracts to identify potentially active extract fractions. (b) The second round was confirmatory examining dose-responsiveness and non-flagellin stimulated samples were used to investigate false positive results due to compound toxicity. X denotes extracts excluded from further analysis due to potential toxicity. (c–d) The confirmed positive crude extracts underwent further assay-guided fractionation until a pure compound was identified (e). Flg: flagellin.

Figure 3

Structure-activity relationship studies examining the inhibitory activity of girolline derivatives. (a) girolline; (b) diastereomer; (c) enantioner; (d) des-amino girolline; (e) des-chloro girolline; (f) des-chlorohydroxy girolline. Flg: flagellin.

Figure 4

Inhibitory activity of girolline on flagellin stimulated THP1 derived macrophages and fresh human PBMCs. Girolline pre-treatment decreased IL-8 secretion (a) and NF-κB activity (b) after flagellin stimulation. The same inhibitory activity was observed with PBMCs, where both IL-8 (c) and IL-6 (d) levels were significantly reduced after girolline pre-treatment. Flg: flagellin.

Figure 5

Cytotoxicity of girolline examined in a number of cell types. Dose-dependent toxicity of girolline on CHO cells examined by (a) LDH release assay and (b) MTS assay. Same MTS assay was performed on HEK cells (c), THP1 derived macrophages (d) and fresh PBMCs (e). Flg: flagellin.

Figure 6

The inhibitory activity of girolline on TLR signaling. Dose-dependent inhibition profiles were examined following stimulation of a variety of TLRs, as well as IL-1R and TNFR, using HEK-TLR2 (a), HEK-TLR3 (b) HEK-TLR4 (c) and HEK-TLR7 (d) reporter cells. A simplified TLR signaling pathway was illustrated in (e).

Figure 7

Girolline acts as a protein synthesis inhibitor targeting Elongation Factor 2. (a) Chemical genomic profile of girolline showing that Elongation Factor 2 (Eft2) is the predicted target of girolline; the deletion mutants of genes that had described genetic interactions with EFT2 had significant correlation with the gene mutants that had chemical genomic interactions with girolline. (b) Protein synthesis inhibition of girolline (2 µg ml⁻¹, 4 h) in comparison with cycloheximide (1 µM, 30 min); total new synthesized proteins in the cells are stained in green (Alex488) while nuclei are stained in blue (Hoechst 33342). (c) Quantitative analysis of Alex488 signals per cells in (b); analysis of 250–350 cells per sample in each experiment; n = 4, *** p < 0.001 vs. medium control. 116×76mm (300 × 300 DPI)