Structure-Antifouling Activity Relationship and Molecular Targets of Bio-Inspired(thio)xanthones

Abstract

The development of alternative ecological and effective antifouling technologies is still challenging. Synthesis of nature-inspired compounds has been exploited, given the potential to assure commercial supplies of potential ecofriendly antifouling agents. In this direction, the antifouling activity of a series of nineteen synthetic small molecules, with chemical similarities with natural products, were exploited in this work. Six (4, 5, 7, 10, 15 and 17) of the tested xanthones showed in vivo activity toward the settlement of Mytilus galloprovincialis larvae (EC₅₀: 3.53-28.60 µM) and low toxicity to this macrofouling species (LC₅₀ > 500 µM and LC₅₀/EC₅₀: 17.42-141.64), and two of them (7 and 10) showed no general marine ecotoxicity (<10% of Artemia salina mortality) after 48 h of exposure. Regarding the mechanism of action in mussel larvae, the best performance compounds 4 and 5 might be acting by the inhibition of acetylcholinesterase activity (in vitro and in silico studies), while 7 and 10 showed specific targets (proteomic studies) directly related with the mussel adhesive structure (byssal threads), given by the alterations in the expression of Mytilus collagen proteins (PreCols) and proximal thread proteins (TMPs). A quantitative structure-activity relationship (QSAR) model was built with predictive capacity to enable speeding the design of new potential active compounds.

Keywords: antifouling activity; chemical synthesis; invertebrates; molecular targets; xanthones.

Figure 1

Library of xanthones investigated for antifouling activity.

Figure 2

Dose-response of anti-settlement activity of the promising antifouling compounds 4, 5, 7, 10, 15, and 17 toward plantigrades of the mussel M. galloprovincialis. B: negative control; C: CuSO₄ 5 µM as positive control.

Figure 3

Quantitative structure–activity relationship (QSAR) model obtained with the heuristic method for 15 xanthones with the CODESSA software (R² = 0.7335, F = 10.09, and s = 0.0085). X, ΔX and t-test are the regression coefficient of the linear model, standard errors of the regression coefficient, and the t significance coefficient of the determination, respectively. AF = antifouling activity.

Figure 4

(a) Acetylcholinesterases (AChE) activity of 19 pure xanthones (50 µM). * significant differences (p ≤ 0.05, Dunnett’s test) compared to the negative control (B); eserine was used as positive control (C). (b) Dose-response of AChE activity of selected xanthones 4 and 5.

Figure 5

(a) Capped surface of AChE, showing the ACh binding pocket with AChE inhibitors pulmonarin A and B (blue sticks); (b) detailed view of known antifouling AChE inhibitors: pulmonarin A (light blue sticks) and pulmonarin B (dark blue sticks); (c) detailed view of test compounds 4 (purple sticks) and 5 (magenta sticks). Hydrogen interactions are represented with yellow broken line and stacking interactions with a double edge yellow arrow. Residues involved on those interactions are represented as green sticks and labeled. AChE is represented as solid surface, where carbon, hydrogen, oxygen, nitrogen, and sulfur are represented in green, grey, red, blue, and yellow, respectively.

Figure 6

(a) Tyrosinase activity of 19 pure xanthones. * significant differences (p ≤ 0.05, Dunnett’s test) compared to the negative control (B); kojic acid was used as positive control (C). (b) Dose-response tyrosinase activity of selected compounds 7 and 8.

Figure 6

(a) Tyrosinase activity of 19 pure xanthones. * significant differences (p ≤ 0.05, Dunnett’s test) compared to the negative control (B); kojic acid was used as positive control (C). (b) Dose-response tyrosinase activity of selected compounds 7 and 8.

Figure 7

Hierarchical cluster analysis of the differential proteins from M. galloprovincialis larvae exposed to three different antifouling compounds, 7, 10, and 17. On the vertical axis of the dendrogram: clustering of proteins with similar abundance profiles. On the horizontal axis: grouping of samples with similar proteome. Only proteins with significant changes (p < 0.05) in abundance (based on NSAF values) are presented in the dendogram. The relative abundance values (NSAF) are represented in color gradient from 0.0 to 0.025. Further information on NSAF values, significant p-values, and names of all differentially abundant proteins is included in Supplementary Table S2.

Figure 7

Hierarchical cluster analysis of the differential proteins from M. galloprovincialis larvae exposed to three different antifouling compounds, 7, 10, and 17. On the vertical axis of the dendrogram: clustering of proteins with similar abundance profiles. On the horizontal axis: grouping of samples with similar proteome. Only proteins with significant changes (p < 0.05) in abundance (based on NSAF values) are presented in the dendogram. The relative abundance values (NSAF) are represented in color gradient from 0.0 to 0.025. Further information on NSAF values, significant p-values, and names of all differentially abundant proteins is included in Supplementary Table S2.

Figure 8

Mortality rate of Artemia salina exposed to the compounds 4, 5, 7, 10, 15, and 17 at 25 and 50 µM.