Glutathione Transferases: Surrogate Targets for Discovering Biologically Active Compounds.

Glutathione transferases comprise a large class of multifunctional enzymes, some involved in detoxification pathways. Since these enzymes are able to interact with potentially toxic molecules, they could be used as targets to screen for compounds with biological activity. To test this hypothesis, glutathione transferases (GSTs) from the white-rot fungus Trametes versicolor have been used to screen for antifungal molecules from a library of tropical wood extracts. The interactions between a set of six GSTs from the omega class and 116 extracts from 21 tropical species were quantified using a high-throughput thermal shift assay. A correlation between these interactions and the antifungal properties of the tested extracts was demonstrated. This approach has been extended to the fractionation of an Andira coriacea extract and led to the detection of maackiain and lapachol in this wood. Altogether, the present results supported the hypothesis that such detoxification enzymes could be used to detect biologically active molecules.

A reverse chemical ecology approach to explore wood natural durability.

The natural durability of wood species, defined as their inherent resistance to wood-destroying agents, is a complex phenomenon depending on many biotic and abiotic factors. Besides the presence of recalcitrant polymers, the presence of compounds with antimicrobial properties is known to be important to explain wood durability. Based on the advancement in our understanding of fungal detoxification systems, a reverse chemical ecology approach was proposed to explore wood natural durability using fungal glutathione transferases. A set of six glutathione transferases from the white-rot Trametes versicolor were used as targets to test wood extracts from seventeen French Guiana neotropical species. Fluorescent thermal shift assays quantified interactions between fungal glutathione transferases and these extracts. From these data, a model combining this approach and wood density significantly predicts the wood natural durability of the species tested previously using long-term soil bed tests. Overall, our findings confirm that detoxification systems could be used to explore the chemical environment encountered by wood-decaying fungi and also wood natural durability.

The antifungal potential of (Z)-ligustilide and the protective effect of eugenol demonstrated by a chemometric approach.

Mankind is on the verge of a postantibiotic era. New concepts are needed in our battle to attenuate infectious diseases around the world and broad spectrum plant-inspired synergistic pharmaceutical preparations should find their place in the global fight against pathogenic microorganisms. To progress towards the discovery of potent antifungal agents against human pathologies, we embarked upon developing chemometric approach coupled with statistical design to unravel the origin of the anticandidal potential of a set of 66 essential oils (EOs). EOs were analyzed by GC-MS and tested against Candida albicans and C. parapsilosis (Minimal Inhibitory Concentration, MIC). An Orthogonal Partial Least Square (OPLS) analysis allowed us to identify six molecules presumably responsible for the anticandidal activity of the oils: (Z)-ligustilide, eugenol, eugenyl acetate, citral, thymol, and ß-citronellol. These compounds were combined following a full factorial experimental design approach in order to optimize the anticandidal activity and selectivity index (SI = IC50(MRC5 cells)/MIC) through reconstituted mixtures. (Z)-Ligustilide and citral were the most active compounds, while (Z)-ligustilide and eugenol were the two main factors that most contributed to the increase of the SI. These two terpenes can, therefore, be used to construct bioinspired synergistic anticandidal mixtures.

Biosynthetic investigation of γ-lactones in Sextonia rubra wood using in situ TOF-SIMS MS/MS imaging to localize and characterize biosynthetic intermediates.

Molecular analysis by parallel tandem mass spectrometry (MS/MS) imaging contributes to the in situ characterization of biosynthetic intermediates which is crucial for deciphering the metabolic pathways in living organisms. We report the first use of TOF-SIMS MS/MS imaging for the cellular localization and characterization of biosynthetic intermediates of bioactive γ-lactones rubrynolide and rubrenolide in the Amazonian tree Sextonia rubra (Lauraceae). Five γ-lactones, including previously reported rubrynolide and rubrenolide, were isolated using a conventional approach and their structural characterization and localization at a lateral resolution of ~400 nm was later achieved using TOF-SIMS MS/MS imaging analysis. 2D/3D MS imaging at subcellular level reveals that putative biosynthetic γ-lactones intermediates are localized in the same cell types (ray parenchyma cells and oil cells) as rubrynolide and rubrenolide. Consequently, a revised metabolic pathway of rubrynolide was proposed, which involves the reaction between 2-hydroxysuccinic acid and 3-oxotetradecanoic acid, contrary to previous studies suggesting a single polyketide precursor. Our results provide insights into plant metabolite production in wood tissues and, overall, demonstrate that combining high spatial resolution TOF-SIMS imaging and MS/MS structural characterization offers new opportunities for studying molecular and cellular biochemistry in plants.

Tandem Mass Spectrometry Imaging and in Situ Characterization of Bioactive Wood Metabolites in Amazonian Tree Species Sextonia rubra.

Driven by a necessity for confident molecular identification at high spatial resolution, a new time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem mass spectrometry (tandem MS) imaging instrument has been recently developed. In this paper, the superior MS/MS spectrometry and imaging capability of this new tool is shown for natural product study. For the first time, via in situ analysis of the bioactive metabolites rubrynolide and rubrenolide in Amazonian tree species Sextonia rubra (Lauraceae), we were able both to analyze and to image by tandem MS the molecular products of natural biosynthesis. Despite the low abundance of the metabolites in the wood sample(s), efficient MS/MS analysis of these γ-lactone compounds was achieved, providing high confidence in the identification and localization. In addition, tandem MS imaging minimized the mass interferences and revealed specific localization of these metabolites primarily in the ray parenchyma cells but also in certain oil cells and, further, revealed the presence of previously unidentified γ-lactone, paving the way for future studies in biosynthesis.

Mapping Dicorynia guianensis Amsh. wood constituents by submicron resolution cluster-TOF-SIMS imaging.

The preparation of tropical wood surface sections for time-of-flight secondary ion mass spectrometry imaging is described, and the use of delayed extraction of secondary ions and its interest for the analysis of vegetal surface are shown. The method has been applied to the study by time-of-flight secondary ion mass spectrometry imaging with a resolution of less than one micron of a tropical wood species, Dicorynia guianensis, which is one of the most exploited wood in French Guiana for its durable heartwood. The heartwood of this species exhibits an economical importance, but its production is not controlled in forestry. Results show an increase of tryptamine from the transition zone and a concomitant decrease of inorganic ions and starch fragment ions. These experiments lead to a better understanding of the heartwood formation and the origin of the natural durability of D. guianensis. Copyright © 2016 John Wiley & Sons, Ltd.

The termiticidal activity of Sextonia rubra (Mez) van der Werff (Lauraceae) extract and its active constituent rubrynolide.

BACKGROUND: Termites are degradation agents that inflict severe damage on wood. Some long-lasting Amazonian trees can resist these insects by producing toxic secondary metabolites. These metabolites could potentially replace synthetic termiticidal products which are becoming more restricted to use. RESULTS: Sextonia rubra is resistant to termite-induced degradation. It has been demonstrated that this species naturally produces an ethyl-acetate-soluble termiticidal metabolite, rubrynolide, to protect its wood. Assays in the presence of tropical and invasive termites established that both rubrynolide and crude ethyl acetate extract from S. rubra wood can be used as a treatment for the protection of sensitive woods against termites. CONCLUSION: Rubrynolide and S. rubra extract are promising candidates for the replacement of synthetic termiticides.