Jean-Luc Wolfender
Induction of natural products for the search of new bioactive compounds
Classical pharmacognosy investigations have always been related to the bioactivity guided fractionation of constitutive secondary metabolites of plants for their isolation and use as possible leads for the development of new drugs. Plants and fungi possess inducible pathways that are activated in responses to stress and that can produce highly bioactive defence compounds upon elicitation. Several projects are related to the induction of natural products in various types of biotic and abiotic stress. This allows a fundamental comprehension of the defence mechanisms involved and lead to the production of original natural products that might have increased chances to be bioactive against various therapeutic targets. These projects include the search for biomarkers of the plant wound response, the search for new antifungal and antimicrobial compounds from pathogenic fungi in confrontation and the phytoalexin production of plants towards human pathogenic fungi.

New antifungals and antimicrobials from pathogenic fungi in confrontation
Besides being common fungal soil saprophytes, species of the genus Fusarium are frequently plant pathogens. In medicine, Fusarium species have not only emerged as major opportunistic fungi in patients with severe immunosuppression, but are also responsible for onychomycosis. Fusarium spp. are especially difficult infectious agents to cure, and the need for new antifungal drugs at both the medicinal and agronomical levels is high. In this respect, a Sinergia SNF project started with partner at ACW Changins (Dr K. Gindro) and CHUV (Pr M. Monod) to study and exploit natural products induced in fungus-fungus and fungus-plant interactions to search for new types of antifungals, particularly effective against Fusarium species. These interactions, largely unexplored from a drug discovery perspective, are at the centre of an intense induction of bioactive secondary metabolites, and co-culture of microorganisms leads to the de novo production of active secondary metabolites. Our approach is expected to yield interesting lead compounds with an enhanced hit rate. In addition, this study will provide important knowledge on the interactions of different species of fungi in confined spaces via signalling events. It will help understanding the phenomena of fungal succession in nails and host-pathogen plant responses to Fusarium spp. showing common traits with fungi involved in onychomycosis.

New biomarkers for the wound response
Physical damage to plants reduces their growth and this has consequences in agriculture. Herbivores damage plant tissues by wounding. A better understanding of the biochemical events triggered by the wound response in plants is thus essential. A lot of research has dealt with this issue and it has been demonstrated that jasmonic acid (JA) and its derivatives play a crucial role in particular in defence signalling. The chemical diversity of oxylipins (oxygenated fatty acids) is intriguing and raises numerous questions about their exact role in the fine tuning of defence gene expression. In this context, we have initiated a project aiming at the comprehensive investigation of all chemicals induced by wounding in the model plant Arabidopsis thaliana in collaboration with Prof. Ted Farmer (UNIL) and Dr Serge Rudaz (UNIGE). Using a mass spectrometry based metabolomic approach, a detailed picture of all observable metabolome changes upon wounding was obtained in spatio-temporal manner. New jasmonates were identified and novel aspects related to the very rapid induction of jasmonates were evidenced. The overall project revealed new aspects of plant hormones induction and enabled the development of innovative metabolomic methods for the search of low biomarkers that can be also applied to the search of biomarkers related to drug metabolism or human disease. The search for new wound biomarkers, the study of their defence gene induction potential and their dynamics is ongoing.

Plant metabolomics
In a short time, plant metabolomics has gone from being just an ambitious concept to a rapidly growing, valuable technology providing a global picture of plant molecular organisation at the metabolite level. Various biological issues have been successfully studied by this holistic approach that includes global metabolite composition assessment, mutant characterization, taxonomy, developmental processes, stress response, interaction with environment, quality control assessment and mode of action of herbal medicine. Several projects in relation with the stress induction of bioactive natural products are studied under a metabolomic view. They include the induction of a new type of antifungals, phytoalexins derived from resveratrol involved in resistance phenomena and of interest for human health, and the search for original phytohormones. With Dr Serge Rudaz (Pharmaceutical Methodology) an original metabolomic strategy has been developed that is based on the concomitant use of mass spectrometry (UHPLC-TOFMS) for plant extract profiling and fingerprinting, as well as MS/MS based determination and microflow NMR techniques for biomarker identification. This platform provides, with the help of advanced data mining (Dr S. Rudaz), an efficient way for the identification of key biomarkers in various biological issues. The use of metabolomics with this strategy and also in conjunction with direct NMR profiling approaches is also foreseen for a better understanding of the mode of action of plants used in phytotherapy where the active principles have not yet been clearly identified.

Rapid lead discovery from natural sources
Miniaturised hit discovery. Over the years, a rather unique analytical platform based on modern hyphenated techniques (LC-MS, LC-MS/MS and LC-NMR) has been built for the rapid analysis and dereplication of natural product in complex matrices, such as crude plant extracts. At the same time various simple bioassays have been developed. Recent advances include the introduction of UHPLC-TOFMS for rapid and precise crude plant extract profiling and microflow NMR (CapNMR) for at-line identification of natural products in the microgram range. In parallel to this chemical characterisation of complex mixtures, bioactivity profiles can be generated based on microfractionation of mg amounts of crude extracts in 96 well plates. This approach ideally provides high quality spectroscopic data for the identification of a given natural product within an extract and a rapid estimation of its activity. This strategy should enhance the efficiency of lead finding from natural sources in a sustainable way. In vitro assays include antifungal activities and targets for cancer chemoprevention. Furthermore, in vivo assays on Zebrafish models (in collaboration with University Leuven) are being implemented in this integrated platform. Large scale isolation of predefined hits. These preliminary results give a very good chemical and biological profile of hits within crude extracts. ADMET data can also be partly extrapolated from chromatographic behaviour and in vivo results. Based on this information, targeted large scale isolation is pursued on the most promising hits for lead finding. In this respect, the most appropriate sustainable source is selected and the best isolation scheme is optimised from analytical chromatographic data to large preparative scale.


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