Study of the mechanism of action of bioactive plants tarpenoids

Abstract

Natural products are small-molecule secondary metabolites displaying considerable structural complexity and “privileged scaffolds”. They are able to bind several endogenous targets eliciting biological effects as chemical weapons or to convey information from one organism to another. Nowadays, medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets. Therefore, the primary purpose of this PhD thesis has been a comprehensive characterization of the interactome profile and then the molecular mechanism of action of bioactive natural molecules. Achieving this in an effective, unbiased and efficient manner subsists as a significant challenge for the new era in drug discovery and optimization. Indeed, the full understanding of the mechanism of action of natural molecules could lead to a number of advantages: first of all, exploit their full therapeutic potential, the identification of side effects or toxicity, or the ability to set up target-based assays and to allow structure activity relationships studies to guide medicinal chemistry efforts towards lead optimization. In my research project, the attention was paid on ent-kaurane diterpenes, a class of natural terpenoids with a great structural variability and a wide spectrum of biological activities. Firstly, I focused on the determination of the interactome of a semi synthetic compound 15-ketoatractyligenin methyl ester. This compound has been previously reported to possess high antiproliferative activity against several solid tumor-derived cell lines. In this regard, I decided to investigate the mechanism of action of this actratylignin derivative researching first of all its molecular targets, responsible for the biological activity. In order to achieve this goal, I used a chemical proteomic approach first. This study led to the identification of PPARγ as the main cellular partner of this compound; achieved results were supported and validated through different biological assays. Subsequently, I studied another diterpene: oridonin. This molecule has been shown to have multiple biological activities. Among them, the anticancer activity has been repeatedly reported by many research groups. With the aim of expanding and validate our knowledge about this molecule, also seen the limitations of the fishing for partners method, I decided to use two orthogonal compound-centric proteomics approaches to define the possible protein target(s) of oridonin. Using this strategy HSP70 and nucleolin were identified. Therefore, several in vitro and in cell tests have been performed to validate the interaction of oridonin with these proteins, and to evaluate its effect on their activity. Some of these tests were developed and optimized during my period of research abroad at the Massachusset General Hospital- Center for System Biology -Harvard Medical School; in that twelve months period I expanded my knowledge into the techniques useful for the study of the mechanism of action of a small molecule, also applying experimental methods complementary to proteomics and focusing on the use of high-resolution intravital microscopy imaging for drug pharmacology. [edited by Author]