Project description:We conducted a comprehensive screening of bioactive compounds sourced from natural products, specifically targeting those capable of inducing apoptosis while inhibiting tyrosine kinase activity. Our investigation encompassed a diverse range of compounds, including pure compounds, compound mixtures, and peptides, all evaluated within the A549 cell line. To delve into the underlying molecular mechanisms, we employed phosphoproteomics analysis, which enabled us to comprehensively assess the impact of these bioactive compounds on cellular pathways. Through labeling digested proteins with distinct isobaric tags, we meticulously examined the cellular response to each compound. In our experimental design, we included two established chemical drugs, Afatinib and Osimertinib, serving as positive controls. These drugs, both kinase inhibitors utilized in cancer treatment, operate via distinct mechanisms and target different kinases. By comparing the effects of our test compounds with these controls, we aimed to elucidate their potential therapeutic relevance and mechanisms of action. Among the compounds examined were extracts from Phallus indusiatus and Fomes rimosus (Berk.) Cooke, as well as specific compounds like Chamuangone, Cannabigerol (CBG), Cannabidiol (CBD), and NP1-cyclic peptide
Project description:Various bioactive food compounds may confer health and longevity benefits, possibly through altering or preserving the human epigenome. While bioactive food compounds are widely being marketed as ‘improving health and longevity’ by counteracting harmful effects of poor nutrition and lifestyle, claimed effects are often not adequately documented. Using the honey bee (Apis mellifera) as a model species, we here employed a multi-step screening approach to investigate seven compounds for effects on lifespan and DNA methylation using ELISA and whole genome bisulfite sequencing (WGBS). A positive longevity effect was detected for valproic acid, isovaleric acid, and cyanocobalamin. For curcumin, we found that lifespan shortening caused by ethanol intake, was restored when curcumin and ethanol were co-administered. Furthermore, we identified region specific DNA methylation changes as a result of ethanol intake. Ethanol specific changes in DNA methylation were fully or partially blocked in honey bees receiving ethanol and curcumin together. Ethanol-affected and curcumin-blocked differentially methylated regions covered genes involved in fertility, temperature regulation and tubulin transport. Our results demonstrate fundamental negative effects of low dose ethanol consumption on lifespan and associated DNA methylation changes and present a proof-of-principle on how longevity and DNA methylation changes can be negated by the bioactive food component curcumin. Our findings provide a fundament for further studies of curcumin in mice and humans and offer an avenue to explore regarding possible prevention of health issues related to alcohol consumption.
Project description:Exploring the therapeutic effects of bioactive compounds has been traditionally approaching by phenotypic screenings followed by functional validation by in vitro assays. The thermal proteome profiling (TPP) has been successfully applied to study drug targets and off-target. We applied a modified protocol based on TPP to elucidate the mechanism of actions (MOA)s of novel bioactives compounds from marine biodiscovery. We have modified the method to gain the specificity for its application to compounds with limited chemical or structural characterization. Method implementation includes increasing the centrifugation force to precipitate the microsomal fraction previous to the thermal shift assay. The range of temperatures has been reduced to optimize the analysis without compromising resolution. Finally, the mass spectrometry analysis was based on label-free quantitative proteomics. Comparison of the targets among methodologies confirmed that the precipitation of the microsomal membranes before TPP is an essential step to discriminate between true targets from proteins precipitates after subcellular fractionation by centrifugation force. As a probe of concept, a novel marine bioactive compound has been analyzed on the hepatic cell line HepG2. We found that 2 targets proteins, aldehyde dehydrogenase and isocitrate dehydoregenase, can be related with beneficial properties towards obesity and obesity-related comorbidities. Identification of targets is key to decipher the MOAs of bioactive compound, predict the mode of action as well as possible harmful effects.