Skip to main content

Development of metabolomics and fluxomics methods for metabolic drug target and toxicity elucidation using yeast

Final Activity Report Summary - CHEMOFLUXOME (Development of metabolomics and fluxomics methods for metabolic drug target and toxicity elucidation using yeast .)

The ability to assess global gene expression of drug-treated yeast cells has greatly contributed to uncover the mode of action of bioactive compounds. Altered levels of gene expression however, do not monitor the actual biological function. For a more direct functional readout of the large set of metabolic functions, we determine here intracellular fluxes (i.e. fluxome) and metabolite concentrations (i.e. metabolome) to screen drug interactions with the complex metabolic network of the model eukaryote Saccharomyces cerevisiae. As a proof of concept, we profiled dose dependent effect of 41 drugs with known specific targets in metabolism that are highly relevant for metabolic disease and toxicity.

To achieve high-throughput metabolome analysis, we developed a rapid quenching and extraction procedure for microtiter plate-grown cells. Using direct sample injection, we then (semi) quantified 55 intracellular metabolites within central metabolism by ESI-MS/MS analysis. Additionally, metabolic fluxes were calculated from GC-MS-detected 13C-pattern in protein-bound amino acids. The results demonstrate that the metabolic function - monitored as fluxes - remains relatively robust to the drug perturbations, but that this robustness is either achieved by or is not yet affected by significantly altered pattern of intracellular metabolite concentrations.

As the key result, we develop here a new high-throughput method for sensitive metabolomics. To the best of our knowledge, this is the first HT method for INTRACELLULAR metabolomics and it is at least 100-fold faster than any other global omics method that we are aware of. Our biological results demonstrate that this method has great potential in functional drug toxicity testing because we can effectively differentiate drugs with primarily local, specific metabolic responses and those with global, mostly off-target responses.