Spying on drugs and metabolites in living cells

In my talk I will discuss our recent efforts to characterize and visualize the biochemical activity of small molecules in cells. In the first part of the talk I will introduce an approach for the detection of drug-protein interactions which combines a new yeast three-hybrid screening for
identification of interactions with affinity chromatography for their unambiguous validation. Using this approach, we discovered that the anti-inflammatory drug sulfasalazine and its metabolites, sulfapyridine and mesalamine, are inhibitors of the enzyme catalyzing the final step in the
biosynthesis of the cofactor tetrahydrobiopterin. The interference with tetrahydrobiopterin metabolism provides an explanation for some of the beneficial and deleterious properties of sulfasalazine and furthermore suggests new and improved therapies for the drug. In the second part of my talk I will introduced a general concept for the generation of semisynthetic fluorescent sensor proteins for metabolites and drugs. An implementation of our sensor approach on the cell surface and in the cytosol of mammalian cells is demonstrated. Our sensor proteins display large FRET ratio changes and their modular design permits the generation of sensor proteins at wavelengths not covered by autofluorescent proteins. This work establishes a generally applicable strategy for the
generation of fluorescent sensor proteins for the detection of previously inaccessible metabolites and drugs.