Recognition of some of the limitations of target-based drug discovery has recently led to the renaissance of a more holistic approach in which complex biological systems are investigated for phenotypic changes upon exposure to small molecules. The subsequent identification of the molecular targets that underlie an observed phenotypic response--termed target deconvolution--is an important aspect of current drug discovery, as knowledge of the molecular targets will greatly aid drug development. Here, the broad panel of experimental strategies that can be applied to target deconvolution is critically reviewed.

Riboflavin (vitamin B2), essential in tiny amounts as a precursor for oxidoreductase coenzymes, is a yellow pigment. Although it causes cytotoxicity via photoinduced damage of macromolecules, several microorganisms are striking overproducers. A question, unanswered for decades, is whether riboflavin overproducers can benefit from this property. Here, we report an ultraviolet (UV) protective effect of riboflavin. The spores of Ashbya gossypii, a riboflavin-overproducing fungus, are more sensitive to UV than those of Aspergillus nidulans. The addition of riboflavin to suspensions improves the UV resistance of both spore types. Interestingly, we show that regulation of sporulation and riboflavin overproduction in A. gossypii are linked. In batch culture, both were elevated when growth ceased. At constant growth rates, obtained in a chemostat culture, neither was elevated. Supplementation of cultures by cAMP, a known stress signal, negatively affected sporulation as well as riboflavin overproduction, establishing a second, independent argument for the linkage.

Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments. When 500 microM 99% [1-(13)C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux.