Mode of Action studies with bioactive natural products
Compound development and optimization require the elucidation of the mode-of-action, i.e., identification of the molecular target of the active compound. This is a prerequisite for detailed analysis of drug – target interactions with purified proteins, which could also include structural analysis of the compound – target complex and subsequently rational compound optimization in cooperation with respective experts. Especially a new mode-of-action makes a compound interesting for further development to finally progress to clinical application.
We have implemented different experimental approaches to get hints about the molecular targets of active compounds. Two methods based on High Content Image Analysis and Impedance Monitoring are described in the High Content Screening Project. A method that can directly show the binding partner of a compound is DARTS, the drug affinity responsive target stability . DARTS is based on the fact that small molecules when binding to a protein protect it from degradation by proteases, at least to a certain degree. Protected protein bands can be identified by PAGE and MS. The method was already successfully applied by us to identify the target of bioactive small molecules, e.g. , .
Protein targets can also be identified by so-called target fishing approaches, which, however, require chemical modification of the compound. Consequently, pull down assays based on affinity labelled compounds are performed and bound proteins are separated by PAGE and identified by MS and Western Blotting.
Recently, whole genome sequencing was introduced as a technology to uncover the molecular mechanism and target of an anti-infective compound via identification of the locus of resistance developed against its activity. We therefore raise resistant pathogens and sequence their genomes to identify mutated proteins that render resistance which could be the direct target of the compound.
 Lomenick, B., Hao, R., Jonai, N., Chin, R.M., Aghajan, M., Warburton, S., Wang, J., Wu, R.P., Gomez, F., Loo, J.A., Wohlschlegel, J.A., Vondriska, T.M., Pelletier, J., Herschman, H.R., Clardy, J., Clarke, C.F., Huang, J. (2009) Target identification using drug affinity responsive target stability (DARTS). Proc Natl Acad Sci U S A. 106, 21984-9.
 Schneider, T., Muthukumar, Y., Hinkelmann, B., Franke, R., Döring, M., Jacob, C., Sasse, F. (2012) Deciphering intracellular targets of organochalcogen based redox catalysts. MedChemComm 3, 784-787.
 Muthukumar, Y., Roy, M., Raja, A., Taylor, R.E., Sasse, F. (2013) The marine polyketide myriaporone 3/4 stalls translation by targeting the elongation phase. ChemBioChem 14, 1439-7633.