Protein targets of rugulactone and illudin S: An analysis of their function and mechanism of action
Organic Molecular Chemistry - Synthesis and Characterisation
Final Report Abstract
This project aimed to investigate the targets of two natural products, illudin and rugulactone, and elucidate their mode of action. While the rugulactone part focussed more on target validation and mechanistic studies, the illudin project started at an earlier stage with a focus on probe synthesis and target identification. Previous experiments with rugulactone and target identification via activity based protein profiling revealed an enzyme of the ribokinase family, termed ThiD as a target. Surprisingly the binding site of the reactive Michael acceptor of rugulactone was not the active site Cys214 but a so far uncharacterized Cys110. Although we initially aimed to perform further characterization with Mycobacterium tuberculosis ThiD, insolubility of all different protein constructs prompted us to focus on Staphylococcus aureus ThiD, which is of similar relevance. Structural studies performed here unravelled several conformational states in presence and absence of the pyridoxal substrate. Strikingly, Cys110 was identified as a crucial residue for catalysis as it forms a hemithioacetal with the 4´aldehyde of pyridoxal. Accordingly, mutational and kinetic studies with Cys110 and Cys214 mutants revealed their role in the turnover of pyridoxal and related vitamers. Based on these results a detailed mechanism of pyridoxal phosphorylation was postulated. Moreover, sequence comparisons and recombinant expression of related enzymes in different organisms revealed that this new family of enzymes utilizing dual Cys for catalysis is widely distributed. We thus renamed this family as CC-PLKs. Synthesis of simplified rugulactone analogues showed that even simple Michael acceptors were able to address ThiD suggesting that Cys110 is of heightened nucleophilicity and reacts readily with Michael acceptors. This is also supported by our crystal structures showing that Cys110 is surface exposed and lacks a defined binding pocket. The design of specific inhibitors via this strategy is therefore complicated. Finally, due to the lack of specific rugulactone-based ThiD inhibitors we adjusted our work plan and performed experiments in M. tuberculosis with lalistat, a protein reactive lipase inhibitor. Synthesis of a corresponding probe and whole proteome analysis revealed several crucial enzymes of the Lip family. These play important roles in the bacterial metabolism and thus provide a link to lalistat´s anti-mycobacterial activity. Illudins are natural products exhibiting a characteristic electrophilic cyclopropyl group. Previous studies demonstrated low nanomolar anti-cancer activity as a consequence of DNA binding. This mode of action was already known from related cyclopropyl-containing natural products such as the duocarmycins. In case of duocarmycin, we previously demonstrated the selective targeting of a protein in addition to DNA binding. Thus we were wondering if illudins exhibit a similar dual reactivity. We therefore synthesized a variety of different probes including one derivative with a photocrosslinking moiety and tested these for cellular toxicity. While some probes only exhibited a minor reduction in potency, others largely dropped in activity. Gel-based labelling of human cell lines revealed that low micromolar concentrations were needed in order to obtain protein labelling. Based on their labelling intensity a couple of probes were selected for gel-free quantitative proteomics. Surprisingly, illudins showed a rather unspecific targeting of diverse proteins without a clear hit. Thus we conclude form these experiments that illudins, contrary to duocarmycin, prefer DNA binding as main mode of action and promiscuously react with proteins at higher concentrations above their cell toxicity. These results demonstrate the exciting variability of natural product targets and reveals insights into different principles of activity design.
Publications
- "A Subfamily of bacterial ribokinases utilizes a hemithioacetal for pyridoxal phosphate salvage", J. Am. Chem. Soc., 2014, 136, 4992-4999
Nodwell, M. B., Koch, M. F., Alte, F., Schneider, S., Sieber, S. A.
(See online at https://doi.org/10.1021/ja411785r) - "Human lysosomal acid lipase inhibitor lalistat impairs Mycobacterium tuberculosis growth by targeting bacterial hydrolases", MedChemComm. 2016
Lehmann, J., Vomacka, J., Esser, K., Nodwell, M., Kolbe, K., Rämer, P., Protzer, U., Reiling, N., Sieber, S.A.
(See online at https://doi.org/10.1039/c6md00231e)
