The development of chemical probes to study rhomboid proteases
Final Report Abstract
Proteases cleave proteins and are dynamically regulated in their activity. Deregulation of proteases can cause disease. Activity-based probes (ABPs) are able to detect active proteases, but not their inactive counterparts. Therefore, ABPs are valuable tools in the study of proteases and their confirmation as potential biomarkers and drug targets. In this Emmy Noether project we have focused on the development and application of new ABPs for (intramembrane) serine proteases. Part of this project dealt with the synthesis of new serine protease ABPs. We have based these on different type of electrophiles that covalently react with the active site serine: 4-chloro-isocoumarins, mixed alkyl aryl phosphonates, diphenyl phosphonates, and diphenyl phosphoramidates. The last two types were the first serine protease ABPs that were constructed on solid support. The solid phase synthesis eases their synthesis and structural diversification. The mixed alkyl aryl phosphonates represent the first fluorescently quenched ABPs for serine proteases. We have shown that all these ABPs react with the main classes of serine proteases, and that their selectivity depends on the nature of the substituent adjacent to the reactive group. Some 4-chloro-isocoumarin analogs induced decreased invasion of the human parasite Toxoplasma gondii in cell culture, and identification of the protein target(s) is subject of future studies. For clean identification of ABP targets we developed cleavable linkers that facilitate selective enrichment from whole proteomes. Ideally, a cleavable linker is easy to synthesize from inexpensive starting materials, readily incorporated into probes, and is efficiently cleaved under mild reaction conditions compatible with proteins. With the development of a vicinal diol cleavable linker derived from tartrate, we have realized all of these requirements. We illustrated the usage of this linker by identification of the targets of a natural product-based cysteine protease inhibitor using lysates and live cells. With usage of the linker, background protein identifications were reduced by 80-90% compared to commonly used ‘on-bead digestion’ protocols. We expanded this work to cleavable trifunctional reagents that can be used for the detection and identification of any alkyne-functionalized protein. The most challenging targets in this project were the intramembrane proteases called rhomboids. These serine proteases are membrane-embedded and cleave their substrate inside the lipid bilayer. We developed a MALDI-TOF mass spectrometry-based method in order to detect the processing of a protein substrate by bacterial rhomboids. We used this assay to screen small molecules and found several isocoumarins as new inhibitors of bacterial rhomboids. These molecules are more potent than the few previously reported isocoumarin inhibitors. They also included alkynylated isocoumarins which were used as ABPs for rhomboid. A crystal structure of the E coli rhomboid GlpG in complex with the most potent isocoumarin showed an unusual binding mode and gave clues about possible substrate-enzyme interactions. For a higher throughput screening method, we applied fluorescent rhomboid ABPs, which upon binding to their protease target, show an increase in fluorescent polarization. Using this assay, we found a novel class of rhomboid inhibitors: β-lactones. We also utilized rhomboid ABPs to obtain inhibitor fingerprints of 13 different rhomboids from eukaryotes, bacteria and archaea. We found some pan rhomboid inhibitors and some inhibitors that display selectivity. Furthermore, we found that some bacterial rhomboids are capable of autoprocessing. Overall, this project has yielded various new chemical proteomics tools for the study of proteases and novel methods for the study of intramembrane proteases. In future projects, these tools will be applied to the detection of probe-modified proteins and to uncover functional aspects of the intriguing class of rhomboid proteases.
Publications
- (2010) The structural basis for catalysis and substrate specificity of a rhomboid protease. EMBO J., 29: 3797-3809
Vinothkumar, K. R.; Strisovsky, K.; Andreeva, A.; Christova, Y.; Verhelst, S.; Freeman, M.
- (2012) Alkyne derivatives of isocoumarins as clickable activity-based probes for serine proteases. Bioorg. Med. Chem., 20: 633-640
Haedke, U.; Götz, M.; Baer, P.; Verhelst, S. H. L.
(See online at https://doi.org/10.1016/j.bmc.2011.03.014) - Activity-based probes for the study of proteases: recent advances and developments. ChemMedChem.,7: 1146-1159
Serim, S.; Haedke, U.; Verhelst, S. H. L.
(See online at https://doi.org/10.1002/cmdc.201200057) - (2013) A Simple and Effective Cleavable Linker for Chemical Proteomics Applications. Mol. Cell. Proteomics, 12: 237-244.
Yang, Y.; Hahne, H.; Küster, B.; Verhelst, S. H. L.
(See online at https://doi.org/10.1074/mcp.M112.021014) - (2013) Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay. Proc. Natl. Acad. Sci. USA, 110: 2472-2477
Vosyka, O.; Vinothkumar, K. R.; Küttler, E. V., Brouwer, A. J.; Liskamp, R. J. M.; Verhelst, S. H. L.
(See online at https://doi.org/10.1073/pnas.1215076110) - (2014) Phosphoramidates as novel activity-based probes for serine proteases. ChemBioChem, 15: 1106-1110
Haedke, U. R.; Frommel, S. C.; Hansen, F.; Hahne, H.; Kuster, B.; Bogyo, M.; Verhelst, S. H. L.
(See online at https://doi.org/10.1002/cbic.201400013) - (2015) Mixed Alkyl Aryl Phosphonate Esters as Quenched Fluorescent Activity-Based Probes for Serine Proteases. Org. Biomol. Chem., 13: 2293-2299
Serim, S.; Baer, P.; Verhelst, S. H. L.
(See online at https://doi.org/10.1039/c4ob02444c)
