Wroclaw University of Science and Technology
The Chemical Biology team is led by Professor Marcin Drąg. The main interests here include the design and synthesis of substrates, inhibitors and ABPs to decipher the mechanism of action and the function of proteolytic enzymes in health and disease. Our focus is on interdisciplinary research, including organic chemistry, combinatorial chemistry, biochemistry and molecular biology in collaboration with partners, mainly from the EU, USA and Australia.
- Design of combinatorial libraries with unnatural amino acids: The focus here is on the development of new strategies for solid and solution phase synthesis of combinatorial substrate libraries for each family of proteolytic enzymes. We have recently introduced the Hybrid Combinatorial Substrate Library (HyCoSuL) strategy, which can be practically applied to each type of endopeptidases. The advantage over all other existing substrate specificity profiling strategies is application by us of a broad range of unnatural amino acids, which results in the extensive analysis of protease chemical space. This knowledge allows us to develop optimal substrates, inhibitors and activity-based probes.
- Proteases substrate specificity: We focus on the determination of substrate specificity of all types of exopeptidases and endopeptidases. This includes the development of new protocols, screening of large libraries of fluorogenic or internally quenched substrates, validation of screening data and selection of optimal hits, solid phase synthesis of champion molecules, enzymes kinetics and data analysis. Key aspects are finding the selective or most active peptide sequences for each protease by direct comparison of enzyme orthologs, homologs or whole protease families.
- Activity-based probes (ABPs) for proteolytic enzymes: We focus here on the development of most selective probes in order to investigate the function of proteolytic enzymes in health and disease. This approach is currently being investigated by Dr Marcin Poreba and includes the development of new strategies of solid and solution phase synthesis, structure and purity analysis by high-performance liquid chromatography and mass spectrometry, selection of optimal reactive groups (warheads) for each family of proteases, selection of optimal reporter groups (fluorophores, affinity tags), and development of new protocols for probe visualisation in biological samples. Key aspects are understanding and prediction of ABP stability, selectivity, membrane permeability and affinity.
- Direct visualisation of protease activity in living cells: This approach is currently being developed by Dr Paulina Kasperkiewicz, who focuses here on the development of new techniques and tools for ABP imaging in vitro to demonstrate the preferential localisation of enzymatically active proteases in biological samples. The main aspects of our work include selecting optimal (selectivity, activity factors) ABPs for protease of interests, the development of protocols and assays for each new probe and the optimisation of visualisation techniques in biological samples. Using both fluorescence and confocal microscopy supported with advanced imaging software, we measure the level of active proteases in single cells in both health and disease. Finally, our major goal is the application of probes directly on human samples of blood or urine.