Super-sensitive Markers Will Help Study Medically Important Enzymes

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Marcin Drąg, PhD hab. (a beneficiary of the START and FOCUS programmes) and his PhD students (Paulina Kasperkiewicz, MSc Eng., and Marcin Poręba, MSc Eng.) have developed an innovative technology for studying proteolytic enzymes: HyCoSuL (from Hybrid Combinatorial Substrate Library). The results of their research conducted under the FOCUS grant have been published in the prestigious journal Proceedings of the National Academy of the United States of America (PNAS).

Proteolytic enzymes (proteases, peptidases) play a very important role in the functioning of single cells as well as entire organisms. Scientists have long known that disturbances in these enzymes’ activity lead to pathophysiological states which can cause lifestyle diseases such as cancer, diabetes, hypertension and viral and bacterial infections. Considering the above, proteases offer a very attractive experimental “training ground” in the context of designing new, more sensitive and specific markers for studying their activity in complex biological systems. However, the road to finding this kind of chemical particle can be very long.

“Before we develop an effective marker for studying one specific enzyme, we first need to learn about its catalytic preferences – its substrate specificity”, explains Marcin Drąg as the project’s principal investigator. “Existing tools of complex profiling of proteolytic enzymes were based on using exclusively natural amino acids found in proteins. The uniqueness of our research consists in using a very wide range of unnatural amino acids (not coded in DNA, mostly products of organic synthesis) to build a hybrid library (i.e. containing natural and unnatural amino acids) of fluorogenic substrates (HyCoSuL). This tool enables us to develop new, more sensitive substrates and chemical markers for a very large group of proteolytic enzymes”.

The first enzyme studied using the HyCoSuL technology was human neutrophil elastase which is involved in the development of cancer as well as taking part in combating pathogens. The research yielded a new peptide fluorogenic substrate that is identified by the enzyme several thousand times more successfully than commercially available particles. In the next stage, the substrate is turned into a chemical marker that binds very well to elastase.

“We have confirmed the usefulness of our marker (and the whole technology) with biological studies in which we performed imaging of elastase activity in the process of Neutrophil Extracellular Trap (NET) formation. These traps are fibres primarily composed of DNA threads derived from neutrophils, their task being to trap life-threatening pathogens”, explains Paulina Kasperkiewicz. The biological studies were conducted in cooperation with the group of Prof. Guy Salvesen (Sanford-Burnham Medical Research Institute, La Jolla, United States) and the group of Prof. Christine Winterbourn (University of Otago, Christchurch, New Zealand).

The project’s authors believe that the technology could be used to find new, more sensitive and specific chemical markers or structures of leading medications preventing disorders caused by proteases. The research results have already garnered interest from several chemical and pharmaceutical companies. At present the scientists from Marcin Drąg’s team are working on using the HyCoSuL technology to study other proteases. The results will be published soon.

Congratulations on this success!

Link to the paper published in PNAS