Intervention with Serine Protease Activity with Small Peptides

Research output: Book/anthology/dissertation/reportPh.D. thesisResearch


  • Peng Xu, Denmark
Serine proteases perform proteolytic reactions in many physiological and metabolic processes and have been certified as targets for therapeutics. Small peptides can be used as potent antagonists to target serine proteases and intervene with their activities. Urokinase-type plasminogen activator (uPA) plays an important role in plasminogen activation system, which has many physiological and pathological functions and is closely associated with the metastasis of tumor cells. Based on a mono-cyclic peptidic inhibitor of murine uPA (muPA), mupain-1, which was screened out from a phage-display library before, we elucidated the binding and inhibitory mechanism by using multiple techniques, like X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and surface plasmon resonance analysis. By studying the peptide-enzyme interaction, we discovered an unusual inhibitor-protease inhibitory model. In the following work, together with the contribution of my colleagues we finally generated a mupain-1 variant inhibiting muPA with a Ki value around 0.2 nM, which is the highest-affinity small molecular inhibitor of uPAs in different species reported until now.
In addition, we also discovered that the mupain-1 scaffold is highly versatile, based on which mupain-1 is potentially able to be retargeted to other serine proteases in the trypsin-like clan. With the scaffold of mupain-1, we rationally designed three inhibitors with high affinity and specificity for another serine protease, plasma kallikrein, which contributes to the pathogenesis in hereditary angioedema. According to the X-ray crystal structure analysis, we proposed a principle for designing inhibitors of other serine proteases from mupain-1.
In order to be able to evaluate the inhibitory activities of our peptides in vivo, we conjugated polyethylene glycol (PEG) to one of our peptidic muPA inhibitor in order to prolonging the circulating life in animals. The PEGylation prevented the peptides from degradation in murine plasma. Furthermore, by estimating the activities of peptides after intravenous injection, we proved the circulating time of the peptides in vivo was significantly improved after coupling with PEG.
Original languageEnglish
Number of pages212
Publication statusPublished - 11 Sep 2015

Bibliographical note

During his studies, Peng Xu developed peptide inhibitors targeting two serine proteases, urokinase and plasma kallikrein. Unregulated activity of both has been implicated in serious diseases, including cancer and heretidary angioedema. Using a variety of biochemical and biophysical methods, Peng Xu developed inhibitors with high affinity as well as high specificity for the respective enzymes and provided an in depth understanding of their inhibitory mechanism. Peng Xu also chemically modified the peptides, thus preparing them for testing in in vivo disease models. Peng Xu work in this PhD programme resulted in 4 scientific articles, among which he is the first author of two.

The PhD degree was completed at the Department of Molecular Biology and Genetics, Graduate School of Science and Technology, Aarhus University.

This résumé is prepared by the PhD student.

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