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Evaluation of Peptides as Protease Inhibitors and Stimulators

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1088)

Abstract

Changes in proteolytic activity are associated with several diseases, including cancer. Proteases are potential drug targets and targeting of proteases is used for treatment of various conditions/diseases, like high blood pressure and HIV. We present here detailed protocols for basic evaluation of the effects of peptides on the activity of proteases, using kallikrein-related peptidases KLK2 and KLK3 (also known as hK2 and PSA), and trypsin as examples. KLK2 and KLK3 are major prostatic proteases, and they are potential targets for prostate cancer treatment. KLK2 has trypsin-like activity and KLK3 chymotrypsin-like activity. By phage display technology, we have developed peptides that specifically stimulate KLK3-activity and other peptides that inhibit KLK2 or trypsin. The effect of the peptides on the proteolytic activity of proteases can be studied using substrates, the cleavage of which generates detectable signal, allowing rapid evaluation of protease activity. The cleavage of protein substrates can be detected by SDS-PAGE, followed by staining of the proteins. We also describe graphical analysis of the IC50-value, the effect of a peptide on Michaelis-Menten constant (K m) and the maximal reaction rate (V max).

Key words

Protease Peptide Inhibition Kinetics SDS-PAGE Kallikrein hK2 PSA Phage display 
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Notes

Acknowledgments

The authors wish to thank Dr Leena Valmu for the silver staining protocol and Ms. Helena Taskinen for technical assistance. Our original studies referred to have been supported by National Technology Agency of Finland, University of Helsinki, Helsinki University Central Hospital, the Finnish Cancer Foundation, the Academy of Finland, Juselius Foundation and Finska Läkaresällskapet.

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© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  1. 1.Department of Clinical Chemistry, BiomedicumUniversity of Helsinki and Helsinki University Central HospitalHelsinkiFinland
  2. 2.Department of UrologyThe University of Texas Health Science Center at San AntonioSan AntonioUSA
  3. 3.School of PharmacyUniversity of Eastern FinlandKuopioFinland

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