In Vitro Selection of Cathepsin E-Activity-Enhancing Peptide Aptamers at Neutral pH.

Madhu Biyani,Masae Futakami,Koichiro Kitamura,Koichi Nishigaki,Kenji Yamamoto,Tomoyo Kawakubo,Miho Suzuki

doi:10.1155/2011/834525

Abstract

The aspartic protease cathepsin E has been shown to induce apoptosis in cancer cells under physiological conditions. Therefore, cathepsin E-activity-enhancing peptides functioning in the physiological pH range are valuable potential cancer therapeutic candidates. Here, we have used a general in vitro selection method (evolutionary rapid panning analysis system (eRAPANSY)), based on inverse substrate-function link (SF-link) selection to successfully identify cathepsin E-activity-enhancing peptide aptamers at neutral pH. A successive enrichment of peptide activators was attained in the course of selection. One such peptide activated cathepsin E up to 260%, had a high affinity (KD; ∼300 nM), and had physiological activity as demonstrated by its apoptosis-inducing reaction in cancerous cells. This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers.

Highlights

Besides other targets like kinases and membrane receptors, proteases are one of the most effective targets for drug discovery [1, 2]
To address the above difficulty, we have recently developed a systemic in vitro evolution method (called eRAPANSY) [9] for screening of protease-regulating peptide aptamers based on mRNA display [10,11,12] and a substrate-function link (SF-link) method [13]
We describe here the successful development of one promising peptide aptamer (S3) that enhanced cathepsin E- (CatE-)activity by up to 260% in vitro and which induced apoptosis in cancer cells (HeLa)

Summary

Introduction

Besides other targets like kinases and membrane receptors, proteases are one of the most effective targets for drug discovery [1, 2]. To address the above difficulty, we have recently developed a systemic in vitro evolution method (called eRAPANSY (evolutionary rapid panning analysis system)) [9] for screening of protease-regulating peptide aptamers based on mRNA (and cDNA) display [10,11,12] and a substrate-function link (SF-link) method [13]. This method was described in the successful identification of cathepsin E- (CatE-) inhibitory peptide aptamers [9].

Methods

Results

Conclusion