Discovery and Rational Design of a Novel Bowman-Birk Related Protease Inhibitor.

Yuxi Miao,Jinao Duan,James F Burrows,Lei Wang,Tianbao Chen,Guanzhu Chen,Chengbang Ma,Mei Zhou,Xinping Xi

doi:10.3390/biom9070280

Yuxi Miao, Jinao Duan + Show 7 more

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https://doi.org/10.3390/biom9070280

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Abstract

Anuran amphibian skin secretions are a rich source of peptides, many of which represent novel protease inhibitors and can potentially act as a source for protease inhibitor drug discovery. In this study, a novel bioactive Bowman-Birk type inhibitory hexadecapeptide of the Ranacyclin family from the defensive skin secretion of the Fukien gold-striped pond frog, Pelophlax plancyi fukienesis, was successfully isolated and identified, named PPF-BBI. The primary structure of the biosynthetic precursor was deduced from a cDNA sequence cloned from a skin-derived cDNA library, which contains a consensus motif representative of the Bowman-Birk type inhibitor. The peptide was chemically synthesized and displayed a potent inhibitory activity against trypsin (Ki of 0.17 µM), as well as an inhibitory activity against tryptase (Ki of 30.73 µM). A number of analogues of this peptide were produced by rational design. An analogue, which substituted the lysine (K) at the predicted P1 position with phenylalanine (F), exhibited a potent chymotrypsin inhibitory activity (Ki of 0.851 µM). Alternatively, a more potent protease inhibitory activity, as well as antimicrobial activity, was observed when P16 was replaced by lysine, forming K16-PPF-BBI. The addition of the cell-penetrating peptide Tat with a trypsin inhibitory loop resulted in a peptide with a selective inhibitory activity toward trypsin, as well as a strong antifungal activity. This peptide also inhibited the growth of two lung cancer cells, H460 and H157, demonstrating that the targeted modifications of this peptide could effectively and efficiently alter its bioactivity.

Highlights

Serine proteases are a widely studied group of proteins as they play various roles in healthy and diseased tissues
The open-reading frame consisted of 65 amino acid residues. The alignment of this peptide with other members of the Ranacyclin family of Bowman-Birk-type protease inhibitors indicates that it is structurally related as its sequence shows a high degree of conservation, as well as including a typical inhibition loop (Figure 1), which started with a 22-residue putative signal peptide at the N-terminus
Bowman-Birk inhibitor from the skin secretion of the Fukien gold-striped frog, In this type study,protease we report the identification and bioactivity evaluations of PPF-Birk family inhibitors (BBIs), pond a novel

Summary

Introduction

Serine proteases are a widely studied group of proteins as they play various roles in healthy and diseased tissues. Serine protease inhibitors can modulate a series of important biological processes, such as coagulation and inflammation, making them a focus for biomedical studies [1,2]. Plants are remarkable sources of the serine protease inhibitor, which can be grouped into at least. The Bowman-Birk family inhibitors (BBIs) are the best studied and most widely known among them. Identified in and isolated from soybean, they were the first to often be referred to as “classical BBI”. Subsequently multiple BBIs have been isolated from plants such as legumes and Gramineae [3,4,5]

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