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

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.

Ranacyclin-NF, a Novel Bowman–Birk Type Protease Inhibitor from the Skin Secretion of the East Asian Frog, Pelophylax nigromaculatus

“…Some publications believe that protease inhibitors cannot work effectively when it comes to human proteases [36]. However, RNF and its designed analogues, as well as some other reported BBI peptides such as OSTI and PPF-BBI, show obvious tryptase inhibitory activity, which was different to previous reports [24,32]. It could be that the larger protease inhibitors might be too large to approach the reactive sites on tryptase and if this should be the case, then some smaller protease inhibitors, such as peptides of the BBI family, may have a great advantage in accessing these active sites.…”

“…RNF GAPRGCWTKSYPPQPCF-NH 2 3 447 Ranacyclin-T [10] GALRGCWTKSYPPKPCK-NH 2 5 116 HJTI [31] GAPKGCWTKSYPPQPCS-NH 2 3 388 OSTI [24] AALKGCWTKSIPPKPCF-NH 2 4 0.3 PPF-BBI [32] ALRGCWTKSIPPKPCP-NH 2 4 170…”

Identification and Target-Modification of SL-BBI: A Novel Bowman–Birk Type Trypsin Inhibitor from Sylvirana latouchii

“…It is well known that rich in positively charged amino acids is one of the characteristics of antimicrobial peptides [35], which means that trypsin-like proteases that regard Lys and Arg as cleavage sites are easy to hydrolyse them. The antimicrobial effect of Ranacyclin family peptides is attributed to the increase in permeability of cytoplasmic phospholipid membranes and the formation of transmembrane pores by inserting hydrophobic cores [2,13]. This mechanism of maintaining the integrity of the cell membrane is different from most antimicrobial peptides.…”

“…Although the specific mechanism of BBI as an anticancer factor is unclear, protease inhibition has been shown to reverse the development in the early stages of tumorigenesis. The broad-spectrum anti-proliferation effects by BBIs act on cancer types such as lung [5,13], prostate [5,8], colon [6], breast [9,14,15], and oral [16]. It is particularly worth mentioning that the few side effects [5,13,15,17] and radioprotection [18,19] to normal mammalian cells establish the cancer chemoprevention properties of such peptides.…”

“…The broad-spectrum anti-proliferation effects by BBIs act on cancer types such as lung [5,13], prostate [5,8], colon [6], breast [9,14,15], and oral [16]. It is particularly worth mentioning that the few side effects [5,13,15,17] and radioprotection [18,19] to normal mammalian cells establish the cancer chemoprevention properties of such peptides. Many types of researched protease inhibitors also have gained huge significance as potential pharmacological tools in cancer treatment [15,[20][21][22] may be through inhibiting the activity of proteasome 20S and promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition [14].…”