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

Abstract

<jats:p>Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity.</jats:p>

Bowman-Birk Inhibitors: Insights into Family of Multifunctional Proteins and Peptides with Potential Therapeutical Applications

“…The relationship between the net charge and activity has also been observed in the case of very recently isolated ranacyclin NF (RNF) (from East Asian frog, Pelophylax nigromaculatus) [30] and SL-BBI (broad-folded frog Sylvirana latouchii) [169]. Similar to HJTI, RNF and SL-BBI (Figure 1) are moderate trypsin inhibitors.…”

“…Moreover, the increased α-helical content has contributed to better antimicrobial abilities of K-SL [169]. It has also been observed for RNF and its analogs that these with limited possibility to α-helix or β-sheet formation are weaker trypsin inhibitors, similarly to peptides with C-terminal carboxyl group [30]. Interestingly, only RNF has demonstrated weak antimicrobial activity against S. aureus, while its analog with the C-terminal carboxyl group is inactive [30].…”

“…Some BBIs are also blocking proteases produced by pathogens; thus, they have the potential to be used as antimicrobials [29]. As mentioned before, the BBIs family was established as a bunch of plant-derived inhibitors; however, a novel group of peptides originating from animals, which imitates the BBI's trypsin inhibitory loop (TIL), has been recently identified [30][31][32]. These peptides were isolated from frogs' skin, and similarly to plant BBIs, they present strong trypsin inhibitory activity.…”

“…These peptides were isolated from frogs' skin, and similarly to plant BBIs, they present strong trypsin inhibitory activity. Their disulfide-bridged loop contains 11 residues, with the general formula CWTP1SX 1 PPX 2 PC (where P1 is the inhibitory active site usually occupied by Lys, while X 1 and X 2 are variable) [30,32]. This loop is longer than that found in plant BBIs composed of 9-amino acids (CTP1SX 1 PPX 2 C).…”

“…In recent years, the interest in amphibian-derived BBLTIs is growing rapidly, which is reflected in an increasing number of publications reporting newly isolated members of this group [30][31][32]. Recently, a novel I99 family in the MEROPS database dedicated to such Bowman-Birk-like inhibitors has been established [2].…”