Abstract
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.
Highlights
Serine proteases exist in almost all living organisms [1], and, in humans, they are intimately involved in many disease processes, such as in infections, coagulation disorders, cancer and inflammation [2,3]
The alignment of nucleotide and peptide precursor amino acid sequence exhibited a high degree of similarity between ranacyclin-HB1
They believed that structural features, especially for amino acid residues lying outside the trypsin inhibitory loop (TIL), were crucial in modulating the effectiveness of trypsin inhibitory activity [31]
Summary
Serine proteases exist in almost all living organisms [1], and, in humans, they are intimately involved in many disease processes, such as in infections, coagulation disorders, cancer and inflammation [2,3]. Most current serine protease-targeted drugs are small molecules, there is an increasing attention shift to polypeptide inhibitors as these more complex molecules offer an achievable approach for optimising the affinity and specificity of inhibitors. Inhibitor scaffolds are normally contained in natural proteins or peptides which have been optimised and improved through a process of parallel evolution for activities against endogenous targets [4]. Bowman–Birk inhibitors (BBIs) exist in a wide range of plants including sunflowers, soybeans, legumes and so on [5]. They are classical serine proteinase inhibitors that contain a highly-conserved disulphide loop structure consisting of nine residues (CTP1 SXPPXC) [6,7,8]. BBI peptides derived from amphibians are slightly different and typically contain a disulphide-bridged hendecapeptide
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