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

Abstract

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman–Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman–Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2′ site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman–Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

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.…”

“…Similarly, SL-BBI analog, K-SL, with a higher positive charge, has exhibited stronger antimicrobial potency and anti-trypsin activity. 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].…”

“…Importantly, both peptides have demonstrated low cytotoxic and hemolytic activities [167]. Similarly, the analog of recently isolated SL-BBI [169], F-SL with Phe in the P1 position, has presented inhibitory potency towards chymotrypsin and proteasome and exerted antiproliferative effect in a series of cancer cell lines, including non-small cell lungs (H157, H460, H838, and H23), prostate (PC-3), and breast (MCF-7) [169].…”

Structure–Activity Relationship and Molecular Docking of a Kunitz-Like Trypsin Inhibitor, Kunitzin-AH, from the Skin Secretion of Amolops hainanensis

“…On the one hand, disulfide bonds are of great importance in stabling specific structures and maintaining bioactivities for proteins [ 20 , 21 ]. When those bonds are broken and reduced to sulfhydryl groups, structures of proteins will undoubtedly be rebuilt, and their biological activities will change more or less.…”

Novel Amphibian Bowman–Birk-Like Inhibitor with Antioxidant and Anticoagulant Effects Ameliorates Pancreatitis Symptoms in Mice

“…Further, the cysteine residues at the beginning and end of the motif form a disulfide bond . To date, at least 14 of BBI peptides possessing trypsin inhibitory activity and belonging to the Ranacyclin family of antimicrobial peptides have been identified in several frogs. − It is worth mentioning that the consensus sequence of the trypsin inhibitory loop (TIL) within these BBIs corresponds to CWTP 1 SX 1 PPX 2 PC and its P 1 is usually occupied by Lys. Nevertheless, despite their structural similarity and common trypsin inhibitory activity, some of these BBIs show some different biological functions. , For example, most of them, with the exception of HJTI, pLR-HL, OSTI, and PE-BBI, exhibit antimicrobial activity.…”