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

Abstract

Kunitz-like trypsin inhibitors are one of the most noteworthy research objects owing to their significance in pharmacological studies, including anticarcinogenic activity, obesity regulation and anticoagulation. In the current study, a novel Kunitz-like trypsin inhibitor, Kunitzin-AH, was isolated from the skin secretion of Amolops hainanensis. The novel peptide displayed a modest trypsin inhibitory activity with the inhibitor constant (Ki) value of 1.18 ± 0.08 µM without inducing damage to healthy horse erythrocytes. Then, a series of shortened variants of Kunitzin-AH were designed by truncating a peptide loop and site mutation inside the loop to illustrate the structure–activity relationship of the trypsin inhibition function. Among the variants, a significant decrease was observed for the Cys-Cys loop domain, while the extension of an Arg at N-terminus (RCKAAFC) retained the inhibitory activity, indicating that the -RCK-motif is essential in forming the reactive domain for exerting the inhibitory activity. Furthermore, substitutions of Ala by hydrophobic or hydrophilic residues decreased the activity, indicating suitable steric hindrance provides convenience for the combination of trypsin. Additionally, the conformational simulation of the analogues processed with Chimera and Gromacs and further combination simulations between the peptides and trypsin conducted with HDOCK offered a potential opportunity for the natural trypsin inhibitory drug design. The truncated sequence, AH-798, may be a good replacement for the full-length peptide, and can be optimized via cyclization for further study.

Structural, biochemical characterization and molecular mechanism of Cerastokunin: A new Kunitz-type peptide with potential inhibition of thrombin, factor Xa and platelets

“…AH-884 is a variant of kunitz-like trypsin inhibitor peptide from the skin secretion of Amolops hainanensis, named Kunitzin-AH presenting trypsin inhibitory activity. The docking simulation of Trypsin with AH-884 has demonstrated that four (4) amino acid residues Ser789, Asp792, Lys702, and Tyr 681 trypsin bond to Arg1, Asn5 and Cys7 residues of the AH-884 [53]. These reported results matched up with the docking data of the current study that illustrated the involvement of Cerastokunin Cys16 and Asn17 in the interaction with trypsin.…”

Expression and Purification of Recombinant Bowman–Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo

“…The total protein concentration was quantified using a BCA assay, and the yield of rFMB-BBTI was approximately 11.30 mg/L. The biological function of a protein is closely related to its conformation, and CD is often used to evaluate the structure and stability of designed proteins. , To evaluate the effect of recombinant expression on the structure of FMB-BBTI protein, the secondary structure of FMB-BBTI was predicted by PSIPRED platform (Figure E). Further, the secondary structures of FMB-BBTI and rFMB-BBTI were analyzed by CD (Figure F).…”

In Vitro & In Vivo Studies on Identifying and Designing Temporin-1CEh from the Skin Secretion of Rana chensinensis as the Optimised Antibacterial Prototype Drug

“…The shot-gun cloning was performed to isolate the prepropeptide encoding RNA, as described before [ 21 ]. In the 3′-RACE reaction, a nested universal primer (NUP) and a degenerate primer specially designed according to the highly conserved 5′-untranslated region of previously characterised peptide precursor cDNAs from closely-related Rana species (5′-ACTTTCYGAWTTRYAAGMCCAAABATG-3′, Y = C + T, W = A + T, R = A + G, M = A + C, B = T + C + G) were used in a segment of the 5′-untranslated region.…”