Abstract
Background:Proteases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated with several pathologies. Therefore, proteases have become potential therapeutic targets regarding the search for inhibitors. Snake venoms are complex mixtures of molecules that can feature a variety of functions, including peptidase inhibition. Considering this, the present study reports the purification and characterization of a Kunitz-type peptide present in the Dendroaspis polylepis venom as a simultaneous inhibitor of elastase-1 and cathepsin L. Methods:The low molecular weight pool from D. polylepis venom was fractionated in reverse phase HPLC and all peaks were tested in fluorimetric assays. The selected fraction that presented inhibitory activity over both proteases was submitted to mass spectrometry analysis, and the obtained sequence was determined as a Kunitz-type serine protease inhibitor homolog dendrotoxin I. The molecular docking of the Kunitz peptide on the elastase was carried out in the program Z-DOCK, and the program RosettaDock was used to add hydrogens to the models, which were re-ranked using ZRANK program. Results:The fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 μM. The docking analysis led us to synthesize two peptides: PEP1, which was substrate for both elastase-1 and cathepsin L, and PEP2, a 30-mer cyclic peptide, which showed to be a cathepsin L competitive inhibitor, with a Ki of 1.96 µM, and an elastase-1 substrate. Conclusion:This work describes a Kunitz-type peptide toxin presenting inhibitory potential over serine and cysteine proteases, and this could contribute to further understand the envenomation process by D. polylepis. In addition, the PEP2 inhibits the cathepsin L activity with a low inhibition constant.
Highlights
Proteases play an important role for the proper physiological functions of the most diverse organisms
The fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 μM
This fact can be well exemplified by bradykinin-potentiating peptides (BPPs), the first natural inhibitors described for the Angiotensin Converting Enzyme (ACE) [4]
Summary
Proteases play an important role for the proper physiological functions of the most diverse organisms. According to World Health Organization (WHO) data, globally, around 5.4 million accidents are reported per year, causing more than 435,000 amputations and 81,000 deaths. Due to this high number of accidents, and deaths, snakebite envenomation is considered a tropical neglected disease since 2017 by the WHO [1]. Toxins can be used as drugs (or prototypes for the development of new drugs) both for their selectivity and potency This fact can be well exemplified by bradykinin-potentiating peptides (BPPs), the first natural inhibitors described for the Angiotensin Converting Enzyme (ACE) [4]. Other features of the small molecules present in snake venoms, studies and research of these compounds are relevant
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