Abstract
This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC50 = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC50 = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.
Highlights
Snake venoms comprise a myriad of bioactive peptides and proteins causing multiple physiological reactions upon envenomation of a prey organism
Prior to optimization of the analytical methodology and the screening of snake venoms, the angiotensin-converting enzyme (ACE) bioassay used in 384well plate was optimized regarding the substrate and enzyme concentration
A total of 30 snake venoms were screened for the presence of ACE inhibitors using at-line nanofractionation approach
Summary
Snake venoms comprise a myriad of bioactive peptides and proteins causing multiple physiological reactions upon envenomation of a prey organism. The diversity of effects involving varying mechanisms of action, together with the high target specificity of each single constituent, makes snake venoms very attractive materials in the discovery of new drugs for the treatment of different diseases. ACE is a zinc-dependent metallopeptidase which plays an important role in regulation of blood pressure in the organism. It is responsible for the conversion of angiotensin I into angiotensin II, a very potent vasoconstrictor, and for the deactivation of bradykinin, an endogenous vasodilating peptide
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