Date Published: August 11, 2017
Publisher: Springer Berlin Heidelberg
Author(s): Marija Mladic, Tessa de Waal, Lindsey Burggraaff, Julien Slagboom, Govert W. Somsen, Wilfried M. A. Niessen, R. Manjunatha Kini, Jeroen Kool.
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.
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. Animal venoms have been the origin of several major drugs or drug classes. One of the best known examples is provided by angiotensin-converting enzyme (ACE) inhibitors . Other snake venom-derived drugs include antiplatelet agents, tirofiban , and eptifibatide , from the venoms of the saw-scaled viper, Echis carinatus, and the southeastern pygmy rattlesnake, Sistrurus miliarius barbouri, respectively.
A total of 30 snake venoms were screened for the presence of ACE inhibitors using at-line nanofractionation approach. Five major bioactive peaks in the venoms of three different species were detected using the newly developed method. The high-resolution at-line nanofractionation allowed to keep the resolution of LC separation and thereby allowed to accurately correlate peak shape and retention time of negative bioactive peaks observed in the bioactivity chromatograms with the XICs from the parallelly obtained MS data. A complementary HILIC separation of a crude snake venom was introduced to successfully narrow down the number of candidate m/z values for the bioactive peptides. The (partial) peptide sequence was determined by direct analysis of a bioactive well using accurate mass nanoLC–MS/MS. The developed methodology is therefore an excellent tool for rapid screening of snake venoms for ACE inhibitors followed by straightforward identification of the hits found. Furthermore, the use of this method can be extended to other drug targets and natural sources.