Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A 2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling PLOS NEGLECTED TROPICAL DISEASES

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This research presents an analytical technology for highly efficient, high-resolution, and high-yield fractionation of compounds after gas chromatography (GC) separations. The technology is straightforward, does not require sophisticated cold traps or adsorbent traps, and allows collecting large numbers of fractions during a GC run. The technology is based on direct infusion of a carrier solvent at the end of the GC column, where infusion takes place in the GC oven. Pentane and hexane used as carrier solvent showed good results. Acetonitrile also showed good results as a more polar carrier solvent. Development and optimization of the technology is described, followed by demonstration in a high-throughput effect directed analysis setting toward dioxin receptor bioactivity. The GC fractionation setup was capable of collecting fractions in the second range. As a result, fractionated compounds could be collected into one or two fractions when 6.5 s resolution fractionation was performed. Subsequently, mixtures containing polycyclic aromatic hydrocarbons, of which some are bioactive toward the dioxin receptor, were profiled with a mammalian gene reporter assay. After fractionation into 96-well plates, we used our new approach for direct cell seeding onto the fractions prior to assaying which allowed dioxin receptor bioactivity to be measured directly after fractionation. The current technology represents a great advance in effect directed analysis for environmental screening worldwide as it allows combining the preferred analytical separation technology for often non-polar environmental pollutants with environmentally relevant bioassays, in high resolution.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-017-0531-3) contains supplementary material, which is available to authorized users.

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in different regions of the central nervous system (CNS). The α7-nAChR has been associated with Alzheimer's disease, epilepsy, and schizophrenia, and therefore is extensively studied as a drug target for the treatment of these diseases. Important sources for new compounds in drug discovery are natural extracts. Since natural extracts are complex mixtures, identification of the bioactives demands the use of analytical techniques to separate a bioactive from inactive compounds. This study describes screening methodology for identifying bioactive compounds in mixtures acting on the α7-nAChR. The methodology developed combines liquid chromatography (LC) coupled via a split with both an at-line calcium (Ca 2+ )-flux assay and highresolution mass spectrometry (MS). This allows evaluation of α7-nAChR responses after LC separation, while parallel MS enables compound identification. The methodology was optimized for analysis of agonists and positive allosteric modulators, and was successfully applied to screening of the hallucinogen mushroom Psilocybe Mckennaii. The crude mushroom extract was analyzed using both reversed-phase and hydrophilic interaction liquid chromatography. Matching retention times and peak shapes of bioactives found with data from the parallel MS measurements allowed rapid pinpointing of accurate masses corresponding to the bioactives.

17Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in 18 envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause 19 severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The 20 mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant 21 and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and 22 peptide components, high throughput characterizations of specific target bioactives is challenging. In this 23 study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins 24 from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput 25 screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom 26 nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid 27 chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, 28 while the larger fraction was collected and dispensed onto 384-well plates before direct analysis using a 29 plasma coagulation assay. Our results demonstrate that many snake venoms simultaneously contain both 30 procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis 31 from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that 32 phospholipase A 2 toxins are frequently identified in anticoagulant venom fractions, while serine protease 33 and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation 34 and proteomics approach applied herein seems likely to be a valuable tool for the rational development of 35 next-generation snakebite treatments by facilitating the rapid identification and fractionation of 36 coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as 37 monoclonal antibodies and small molecule inhibitors. Classified Personnel Information 39Author summary 40 Snakebite is a neglected tropical disease that results in more than 100,000 deaths every year. 41Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and 42 many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to 43 suffering life-threating hemorrhage. In this study, we applied a combination of analytical and 44 pharmacological methods to identify snake venom toxins from a wide diversity of snake species that 45 interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood 46 coagulation cascade and based on the initial results and the medical relevance of the species, seven 47 venoms were selected for in-depth analysis of the responsible toxins using advanced identification 48 techniques. Our findings reveal a number ...

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With early assessment of inhibitory properties of drug candidates and their circulating metabolites toward cytochrome P450 enzymes, drug attrition, especially later in the drug development process, can be decreased. Here we describe the development and validation of an at-line nanofractionation platform, which was applied for screening of CYP1A2 inhibitors in Phase I metabolic mixtures. With this platform, a metabolic mixture is separated by liquid chromatography (LC), followed by parallel nanofractionation on a microtiter well plate and mass spectrometry (MS) analysis. After solvent evaporation, all metabolites present in the nanofractionated mixture are assayed utilizing a fluorescence CYP1A2 inhibition bioassay performed on the plate. Next, a bioactivity chromatogram is constructed from the bioassay results. By peak shape and retention time correlation of the bioactivity peaks with the obtained MS data, CYP1A2-bioactive inhibiting metabolites can be identified. The method correctly evaluated the potency of five CYP1A2 inhibitors. Mixtures comprising potent inhibitors of CYP1A2 or in vitro-generated metabolites of ellipticine were evaluated for their inhibitory bioactivities. In both cases, good LC separation of all compounds was achieved and bioactivity data could be accurately correlated with the parallel recorded MS data. Generation and evaluation of Phase II metabolites of hydroxylated ellipticine was also pursued.