nanoRAPIDS as an analytical pipeline for the discovery of novel bioactive metabolites in complex culture extracts at the nanoscale.

Microbial natural products form the basis of most of the antibiotics used in the clinic. The vast majority has not yet been discovered, among others because the hidden chemical space is obscured by previously identified (and typically abundant) antibiotics in culture extracts. Efficient dereplication is therefore key to the discovery of our future medicines. Here we present an analytical platform for the efficient identification and prioritization of low abundance bioactive compounds at nanoliter scale, called nanoRAPIDS. NanoRAPIDS encompasses analytical scale separation and nanofractionation of natural extracts, followed by the bioassay of interest, automated mass spectrometry identification, and Global Natural Products Social molecular networking (GNPS) for dereplication. As little as 10 µL crude extract is fractionated into 384 fractions. First, bioactive congeners of iturins and surfactins were identified in Bacillus, based on their bioactivity. Subsequently, bioactive molecules were identified in an extensive network of angucyclines elicited by catechol in cultures of Streptomyces sp. This allowed the discovery of a highly unusual N-acetylcysteine conjugate of saquayamycin, despite low production levels in an otherwise abundant molecular family. These data underline the utility and broad application of the technology for the prioritization of minor bioactive compounds in complex extracts.

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches.

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 A2 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 specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.

Analytics for Bioactivity Profiling of Complex Mixtures with a Focus on Venoms.

This chapter introduces bioactivity and bioaffinity terms in relation to mixture profiling and gives the significance of bioactivity and/or bioaffinity profiling of biologically active mixtures in general, and for bioactive mixtures in drug discovery research in particular. Further, the chapter gives an overview of the common and less common analytical approaches for bioactivity profiling of bioactive mixtures. Special focus is put on bioassay-guided fractionation as the standard technique employed (in identification and purification of bioactive molecules from a bioactive mixture), and on state-of-the-art post-column bioactivity profiling approaches, also providing examples and limitations of these analytical methods. On-column and pre-column bioactivity profiling analytics is also discussed. Examples of bioactive molecules identified and purified from different natural products are given with emphasis on molecules isolated from animal venoms. Finally, this chapter briefly discusses the importance of bioactivity profiling of metabolic mixtures in drug discovery.

Detection and identification of antibacterial proteins in snake venoms using at-line nanofractionation coupled to LC-MS.

This study describes the application of at-line nanofractionation to the screening of snake venoms for antibacterial activity against Gram-positive and Gram-negative bacteria, the detection of proteins of interest, and their partial or full identification. A method was developed to identify bioactive peptides in crude snake venoms based on reversed-phase liquid chromatography (LC), with parallel nanofractionation onto 384-well plates and mass spectrometry (MS). Bioactivity assays were based on a resazurin-reduction assay. Accurate masses of the bioactive peptides were determined, and peptides were then identified via nanoLC-MS/MS analysis of tryptic digests, allowing full or partial identification of the bioactive proteins. Crude venoms from 41 species were screened for their antibacterial bioactivity. Venoms showing the highest activity were further screened using at-line nanofractionation, which resulted in the elucidation of 28 bioactive proteins.

Nanofractionation Platform with Parallel Mass Spectrometry for Identification of CYP1A2 Inhibitors in Metabolic Mixtures.

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.

Rapid screening and identification of ACE inhibitors in snake venoms using at-line nanofractionation LC-MS.

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.

At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor.

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 high-resolution 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.

At-line nanofractionation with parallel mass spectrometry and bioactivity assessment for the rapid screening of thrombin and factor Xa inhibitors in snake venoms.

Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease bioactivity fingerprints in biological studies on venoms.

At-line coupling of LC-MS to bioaffinity and selectivity assessment for metabolic profiling of ligands towards chemokine receptors CXCR1 and CXCR2.

This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation. The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40pmol in CXCR1 binding and 8pmol in CXCR2 binding. For CXCL8, the LOD was 5pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314±59nM (CXCR1 binding) and 38±11nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9±1.4nM (CXCR1 binding) and 2.7±1.3nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC-MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.

Metabolic profiling of ligands for the chemokine receptor CXCR3 by liquid chromatography-mass spectrometry coupled to bioaffinity assessment.

Chemokine receptors belong to the class of G protein-coupled receptors and are important in the host defense against infections and inflammation. However, aberrant chemokine signaling is linked to different disorders such as cancer, central nervous system and immune disorders, and viral infections [Scholten DJ et al. (2012) Br J Pharmacol 165(6):1617-1643]. Modulating the chemokine receptor function provides new ways of targeting specific diseases. Therefore, discovery and development of drugs targeting chemokine receptors have received considerable attention from the pharmaceutical industry in the past decade. Along with that, the determination of bioactivities of individual metabolites derived from lead compounds towards chemokine receptors is crucial for drug selectivity, pharmacodynamics, and potential toxicity issues. Therefore, advanced analytical methodologies are in high demand. This study is aimed at the optimization of a new analytical method for metabolic profiling with parallel bioaffinity assessment of CXCR3 ligands of the azaquinazolinone and piperazinyl-piperidine class and their metabolites. The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures. The bioaffinity assessment is performed "at-line" via high-resolution nanofractionation onto 96-well plates allowing direct integration of radioligand binding assays. This new method enables identification of metabolites from lead compounds with associated estimation of their individual bioaffinity. Moreover, the identification of the metabolite structures via accurate mass measurements and MS(2) allows the identification of liable metabolic "hotspots" for further lead optimization. The efficient combination of chemokine receptor ligand binding assays with analytical techniques, involving nanofractionation as linking technology, allows implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

High-resolution fractionation after gas chromatography for effect-directed analysis.

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.