Drug Discovery on Natural Products: From Ion Channels to nAChRs, from Nature to Libraries, from Analytics to Assays.

Natural extracts are complex mixtures that may be rich in useful bioactive compounds and therefore are attractive sources for new leads in drug discovery. This review describes drug discovery from natural products and in explaining this process puts the focus on ion-channel drug discovery. In particular, the identification of bioactives from natural products targeting nicotinic acetylcholine receptors (nAChRs) and serotonin type 3 receptors (5-HT3Rs) is discussed. The review is divided into three parts: "Targets," "Sources," and "Approaches." The "Targets" part will discuss the importance of ion-channel drug targets in general, and the α7-nAChR and 5-HT3Rs in particular. The "Sources" part will discuss the relevance for drug discovery of finding bioactive compounds from various natural sources such as venoms and plant extracts. The "Approaches" part will give an overview of classical and new analytical approaches that are used for the identification of new bioactive compounds with the focus on targeting ion channels. In addition, a selected overview is given of traditional venom-based drug discovery approaches and of diverse hyphenated analytical systems used for screening complex bioactive mixtures including venoms.

Neurotoxicity fingerprinting of venoms using on-line microfluidic AChBP profiling.

Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins.

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT3 A receptor.

BACKGROUND AND PURPOSE: The 5-HT3 receptor is a prototypical member of the Cys-loop ligand-gated ion channel (LGIC) superfamily and an established therapeutic target. In addition to activation via the orthosteric site, receptor function can be modulated by allosteric ligands. We have investigated the pharmacological action of Cl-indole upon the 5-HT3 A receptor and identified that this positive allosteric modulator possesses a novel mechanism of action for LGICs. EXPERIMENTAL APPROACH: The impact of Cl-indole upon the 5-HT3 receptor was assessed using single cell electrophysiological recordings and [3 H]-granisetron binding in HEK293 cells stably expressing the 5-HT3 receptor. KEY RESULTS: Cl-indole failed to evoke 5-HT3 A receptor-mediated responses (up to 30 µM) or display affinity for the [3 H]-granisetron binding site. However, in the presence of Cl-indole, termination of 5-HT application revealed tail currents mediated via the 5-HT3 A receptor that were independent of the preceding 5-HT concentration but were antagonized by the 5-HT3 receptor antagonist, ondansetron. These tail currents were absent in the 5-HT3 AB receptor. Furthermore, the presence of 5-HT revealed a concentration-dependent increase in the affinity of Cl-indole for the orthosteric binding site of the human 5-HT3 A receptor. CONCLUSIONS AND IMPLICATIONS: Cl-indole acts as both an orthosteric agonist and an allosteric modulator, but the presence of an orthosteric agonist (e.g. 5-HT) is a prerequisite to reveal both actions. Precedent for ago-allosteric action is available, yet the essential additional presence of an orthosteric agonist is now reported for the first time. This widening of the pharmacological mechanisms to modulate LGICs may offer further therapeutic opportunities.

Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry.

This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC-FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist-agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist-agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup.

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.

Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa.

Thrombin and factor Xa (FXa) are critical enzymes of the blood coagulation cascade and are excellent targets of anticoagulant agents. Natural sources present an array of anticoagulants that can be developed as antithrombotic drugs. High-resolution, online screening techniques have been developed for the identification of drug leads from complex mixtures. In this study, we have developed and optimized a microfluidic online screening technique coupled to nano-liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are suitable for the identification of inhibitors from sample amounts as low as sub-microliter volumes.

Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms.

The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach.

Miniaturized bioaffinity assessment coupled to mass spectrometry for guided purification of bioactives from toad and cone snail.

A nano-flow high-resolution screening platform, featuring a parallel chip-based microfluidic bioassay and mass spectrometry coupled to nano-liquid chromatography, was applied to screen animal venoms for nicotinic acetylcholine receptor like (nAChR) affinity by using the acetylcholine binding protein, a mimic of the nAChR. The potential of this microfluidic platform is demonstrated by profiling the Conus textile venom proteome, consisting of over 1,000 peptides. Within one analysis (<90 min, 500 ng venom injected), ligands are detected and identified. To show applicability for non-peptides, small molecular ligands such as steroidal ligands were identified in skin secretions from two toad species (Bufo alvarius and Bufo marinus). Bioactives from the toad samples were subsequently isolated by MS-guided fractionation. The fractions analyzed by NMR and a radioligand binding assay with α7-nAChR confirmed the identity and bioactivity of several new ligands.

Analytical workflow for rapid screening and purification of bioactives from venom proteomes.

Animal venoms are important sources for finding new pharmaceutical lead molecules. We used an analytical platform for initial rapid screening and identification of bioactive compounds from these venoms followed by fast and straightforward LC-MS only guided purification to obtain bioactives for further chemical and biological studies. The analytical platform consists of a nano-LC separation coupled post-column to high-resolution mass spectrometry and parallel on-line bioaffinity profiling for the acetylcholine binding protein (AChBP) in a chip based fluorescent enhancement based bioassay. AChBP is a stable structural homologue of the extracellular ligand binding domain of the α7-nicotinic acetylcholine receptor (α7-nAChR). This receptor is an extensively studied medicinal target, previously associated with epilepsy, Alzheimer's, schizophrenia and anxiety. The workflow is demonstrated with the venom of the Naja mossambica mossambica. Two medium affinity AChBP ligands were found. After subsequent LC-MS guided purification of the respective venom peptides, the purified peptides were sequenced and confirmed as Cytotoxin 1 and 2. These peptides were not reported before to have affinity for the AChBP. The purified peptides can be used for further biological studies.

An efficient analytical platform for on-line microfluidic profiling of neuroactive snake venoms towards nicotinic receptor affinity.

Venomous snakes have evolved their efficient venomous arsenals mainly to immobilize prey. The highly variable toxic peptides in these venoms target a myriad of neurotoxic and haemotoxic receptors and enzymes and comprise highly interesting candidates for drug discovery. Discovery of bioactive compounds from snake venoms, however, is a challenge to achieve. We have developed and applied a methodology to rapidly assess bioactives in a snake venom proteome. Our microfluidic platform opens up efficient and rapid profiling of venomous anti-cholinergic receptor compounds. The key advantages of our methodology are: (i) nano amounts of venom needed; and (ii) a direct correlation of selected bioaffinities with accurate mass. To achieve this, we have for the first time successfully constructed a functional post nano-LC split to MS and bioaffinity profiling. In our method, comprehensive venom profiles with accurate masses and corresponding bioaffinities are obtained in one analytical run and will subsequently allow immediate purification of bioactive peptides with LC-MS, guided by accurate masses of the bioactives only. We profiled several neurotoxic Elapidae snake venoms using our methodology in combination with the acetylcholine binding protein (AChBP) as biological target protein. The latter is a homologue of nicotinic acetylcholine receptors (nAChRs), a drug target in neurodegenerative diseases and cognitive decline such as Parkinson's and Alzheimer's, and in pain related diseases. Our methodology was evaluated and validated with high-affinity α-bungarotoxin and haemotoxic/proteolytic Vipera ammodytes venom spiked with α-bungarotoxin. Thereafter, the methodology was applied to profile the venom proteomes of Dendroaspis jamesoni kaimosae, Naja annulifera and Naja nivea. Gathering comprehensive profiling data took less than 2 h per snake venom measured. The data yielded 20 AChBP ligands of which the corresponding accurate masses were used to retrieve information from literature regarding their function and targeting specificity. We found that from these 20 ligands, 11 were previously reported on, while information on the others could not be found. From these 11 peptides, five have been reported to have nAChR affinity, while the others are reported as cytotoxic, cardiotoxic or as orphan toxin. Our methodology has the potential to aid the field of profiling complex animal venoms for drug discovery.

Click reaction synthesis of carbohydrate derivatives from ristocetin aglycon with antibacterial and antiviral activity.

New sugar derivatives of ristocetin were prepared by copper-catalyzed 1,3-dipolar cycloaddition reaction using azido-ristocetin aglycon and various propargyl glycosides. Some of them were found to be active against gram-positive bacteria and showed favorable antiviral activity against the H1N1 subtype of influenza A virus.