Publications by authors named "Alexander D Crawford"

44 Publications

Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.

J Ethnopharmacol 2021 Nov 22:114867. Epub 2021 Nov 22.

Department of Pharmaceutical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria, 0001, South Africa; SAMRC Herbal Drugs Research Unit, Tshwane University of Technology, Pretoria, South Africa. Electronic address:

Ethnopharmacological Relevance: In South Africa, medicinal plants have a history of traditional use, with many species used for treating wounds. The scientific basis of such uses remains largely unexplored.

Aim Of The Study: To screen South African plants used ethnomedicinally for wound healing based on their pro-angiogenic and wound healing activity, using transgenic zebrafish larvae and cell culture assays.

Materials And Methods: South African medicinal plants used for wound healing were chosen according to literature. Dried plant material was extracted using six solvents of varying polarities. Pro-angiogenesis was assessed in vivo by observing morphological changes in sub-intestinal vessels after crude extract treatment of transgenic zebrafish larvae with vasculature-specific expression of a green fluorescent protein. Subsequently, the in vitro anti-inflammatory, fibroblast proliferation and collagen production effects of the plant extracts that were active in the zebrafish angiogenesis assay were investigated using murine macrophage (RAW 264.7) and human fibroblast (MRHF) cell lines.

Results: Fourteen plants were extracted using six different solvents to yield 84 extracts and the non-toxic (n = 72) were initially screened for pro-angiogenic activity in the zebrafish assay. Of these plant species, extracts of Lobostemon fruticosus, Scabiosa columbaria and Cotyledon orbiculata exhibited good activity in a concentration-dependent manner. All active extracts showed negligible in vitro toxicity using the MTT assay. Lobostemon fruticosus and Scabiosa columbaria extracts showed noteworthy anti-inflammatory activity in RAW 264.7 macrophages. The acetone extract of Lobostemon fruticosus stimulated the most collagen production at 122% above control values using the MRHF cell line, while all four of the selected extracts significantly stimulated cellular proliferation in vitro in the MRHF cell line.

Conclusions: The screening of the selected plant species provided valuable preliminary information validating the use of some of the plants in traditional medicine used for wound healing in South Africa. This study is the first to discover through an evidence-based pharmacology approach the wound healing properties of such plant species using the zebrafish as an in vivo model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jep.2021.114867DOI Listing
November 2021

Anti-seizure activity of African medicinal plants: The identification of bioactive alkaloids from the stem bark of Rauvolfia caffra using an in vivo zebrafish model.

J Ethnopharmacol 2021 Oct 10;279:114282. Epub 2021 Jun 10.

Department of Pharmaceutical Sciences, Tshwane University of Technology, Pretoria, 0001, South Africa. Electronic address:

Ethnopharmacological Relevance: Epilepsy is one of the major chronic diseases that does not have a cure to date. Adverse drug reactions have been reported from the use of available anti-epileptic drugs (AEDs) which are also effective in only two-thirds of the patients. Accordingly, the identification of scaffolds with promising anti-seizure activity remains an important first step towards the development of new anti-epileptic therapies, with improved efficacy and reduced adverse effects. Herbal medicines are widely used in developing countries, including in the treatment of epilepsy but with little scientific evidence to validate this use. In the search for new epilepsy treatment options, the zebrafish has emerged as a chemoconvulsant-based model for epilepsy, mainly because of the many advantages that zebrafish larvae offer making them highly suitable for high-throughput drug screening.

Aim Of The Study: In this study, 20 medicinal plants traditionally used in South Africa to treat epilepsy were screened for anti-epileptic activity using a zebrafish larvae model.

Materials And Methods: Toxicity triaging was conducted on 120 crude extracts, 44 fractions and three isolated compounds to determine the maximum tolerated concentration (MTC) of each extract, fraction or compound. MTC values were used to guide the concentration range selection in bioactivity studies. The effectiveness of crude extracts, fractions and isolated compounds from Rauvolfia caffra Sond. in suppression of pentylenetetrazole (PTZ) induced seizure-like behaviour in a 6-dpf zebrafish larvae model was measured using the PTZ assay.

Results: Following a preliminary toxicity triage and bioactivity screen of crude extracts from 20 African plants used traditionally for the treatment and management of epilepsy, the methanolic extract of Rauvolfia caffra Sond. was identified as the most promising at suppressing PTZ induced seizure-like behaviour in a zebrafish larvae model. Subsequent bioactivity-guided fractionation and spectroscopic structural elucidation resulted in the isolation and identification of two tryptoline derivatives; a previously unreported alkaloid to which we assigned the trivial name rauverine H (1) and the known alkaloid pleiocarpamine (2). Pleiocarpamine was found to reduce PTZ-induced seizures in a dose-dependent manner.

Conclusions: Accordingly, pleiocarpamine represents a promising scaffold for the development of new anti-seizure therapeutic compounds. Furthermore, the results of this study provide preliminary evidence to support the traditional use of Rauvolfia caffra Sond. in the treatment and management of epilepsy. These findings warrant further studies on the anti-epileptic potential of Rauvolfia caffra Sond. using other models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jep.2021.114282DOI Listing
October 2021

Identification of Potential Antiseizure Agents in using Zebrafish and Mouse Epilepsy Models.

ACS Chem Neurosci 2021 05 29;12(10):1791-1801. Epub 2021 Apr 29.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.

The resin of the tree Flueck. (synonym: ; Burseraceae), also known as "frankincense", is a traditional remedy used for central nervous system disorders in East Africa. Here we report the evaluation of its antiseizure activity in zebrafish and mouse epilepsy models to identify novel antiseizure compounds. The resin was extracted by solvents of increasing polarity. The hexane extract demonstrated the strongest antiseizure activity and was therefore subjected to bioactivity-guided isolation, which leaded to the isolation of eight terpene derivatives. A new prenylbicyclogermacrene derivative () was isolated along with seven other compounds (, -). Among them, the triterpene β-boswellic acid () showed the strongest activity and reduced 90% of pentylenetetrazole (PTZ)-induced seizures at 100 μg/mL. In parallel to , a commercial extract of was also evaluated and showed moderate bioactivity (45% reduction at 30 μg/mL). The extract of was subjected to targeted isolation of other boswellic acid derivatives (-), which were evaluated for antiseizure activity in comparison with . In the whole series, β-boswellic acid () was the most active (60% reduction at 200 μM), and its potency was also confirmed with its purchased standard (). Pure nanoparticles of and a commercially formulated extract of were tested in a PTZ-kindling mouse seizure model. This notably revealed that the administration reduced seizures by 50% in this mouse model, which was consistent with its detection and quantification in plasma and brain samples. This study and the preclinical evaluation performed indicate that β-boswellic acid, common to various species of , has some potential as an antiseizure agent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschemneuro.1c00044DOI Listing
May 2021

l-Isoaspartyl Methyltransferase Deficiency in Zebrafish Leads to Impaired Calcium Signaling in the Brain.

Front Genet 2020 21;11:612343. Epub 2021 Jan 21.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.

Isomerization of l-aspartyl and l-asparaginyl residues to l-isoaspartyl residues is one type of protein damage that can occur under physiological conditions and leads to conformational changes, loss of function, and enhanced protein degradation. Protein l-isoaspartyl methyltransferase (PCMT) is a repair enzyme whose action initiates the reconversion of abnormal l-isoaspartyl residues to normal l-aspartyl residues in proteins. Many lines of evidence support a crucial role for PCMT in the brain, but the mechanisms involved remain poorly understood. Here, we investigated PCMT activity and function in zebrafish, a vertebrate model that is particularly well-suited to analyze brain function using a variety of techniques. We characterized the expression products of the zebrafish homologous genes and . Both zebrafish proteins showed a robust l-isoaspartyl methyltransferase activity and highest mRNA transcript levels were found in brain and testes. Zebrafish morphant larvae with a knockdown in both the and genes showed pronounced morphological abnormalities, decreased survival, and increased isoaspartyl levels. Interestingly, we identified a profound perturbation of brain calcium homeostasis in these morphants. An abnormal calcium response upon ATP stimulation was also observed in mouse hippocampal HT22 cells knocked out for . This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time , that PCMT plays a pivotal role in the regulation of calcium fluxes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fgene.2020.612343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859441PMC
January 2021

Phenotypic assays in yeast and zebrafish reveal drugs that rescue deficiency.

Brain Commun 2019 27;1(1):fcz019. Epub 2019 Sep 27.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4367 Belvaux, Luxembourg.

Mutations in () are causally linked to the rare neurodegenerative disorders Kufor-Rakeb syndrome, hereditary spastic paraplegia and neuronal ceroid lipofuscinosis. This suggests that ATP13A2, a lysosomal cation-transporting ATPase, plays a crucial role in neuronal cells. The heterogeneity of the clinical spectrum of -associated disorders is not yet well understood and currently, these diseases remain without effective treatment. Interestingly, ATP13A2 is widely conserved among eukaryotes, and the yeast model for deficiency was the first to indicate a role in heavy metal homeostasis, which was later confirmed in human cells. In this study, we show that the deletion of (the yeast orthologue of ) in leads to growth impairment in the presence of Zn, Mn, Co and Ni, with the strongest phenotype being observed in the presence of zinc. Using the Δ mutant, we developed a high-throughput growth rescue screen based on the Zn sensitivity phenotype. Screening of two libraries of Food and Drug Administration-approved drugs identified 11 compounds that rescued growth. Subsequently, we generated a zebrafish model for deficiency and found that both partial and complete loss of function led to increased sensitivity to Mn. Based on this phenotype, we confirmed two of the drugs found in the yeast screen to also exert a rescue effect in zebrafish--acetylcysteine, a potent antioxidant, and furaltadone, a nitrofuran antibiotic. This study further supports that combining the high-throughput screening capacity of yeast with rapid drug testing in zebrafish can represent an efficient drug repurposing strategy in the context of rare inherited disorders involving conserved genes. This work also deepens the understanding of the role of ATP13A2 in heavy metal detoxification and provides a new model for investigating deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/braincomms/fcz019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425419PMC
September 2019

Metabolite Profiling of Javanese Ginger and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation.

J Agric Food Chem 2020 Jul 19;68(30):7904-7915. Epub 2020 Jul 19.

School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.

The rhizomes of "Bangle", were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (-) were isolated, and two bioactive phenylbutenoids, - () and -banglene (), reduced up to 70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of active compounds, its chemical profile was compared to that of . Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jafc.0c02641DOI Listing
July 2020

Seizing the moment: Zebrafish epilepsy models.

Neurosci Biobehav Rev 2020 09 13;116:1-20. Epub 2020 Jun 13.

Chemical Neuroscience Group, Centre for Molecular Medicine Norway (NCMM), University of Oslo, Gaustadalléen 21, Forskningsparken, 0349, Oslo, Norway. Electronic address:

Zebrafish are now widely accepted as a valuable animal model for a number of different central nervous system (CNS) diseases. They are suitable both for elucidating the origin of these disorders and the sequence of events culminating in their onset, and for use as a high-throughput in vivo drug screening platform. The availability of powerful and effective techniques for genome manipulation allows the rapid modelling of different genetic epilepsies and of conditions with seizures as a core symptom. With this review, we seek to summarize the current knowledge about existing epilepsy/seizures models in zebrafish (both pharmacological and genetic) and compare them with equivalent rodent and human studies. New findings obtained from the zebrafish models are highlighted. We believe that this comprehensive review will highlight the value of zebrafish as a model for investigating different aspects of epilepsy and will help researchers to use these models to their full extent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neubiorev.2020.06.010DOI Listing
September 2020

Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles.

J Ethnopharmacol 2020 Sep 21;259:112954. Epub 2020 May 21.

School of Pharmaceutical Sciences, Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel Servet 1, CH-1211, Geneva 4, Switzerland. Electronic address:

Ethnopharmacological Relevance: Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece.

Aim Of The Study: A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed.

Materials And Methods: The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling.

Results: The RME showed 80% inhibition of PTZ-induced locomotor activity (300 μg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 μM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 μM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35-37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids.

Conclusions: This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jep.2020.112954DOI Listing
September 2020

Zebrafish bioassay-guided isolation of antiseizure compounds from the Cameroonian medicinal plant Cyperus articulatus L.

Phytomedicine 2020 Jan 23;70:153175. Epub 2020 Jan 23.

Institute of Pharmaceutical Sciences of Western Switzerland, CMU - Rue Michel-Servet 1, Geneva 4 CH-1211, Switzerland. Electronic address:

Background: Epilepsy is a chronic neurological disorder affecting more than 50 million people worldwide, of whom 80% live in low- and middle-income countries. Due to the limited availability of antiseizure drugs (ASDs) in these countries, medicinal plants are the first-line treatment for most epilepsy patients. In Cameroon, a decoction of Cyperus articulatus L. rhizomes is traditionally used to treat epilepsy.

Purpose: The aim of this study was to identify and isolate the active compounds responsible for the antiseizure activity of C. articulatus in order to confirm both its traditional medicinal usage and previous in vivo studies on extracts of this plant in mouse epilepsy models.

Methods: The dried rhizomes of C. articulatus were extracted with solvents of increasing polaritie (hexane, dichloromethane, methanol and water). A traditional decoction and an essential oil were also prepared. These extracts were evaluated for antiseizure activity using a larval zebrafish seizure model with seizures induced by the GABA antagonist pentylenetetrazole (PTZ). The hexane extract demonstrated the highest antiseizure activity and was therefore selected for bioassay-guided fractionation. The isolated bioactive compounds were characterized by classical spectroscopic methods. Since they were found to be volatile, they were quantified by GC-FID. In addition, the absorption of the active compounds through the gastrointestinal tract and the blood-brain barrier was evaluated using a hexadecane and a blood-brain barrier parallel artificial membrane permeability assays (HDM-PAMPA and PAMPA-BBB).

Results: The hexane extract of C. articulatus exhibited the highest antiseizure activity with a reduction of 93% of PTZ-induced seizures, and was therefore subjected to bioassay-guided fractionation in order to isolate the active principles. Four sesquiterpenoids were identified as cyperotundone (1), mustakone (2), 1,2-dehydro-α-cyperone (3) and sesquichamaenol (4) and exhibited significant antiseizure activity. These volatile compounds were quantified by GC in the hexane extract, the essential oil and the simulated traditional decoction. In addition, the constituents of the hexane extract including compounds 1 and 2 were found to cross the gastrointestinal barrier and the major compound 2 crossed the blood-brain barrier as well.

Conclusion: These results highlight the antiseizure activity of various sesquiterpene compounds from a hexane extract of C. articulatus dried rhizomes and support its use as a traditional treatment for epilepsy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phymed.2020.153175DOI Listing
January 2020

Fishing for a deeper understanding of nicotine effects using zebrafish behavioural models.

Prog Neuropsychopharmacol Biol Psychiatry 2020 03 26;98:109826. Epub 2019 Nov 26.

Independent Laboratory of Behavioral Studies, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland. Electronic address:

Nicotine, the primary psychoactive component of tobacco, is the most widely used drug of abuse. Although the substance is well-known, there is still a lack of information concerning its long-term neurological and physiological effects and its mechanisms of action. In order to search for new, effective drugs in the therapy of nicotinism, as well as to design new drugs that exert positive nicotine-like effects, further experiments are needed, ideally also using new behavioural models and paradigms. A wide range of complex behaviours - including aggression, anxiety, long- and short-term memory, object discrimination and colour preference - have recently been comprehensively classified and characterized in the zebrafish model. Zebrafish offer an attractive experimental platform, based on a microscale in vivo bioassays, which can be used to investigate psychoactive drugs, their effects on the central nervous system and potential treatments of drug addictions. In this review, we present recent data revealing the potential of the zebrafish model to evaluate the effects and molecular mechanisms of nicotine by taking into consideration its impact on anxiety, learning and memory, addiction and social behaviours.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pnpbp.2019.109826DOI Listing
March 2020

Zebrafish-Based Discovery of Antiseizure Compounds from the North Sea: Isoquinoline Alkaloids TMC-120A and TMC-120B.

Mar Drugs 2019 Oct 25;17(11). Epub 2019 Oct 25.

Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49 box 824, 3000 Leuven, Belgium.

There is a high need for the development of new and improved antiseizure drugs (ASDs) to treat epilepsy. Despite the potential of marine natural products (MNPs), the EU marine biodiscovery consortium PharmaSea has made the only effort to date to perform ASD discovery based on large-scale screening of MNPs. To this end, the embryonic zebrafish photomotor response assay and the larval zebrafish pentylenetetrazole (PTZ) model were used to screen MNP extracts for neuroactivity and antiseizure activity, respectively. Here we report the identification of the two known isoquinoline alkaloids TMC-120A and TMC-120B as novel antiseizure compounds, which were isolated by bioactivity-guided purification from the marine-derived fungus . TMC-120A and TMC-120B were observed to significantly lower PTZ-induced seizures and epileptiform brain activity in the larval zebrafish PTZ seizure model. In addition, their structural analogues TMC-120C, penicisochroman G, and ustusorane B were isolated and also significantly lowered PTZ-induced seizures. Finally, TMC-120A and TMC-120B were investigated in a mouse model of drug-resistant focal seizures. Compound treatment significantly shortened the seizure duration, thereby confirming their antiseizure activity. These data underscore the possibility to translate findings in zebrafish to mice in the field of epilepsy and the potential of the marine environment for ASD discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/md17110607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891649PMC
October 2019

High-performance counter-current chromatography isolation and initial neuroactivity characterization of furanocoumarin derivatives from Peucedanum alsaticum L (Apiaceae).

Phytomedicine 2019 Feb 26;54:259-264. Epub 2018 Oct 26.

Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodźki Str., 20-093 Lublin, Poland. Electronic address:

Background: Medicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity.

Purpose: To accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesterase ELISAs and zebrafish epilepsy models.

Research Methods And Procedure: In this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABA antagonist pentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior.

Results: The solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ± 1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ± 2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ± 1.25%). The DCM extract of P. alsaticum fruits (0.62-1.75 µg/ml) and bergamottin (2-10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10-16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A.

Conclusion: Our findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phymed.2018.10.030DOI Listing
February 2019

Deep learning image recognition enables efficient genome editing in zebrafish by automated injections.

PLoS One 2019 7;14(1):e0202377. Epub 2019 Jan 7.

Life Science Methods BV, Leiden, the Netherlands.

One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 μm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202377PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322765PMC
September 2019

Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy.

ACS Chem Neurosci 2019 04 6;10(4):1992-2003. Epub 2018 Nov 6.

Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences , University of Leuven , Leuven 3000 , Belgium.

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3'-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschemneuro.8b00281DOI Listing
April 2019

Zebrafish-based identification of the antiseizure nucleoside inosine from the marine diatom Skeletonema marinoi.

PLoS One 2018 24;13(4):e0196195. Epub 2018 Apr 24.

Luxembourg Centre for Systems Biomedicine, Université du Luxembourg, Belvaux, Luxembourg.

With the goal of identifying neuroactive secondary metabolites from microalgae, a microscale in vivo zebrafish bioassay for antiseizure activity was used to evaluate bioactivities of the diatom Skeletonema marinoi, which was recently revealed as being a promising source of drug-like small molecules. A freeze-dried culture of S. marinoi was extracted by solvents with increasing polarities (hexane, dichloromethane, methanol and water) and these extracts were screened for anticonvulsant activity using a larval zebrafish epilepsy model with seizures induced by the GABAA antagonist pentylenetetrazole. The methanolic extract of S. marinoi exhibited significant anticonvulsant activity and was chosen for bioassay-guided fractionation, which associated the bioactivity with minor constituents. The key anticonvulsant constituent was identified as the nucleoside inosine, a well-known adenosine receptor agonist with previously reported antiseizure activities in mice and rat epilepsy models, but not reported to date as a bioactive constituent of microalgae. In addition, a UHPLC-HRMS metabolite profiling was used for dereplication of the other constituents of S. marinoi. Structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution spectrometry. These results highlight the potential of zebrafish-based screening and bioassay-guided fractionation to identify neuroactive marine natural products.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196195PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916873PMC
July 2018

CaSiAn: a Calcium Signaling Analyzer tool.

Bioinformatics 2018 09;34(17):3052-3054

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux L-4367, Luxembourg.

Summary: Ca2+ is a central second messenger in eukaryotic cells that regulates many cellular processes. Recently, we have indicated that typical Ca2+ signals are not purely oscillatory as widely assumed, but exhibit stochastic spiking with cell type and pathway specific characteristics. Here, we present the Calcium Signaling Analyzer (CaSiAn), an open source software tool that allows for quantifying these signal characteristics including individual spike properties and time course statistics in a semi-automated manner. CaSiAn provides an intuitive graphical user interface allowing experimentalists to easily process a large amount of Ca2+ signals, interactively tune peak detection, revise statistical measures and access the quantified signal properties as excel or text files.

Availability And Implementation: CaSiAn is implemented in Java and available on Github (https://github.com/mmahsa/CaSiAn) as well as on the project page (http://r3lab.uni.lu/web/casa).

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/bty281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129310PMC
September 2018

Anti-Inflammatory Activity and Cheminformatics Analysis of New Poten t 2-Substituted 1-Methyl-5-Nitroindazolinones.

Curr Top Med Chem 2018 Feb;17(30):3236-3248

Instituto de Quimica Medica, CSIC, c/Juan de la Cierva 3, 28006 Madrid, Spain.

After the identification of the anti-inflammatory properties of VA5-13l (2-benzyl-1- methyl-5-nitroindazolinone) in previous investigations, some of its analogous compounds were designed, synthesized and evaluated in two anti-inflammatory methods: LPS-enhanced leukocyte migration assay in zebrafish; and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema. The products evaluated (3, 6, 8, 9 and 10) showed the lower values of relative leukocyte migration at 30 µM (0.14, 0.07, 0.10, 0.13 and 0.07, respectively), while in ear edema and myeloperoxidase activity methods, all the compounds reduced inflammation, only 4 and 16 yielded unsatisfactory results. The relationship linking structure and activity (SAR analysis) was determinate by using SARANEA software. The importance of the 5-Nitro group of the indazole ring for the activity was evident, and showed modest reduction when benzyl (Bn) is changed by alkyl group. A substituted Bn moiety at N2 (R) is the best substituent (5-10); nevertheless, if methylene group of Bn is deleted, the activity is affected. Also, introduction of halogen atoms mainly at positions 3 or 4 of the benzyl moiety (6 and 10) leads in general to strong activities. In fact, compounds 7 and 8 (R = 4-FBn or 4-ClBn, respectively) exhibit satisfactory results in in vivo tests and appear promising. The production of IL-6 at all doses assayed was significantly reduced, except with 16. Nonetheless, the production of TNF-α was significantly inhibited only by this chemical (16) at concentration of 50 μM. On the other hand, compound 2 was the one that mostly inhibited the expression of COX-2 and iNOS. From these results, it can be concluded that the inhibition in the release of cytokines can be one of the mechanisms of action responsible for the anti-inflammatory effect for 2-benzyl derivates while other 2-alkyl derivatives can inhibit production of NO. Therefore, nitroindazolinone chemical prototype could be an interesting structural group with anti-inflammatory purposes in the therapeutic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1568026618666180119125255DOI Listing
February 2018

Fishing Anti-Inflammatories from Known Drugs: In Silico Repurposing, Design, Synthesis and Biological Evaluation of Bisacodyl Analogues.

Curr Top Med Chem 2017 Aug 17. Epub 2017 Aug 17.

Grupo de Investigación Ambiental (GIA), Fundación Universitaria Tecnológico de Comfenalco, Facultad de Ingenierías, Programa de Ingeniería de Procesos, Cartagena de Indias, Bolívar 130001. Colombia.

Herein is described in silico repositioning, design, synthesis, biological evaluation and structure-activity relationship (SAR) of an original class of anti-inflammatory agents based on a polyaromatic pharmacophore structurally related to bisacodyl (BSL) drug used in therapeutic as laxative. We describe the potential of TOMOCOMD-CARDD methods to find out new anti-inflammatory drug-like agents from a diverse series of compounds using the total and local atom based bilinear indices as molecular descriptors. The models obtained were validated by biological studies, identifying BSL as the first anti-inflammatory lead-like using in silico repurposing from commercially available drugs. Several biological in vitro and in vivo assays were performed in order to understand its mechanism of action. A set of analogues of BSL was prepared using low-cost synthetic procedures and further biologically investigated in zebrafish models. Compound 5c and 7e exhibited the best antiinflammatory activities and represent new promising anti-inflammatory agents for further preclinical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1568026617666170817161953DOI Listing
August 2017

Marine Biodiscovery Goes Deeper: Using In Vivo Bioassays Based on Model Organisms to Identify Biomedically Relevant Marine Metabolites.

Planta Med 2016 Jun 18;82(9-10):754-60. Epub 2016 May 18.

Chemical Biology Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux, Luxembourg.

Secondary metabolites from marine organisms are structurally diverse small molecules with high levels of bioactivity, and represent an underutilized resource for modern drug discovery. To facilitate the identification of drug-like marine metabolites, the significant potential of in vivo models of human disease - in particular those suitable for medium-throughput screening and bioassay-guided fractionation - should be explored in future marine biodiscovery efforts. Here, we explore the advantages of Caenorhabditis elegans, Drosophila, and zebrafish bioassays for marine biodiscovery, and review recent progress in using these in vivo models to identify bioactive marine metabolites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0042-106391DOI Listing
June 2016

Potential of zebrafish as a model for exploring the role of the amygdala in emotional memory and motivational behavior.

J Neurosci Res 2016 Jun 2;94(6):445-62. Epub 2016 Feb 2.

Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Belvaux, Luxembourg.

Emotion is a key aspect of behavior, enabling humans and animals to assign either positive or negative values to sensory inputs and thereby to make appropriate decisions. Classical experiments in mammalian models, mainly in primates and rodents, have shown that the amygdala is essential for appetitive and aversive associative processing and that dysfunction of this brain region leads to various psychiatric conditions, including depression, generalized anxiety disorder, panic disorder, phobias, autism, and posttraumatic stress disorder. In the past 2 decades, the zebrafish (Danio rerio; Cyprinidae) has emerged as a versatile, reliable vertebrate model organism for the in vivo study of development, gene function, and numerous aspects of human pathologies. Small size, high fecundity, rapid external development, transparency, genetic tractability, and high genetic and physiologic homology with humans are among the factors that have contributed to the success with this small fish in different biomedical research areas. Recent findings indicate that, despite the anatomical differences in the brain structure of teleosts and tetrapods, fish possess a structure homologous to the mammalian amygdala, a hypothesis that is supported by the expression of molecular markers, analyses of neuronal projections in different brain areas, and behavioral studies. This Review summarizes this evidence and highlights a number of relevant bioassays in zebrafish to study emotional memory and motivational behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jnr.23712DOI Listing
June 2016

Pharmacological characterization of an antisense knockdown zebrafish model of Dravet syndrome: inhibition of epileptic seizures by the serotonin agonist fenfluramine.

PLoS One 2015 12;10(5):e0125898. Epub 2015 May 12.

Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Leuven, Belgium.

Dravet syndrome (DS) is one of the most pharmacoresistant and devastating forms of childhood epilepsy syndromes. Distinct de novo mutations in the SCN1A gene are responsible for over 80% of DS cases. While DS is largely resistant to treatment with existing anti-epileptic drugs, promising results have been obtained in clinical trials with human patients treated with the serotonin agonist fenfluramine as an add-on therapeutic. We developed a zebrafish model of DS using morpholino antisense oligomers (MOs) targeting scn1Lab, the zebrafish ortholog of SCN1A. Zebrafish larvae with an antisense knockdown of scn1Lab (scn1Lab morphants) were characterized by automated behavioral tracking and high-resolution video imaging, in addition to measuring brain activity through local field potential recordings. Our findings reveal that scn1Lab morphants display hyperactivity, convulsive seizure-like behavior, loss of posture, repetitive jerking and a myoclonic seizure-like pattern. The occurrence of spontaneous seizures was confirmed by local field potential recordings of the forebrain, measuring epileptiform discharges. Furthermore, we show that these larvae are remarkably sensitive to hyperthermia, similar to what has been described for mouse models of DS, as well as for human DS patients. Pharmacological evaluation revealed that sodium valproate and fenfluramine significantly reduce epileptiform discharges in scn1Lab morphants. Our findings for this zebrafish model of DS are in accordance with clinical data for human DS patients. To our knowledge, this is the first study demonstrating effective seizure inhibition of fenfluramine in an animal model of Dravet syndrome. Moreover, these results provide a basis for identifying novel analogs with improved activity and significantly milder or no side effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125898PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428833PMC
February 2016

Cross-species pharmacological characterization of the allylglycine seizure model in mice and larval zebrafish.

Epilepsy Behav 2015 Apr 3;45:53-63. Epub 2015 Apr 3.

Neuroscience TA, UCB Biopharma, Braine-l'Alleud, Belgium. Electronic address:

Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill the need for new medications targeting treatment-resistant seizures, a number of rodent models offer the opportunity to assess a variety of potential treatment approaches. The use of such models, however, has proven to be time-consuming and labor-intensive. In this study, we performed pharmacological characterization of the allylglycine (AG) seizure model, a simple in vivo model for which we demonstrated a high level of treatment resistance. (d,l)-Allylglycine inhibits glutamic acid decarboxylase (GAD) - the key enzyme in γ-aminobutyric acid (GABA) biosynthesis - leading to GABA depletion, seizures, and neuronal damage. We performed a side-by-side comparison of mouse and zebrafish acute AG treatments including biochemical, electrographic, and behavioral assessments. Interestingly, seizure progression rate and GABA depletion kinetics were comparable in both species. Five mechanistically diverse antiepileptic drugs (AEDs) were used. Three out of the five AEDs (levetiracetam, phenytoin, and topiramate) showed only a limited protective effect (mainly mortality delay) at doses close to the TD50 (dose inducing motor impairment in 50% of animals) in mice. The two remaining AEDs (diazepam and sodium valproate) displayed protective activity against AG-induced seizures. Experiments performed in zebrafish larvae revealed behavioral AED activity profiles highly analogous to those obtained in mice. Having demonstrated cross-species similarities and limited efficacy of tested AEDs, we propose the use of AG in zebrafish as a convenient and high-throughput model of treatment-resistant seizures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yebeh.2015.03.019DOI Listing
April 2015

CHD2 variants are a risk factor for photosensitivity in epilepsy.

Brain 2015 May 17;138(Pt 5):1198-207. Epub 2015 Mar 17.

19 Department of Child Neurology, Paediatric Clinic, Clinical Centre Nis, Serbia 20 Department of Paediatric Neurology, Paediatric Clinic, Al Sabah Hospital, Kuwait.

Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as particular electroencephalographic changes, with or without seizures. Photosensitivity is prominent in a very rare epileptic encephalopathy due to de novo CHD2 mutations, but is also seen in epileptic encephalopathies due to other gene mutations. We determined whether CHD2 variation underlies photosensitivity in common epilepsies, specific photosensitive epilepsies and individuals with photosensitivity without seizures. We studied 580 individuals with epilepsy and either photosensitive seizures or abnormal photoparoxysmal response on electroencephalography, or both, and 55 individuals with photoparoxysmal response but no seizures. We compared CHD2 sequence data to publicly available data from 34 427 individuals, not enriched for epilepsy. We investigated the role of unique variants seen only once in the entire data set. We sought CHD2 variants in 238 exomes from familial genetic generalized epilepsies, and in other public exome data sets. We identified 11 unique variants in the 580 individuals with photosensitive epilepsies and 128 unique variants in the 34 427 controls: unique CHD2 variation is over-represented in cases overall (P = 2.17 × 10(-5)). Among epilepsy syndromes, there was over-representation of unique CHD2 variants (3/36 cases) in the archetypal photosensitive epilepsy syndrome, eyelid myoclonia with absences (P = 3.50 × 10(-4)). CHD2 variation was not over-represented in photoparoxysmal response without seizures. Zebrafish larvae with chd2 knockdown were tested for photosensitivity. Chd2 knockdown markedly enhanced mild innate zebrafish larval photosensitivity. CHD2 mutation is the first identified cause of the archetypal generalized photosensitive epilepsy syndrome, eyelid myoclonia with absences. Unique CHD2 variants are also associated with photosensitivity in common epilepsies. CHD2 does not encode an ion channel, opening new avenues for research into human cortical excitability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/brain/awv052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407192PMC
May 2015

Are zebrafish larvae suitable for assessing the hepatotoxicity potential of drug candidates?

J Appl Toxicol 2015 Sep 6;35(9):1017-29. Epub 2015 Feb 6.

Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven Campus Gasthuisberg, O&N 2, 08.4226 Herestraat 49, PB 824, 3000, Leuven, Belgium.

Drug-induced liver injury (DILI) is poorly predicted by single-cell-based assays, probably because of the lack of physiological interactions with other cells within the liver. An intact whole liver system such as one present in zebrafish larvae could provide added value in a screening strategy for DILI; however, the possible occurrence of other organ toxicities and the immature larval stage of the zebrafish might complicate accurate and fast analysis. We investigated whether expression analysis of liver-specific fatty acid binding protein 10a (lfabp10a) was an appropriate endpoint for assessing hepatotoxic effects in zebrafish larvae. It was found that expression analysis of lfabp10a was a valid marker, as after treatment with hepatotoxicants, dose-response curves could be obtained and statistically significant abnormal lfabp10 expression levels correlated with hepatocellular histopathological changes in the liver. However, toxicity in other vital organs such as the heart could impact liver outgrowth and thus had to be assessed concurrently. Whether zebrafish larvae were suitable for assessing human relevant drug-induced hepatotoxicity was assessed with hepatotoxicants and non-hepatotoxicants that have been marketed for human use and classified according to their mechanism of toxicity. The zebrafish larva showed promising predictivity towards a number of mechanisms and was capable of distinguishing between hepatotoxic and non-hepatotoxic chemical analogues, thus implying its applicability as a potential screening model for DILI.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jat.3091DOI Listing
September 2015

Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes.

Nat Genet 2014 Dec 2;46(12):1327-32. Epub 2014 Nov 2.

Section of Complex Genetics, Department of Medical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands.

Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.3130DOI Listing
December 2014

Zebrafish bioassay-guided microfractionation identifies anticonvulsant steroid glycosides from the Philippine medicinal plant Solanum torvum.

ACS Chem Neurosci 2014 Oct 8;5(10):993-1004. Epub 2014 Sep 8.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne , 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.

Medicinal plants used for the treatment of epilepsy are potentially a valuable source of novel antiepileptic small molecules. To identify anticonvulsant secondary metabolites, we performed an in vivo, zebrafish-based screen of medicinal plants used in Southeast Asia for the treatment of seizures. Solanum torvum Sw. (Solanaceae) was identified as having significant anticonvulsant activity in zebrafish larvae with seizures induced by the GABAA antagonist pentylenetetrazol (PTZ). This finding correlates well with the ethnomedical use of this plant in the Philippines, where a water decoction of S. torvum leaves is used to treat epileptic seizures. HPLC microfractionation of the bioactive crude extract, in combination with the in vivo zebrafish seizure assay, enabled the rapid localization of several bioactive compounds that were partially identified online by UHPLC-TOF-MS as steroid glycosides. Targeted isolation of the active constituents from the methanolic extract enabled the complete de novo structure identification of the six main bioactive compounds that were also present in the traditional preparation. To partially mimic the in vivo metabolism of these triterpene glycosides, their common aglycone was generated by acid hydrolysis. The isolated molecules exhibited significant anticonvulsant activity in zebrafish seizure assays. These results underscore the potential of zebrafish bioassay-guided microfractionation to rapidly identify novel bioactive small molecules of natural origin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cn5001342DOI Listing
October 2014

2-Octadecynoic acid as a dual life stage inhibitor of Plasmodium infections and plasmodial FAS-II enzymes.

Bioorg Med Chem Lett 2014 Sep 24;24(17):4151-7. Epub 2014 Jul 24.

Department of Biological and Pharmaceutical Chemistry, University of London, School of Pharmacy, London WC1N 1AX, UK; School of Chemistry, National University of Ireland, Galway, Ireland. Electronic address:

The malaria parasite Plasmodium goes through two life stages in the human host, a non-symptomatic liver stage (LS) followed by a blood stage with all clinical manifestation of the disease. In this study, we investigated a series of 2-alkynoic fatty acids (2-AFAs) with chain lengths between 14 and 18 carbon atoms for dual in vitro activity against both life stages. 2-Octadecynoic acid (2-ODA) was identified as the best inhibitor of Plasmodium berghei parasites with ten times higher potency (IC50=0.34 μg/ml) than the control drug. In target determination studies, the same compound inhibited three Plasmodium falciparum FAS-II (PfFAS-II) elongation enzymes PfFabI, PfFabZ, and PfFabG with the lowest IC50 values (0.28-0.80 μg/ml, respectively). Molecular modeling studies provided insights into the molecular aspects underlying the inhibitory activity of this series of 2-AFAs and a likely explanation for the considerably different inhibition potentials. Blood stages of P. falciparum followed a similar trend where 2-ODA emerged as the most active compound, with 20 times less potency. The general toxicity and hepatotoxicity of 2-AFAs were evaluated by in vitro and in vivo methods in mammalian cell lines and zebrafish models, respectively. This study identifies 2-ODA as the most promising antiparasitic 2-AFA, particularly towards P. berghei parasites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2014.07.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146677PMC
September 2014

A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling.

PLoS One 2013 11;8(12):e83293. Epub 2013 Dec 11.

Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Leuven, Belgium.

Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083293PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859651PMC
March 2015

De novo loss-of-function mutations in CHD2 cause a fever-sensitive myoclonic epileptic encephalopathy sharing features with Dravet syndrome.

Am J Hum Genet 2013 Nov 24;93(5):967-75. Epub 2013 Oct 24.

Neurogenetics group, Department of Molecular Genetics, VIB, 2610 Antwerp, Belgium; Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, 2610 Antwerp, Belgium.

Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2-knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2013.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824114PMC
November 2013

Optimization and pharmacological validation of a leukocyte migration assay in zebrafish larvae for the rapid in vivo bioactivity analysis of anti-inflammatory secondary metabolites.

PLoS One 2013 4;8(10):e75404. Epub 2013 Oct 4.

Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Leuven, Belgium ; Faculty of Chemical Sciences, School of Biochemistry and Pharmacy, University of Cuenca, Cuenca, Ecuador ; Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.

Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075404PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790782PMC
July 2014
-->