CLN3 deficiency leads to neurological and metabolic perturbations during early development.

Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3 Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.

Seizure-induced increase in microglial cell population in the developing zebrafish brain.

Epilepsy is a chronic brain disorder characterized by unprovoked and recurrent seizures, of which 60% are of unknown etiology. Recent studies implicate microglia in the pathophysiology of epilepsy. However, their role in this process, in particular following early-life seizures, remains poorly understood due in part to the lack of suitable experimental models allowing the in vivo imaging of microglial activity. Given the advantage of zebrafish larvae for minimally-invasive imaging approaches, we sought for the first time to describe the microglial responses after acute seizures in two different zebrafish larval models: a chemically-induced epileptic model by the systemic injection of kainate at 3 days post-fertilization, and the didys552 genetic epilepsy model, which carries a mutation in scn1lab that leads to spontaneous epileptiform discharges. Kainate-treated larvae exhibited transient brain damage as shown by increased numbers of apoptotic nuclei as early as one day post-injection, which was followed by an increase in the number of microglia in the brain. A similar microglial phenotype was also observed in didys552-/- mutants, suggesting that microglia numbers change in response to seizure-like activity in the brain. Interestingly, kainate-treated larvae also displayed a decreased seizure threshold towards subsequent pentylenetetrazole-induced seizures, as shown by higher locomotor and encephalographic activity in comparison with vehicle-injected larvae. These results are comparable to kainate-induced rodent seizure models and suggest the suitability of these zebrafish seizure models for future studies, in particular to elucidate the links between epileptogenesis and microglial dynamic changes after seizure induction in the developing brain, and to understand how these modulate seizure susceptibility.

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

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.

New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome.

OBJECTIVE: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine. METHODS: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae. RESULTS: Homozygous mutant (scn1labmut/mut ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1labmut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1labmut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1labmut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1labmut/mut larval brains that were rescued by fenfluramine but not diazepam. SIGNIFICANCE: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1labmut/mut brain, and present first-time evidence for potential disease modification by fenfluramine.

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

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 PCMT homologous genes pcmt and pcmtl. 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 pcmt and pcmtl 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 Pcmt1. This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time in vivo, that PCMT plays a pivotal role in the regulation of calcium fluxes.

Transcriptome profiling data reveals ubiquitin-specific peptidase 9 knockdown effects.

Ubiquitin specific peptidase 9 (USP9) is a deubiquitinase encoded by a sex-linked gene with a Y-chromosomal form (USP9Y) and an X-chromosomal form (USP9X) that escapes X-inactivation. Since USP9 is a key regulatory gene with sex-linked expression in the human brain, the gene may be of interest for researchers studying molecular gender differences and ubiquitin signaling in the brain. To assess the downstream effects of knocking down USP9X and USP9Y on a transcriptome-wide scale, we have conducted microarray profiling experiments using the human DU145 prostate cancer cell culture model, after confirming the robust expression of both USP9X and USP9Y in this model. By designing shRNA constructs for the specific knockdown of USP9X and the joint knockdown of USP9X and USP9Y, we have compared gene expression changes in both knockdowns to control conditions to infer potential shared and X- or Y-form specific alterations. Here, we provide details of the corresponding microarray profiling data, which has been deposited in the Gene Expression Omnibus database (GEO series accession number GSE79376). A biological interpretation of the data in the context of a potential involvement of USP9 in Alzheimer's disease has previously been presented in Köglsberger et al. (2016). To facilitate the re-use and re-analysis of the data for other applications, e.g. the study of ubiquitin signaling and protein turnover control, and the regulation of molecular gender differences in the human brain and brain-related disorders, we provide a more in-depth discussion of the data properties, specifications and possible use cases.

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

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.

Phenotypic assays in yeast and zebrafish reveal drugs that rescue ATP13A2 deficiency.

Mutations in ATP13A2 (PARK9) 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 ATP13A2-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 ATP13A2 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 YPK9 (the yeast orthologue of ATP13A2) in Saccharomyces cerevisiae leads to growth impairment in the presence of Zn2+, Mn2+, Co2+ and Ni2+, with the strongest phenotype being observed in the presence of zinc. Using the ypk9Δ mutant, we developed a high-throughput growth rescue screen based on the Zn2+ 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 ATP13A2 deficiency and found that both partial and complete loss of atp13a2 function led to increased sensitivity to Mn2+. Based on this phenotype, we confirmed two of the drugs found in the yeast screen to also exert a rescue effect in zebrafish-N-acetylcysteine, a potent antioxidant, and furaltadone, a nitrofuran antibiotic. This study further supports that combining the high-throughput screening capacity of yeast with rapid in vivo 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 in vivo model for investigating ATP13A2 deficiency.

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

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.

Gender-Specific Expression of Ubiquitin-Specific Peptidase 9 Modulates Tau Expression and Phosphorylation: Possible Implications for Tauopathies.

Public transcriptomic studies have shown that several genes display pronounced gender differences in their expression in the human brain, which may influence the manifestations and risk for neuronal disorders. Here, we apply a transcriptome-wide analysis to discover genes with gender-specific expression and significant alterations in public postmortem brain tissue from Alzheimer's disease (AD) patients compared to controls. We identify the sex-linked ubiquitin-specific peptidase 9 (USP9) as an outstanding candidate gene with highly significant expression differences between the genders and male-specific underexpression in AD. Since previous studies have shown that USP9 can modulate the phosphorylation of the AD-associated protein MAPT, we investigate functional associations between USP9 and MAPT in further detail. After observing a high positive correlation between the expression of USP9 and MAPT in the public transcriptomics data, we show that USP9 knockdown results in significantly decreased MAPT expression in a DU145 cell culture model and a concentration-dependent decrease for the MAPT orthologs mapta and maptb in a zebrafish model. From the analysis of microarray and qRT-PCR experiments for the knockdown in DU145 cells and prior knowledge from the literature, we derive a data-congruent model for a USP9-dependent regulatory mechanism modulating MAPT expression via BACH1 and SMAD4. Overall, the analyses suggest USP9 may contribute to molecular gender differences observed in tauopathies and provide a new target for intervention strategies to modulate MAPT expression.

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

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.

CHD2 variants are a risk factor for photosensitivity in epilepsy.

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.

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

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.

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

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.

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

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.

Integration of Microfractionation, qNMR and zebrafish screening for the in vivo bioassay-guided isolation and quantitative bioactivity analysis of natural products.

Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation - generic for NP extracts of diverse origin - that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay-guided fractionation can be performed with only 20 mg of NP extract within a few days.

Discovery of novel anti-inflammatory drug-like compounds by aligning in silico and in vivo screening: the nitroindazolinone chemotype.

In this report, we propose the combination of computational methods and in vivo primary screening in zebrafish larvae and confirmatory in mice models as a novel strategy to accelerate anti-inflammatory drug discovery. Initially, a database of 1213 organic chemicals with great structural variability - 587 of them anti-inflammatory agents plus 626 compounds with other clinical uses - was divided into training and test groups. Atom-based quadratic indices - a TOMOCOMD-CARDD molecular descriptors family - and linear discriminant analysis (LDA) were used to develop a total of 13 models to describe the anti-inflammatory activity. The best model (Eq. (13)) shows an accuracy of 87.70% in the training set, and values of Matthews correlation coefficient (C) of 0.75. The robustness of the models was demonstrated using an external test set as validation method, i.e., Eq. (13) revealing classification of 88.44% (C = 0.77) in this series. All models were employed to develop ensemble a QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. The fusion model was used for the identification of novel anti-inflammatory compounds using virtual screening of 145 molecules available in our in-house library of indazole, indole, cinnoline and quinoxaline derivatives. Out of these, 34 chemicals were selected, synthesized and tested in a lipopolysaccharide (LPS)-induced leukocyte migration assay in zebrafish larvae. This activity was evaluated based on leukocyte migration to the injury zone of tail-transected larvae. Compounds 18 (3 µM), 24 (10 µM), 25 (10 µM), 6 (10 µM), 15 (30 µM), 11 (30 µM) and 12 (30 µM) gave the best results displaying relative leukocyte migration (RLM) values of 0.24, 0.27, 0.35, 0.41, 0.17, 0. 26 and 0.27 respectively, date that suggest an anti-inflammatory activity of 76, 73, 65, 59, 83, 84 and 73%, respectively. Compound 18 was the most potent but showed high toxicity together with compound 6. Next, we used the tetradecanoylphorbol acetate (TPA)-induced mouse ear oedema model to evaluate the most potent compounds in the zebrafish larvae tail transection assay. All assayed compounds, with the exception of chemical 15, showed anti-inflammatory activity in mice. Compound 12 (VA5-13l, 2-benzyl-1-methyl-5-nitro-1,2-dihydro-3H-indazol-3-one) was the most active and completely abolished the oedema. Compounds 6, 11 and 24 showed inhibition percentages in the range of the reference drug (indomethacin), whereas compounds 18 and 25 reduced the oedema in a lesser extent (inhibition of 73 and 80%, respectively). In addition, all compounds except chemical 15, significantly reduced neutrophil infiltration, measured as myeloperoxidase activity on TPA application test. Compounds 6, 11, 12 and 18 showed values comparable to indomethacin (inhibition percentage of 61), but compounds 6 and 18 were toxic in zebrafish and showed unspecific cytotoxicity in murine macrophages at 100 µg/mL, while the remaining compounds 11, 12 and 25 were inactive at most levels. Evidently, this study suggests a new support structure (12, 11 and 24; a nitroindazolinone chemotype) that constitutes a novel promising lead and may represent an important therapeutic alternative for the treatment of inflammatory conditions.