Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish

Most neuroactive drugs were discovered through unexpected behavioral observations. Systematic behavioral screening is inefficient in most model organisms. But, automated technologies are enabling a new phase of discovery-based research in central nervous system (CNS) pharmacology. Researchers are using large-scale behavior-based chemical screens in zebrafish to discover compounds with new structures, targets, and functions. These compounds are powerful tools for understanding CNS signaling pathways. Substantial differences between human and zebrafish biology will make it difficult to translate these discoveries to clinical medicine. However, given the molecular genetic similarities between humans and zebrafish, it is likely that some of these compounds will have translational utility. We predict that the greatest new successes in CNS drug discovery will leverage many model systems, including in vitro assays, cells, rodents, and zebrafish

Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABAA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABAA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish

Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation

A major obstacle for the discovery of psychoactive drugs is the inability to predict how small molecules will alter complex behaviors. We report the development and application of a high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found that the multidimensional nature of observed phenotypes enabled the hierarchical clustering of molecules according to shared behaviors. Behavioral profiling revealed conserved functions of psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds. In addition, behavioral profiling implicated new factors such as ether-a-go-go–related gene (ERG) potassium channels and immunomodulators in the control of rest and locomotor activity. These results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors

Synthesis of 4H-Benzo[e][1,3]oxazin-4-ones by a Carbonylation–Cyclization Domino Reaction of ortho-Halophenols and Cyanamide

A mild and convenient one-step preparation of 4H-1,3-benzoxazin-4-ones by a domino carbonylation–cyclization process is developed. Readily available ortho-iodophenols are subjected to palladium-catalyzed carbonylative coupling with Mo(CO)6 and cyanamide, followed by a spontaneous, intramolecular cyclization to afford 4H-1,3-benzoxazin-4-ones in moderate to excellent yields. Furthermore, the scope of the reaction is extended to include challenging ortho-bromophenols. Finally, to highlight the versatility of the developed method, Mo(CO)6 is successfully replaced with a wide array of CO-releasing reagents, such as oxalyl chloride, phenyl formate, 9-methylfluorene-9-carbonyl chloride, and formic acid, making this an appealing strategy for the synthesis of 4H-benzo[e][1,3]oxazin-4-ones

Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures

The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca2+) flux in muscle cells as well as intense locomotor activity. We then screened a library of ∼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities

Establishment of Native Aquatic Vegetation in Conjunction with an Integrated Invasive Aquatic Vegetation Management Program

Invasive aquatic vegetation is problematic in many Texas lakes and reservoirs and requires management for control. Most often an integrated pest management program incorporating biological, mechanical or chemical means is implemented. Establishment of non-native plants can also be deterred by utilizing an ecological method of planting native vegetation into the empty niches of a disturbed ecosystems that do not have a propagule bank. However, native vegetation establishment can be delayed by herbivory resulting in the need for protection of plants. Lake Raven was chosen as the study site because an integrated pest management approach using chemical, mechanical and biological means has been implemented to control invasive aquatic plants. The most recent herbicide treatments were fluridone on June 6, 2014 and glyphosate in May and August 2014. Native plant restoration was conducted in July 2014 in niches opened from the management of invasive aquatic vegetation. Six deep water and six shallow water species of native aquatic plants were planted in protective exclosures along the shoreline. Plants were given one month to establish before half the treatment exclosures were opened to potential herbivory. Analysis of covariance was used to determine if herbicide and herbivory limited native plant survival. The herbicide application had a significant effect on deep water plants, but did not have a significant effect on shallow water plants. There was not a significant effect associated with herbivory for any of the plant species, which is likely due to remaining invasive aquatic vegetation. Future research is needed to develop an integrated pest management program that incorporates ecological method without limitations from herbicide application

Neurodegeneration and Epilepsy in a Zebrafish Model of CLN3 Disease (Batten Disease)

The neuronal ceroid lipofuscinoses are a group of lysosomal storage disorders that comprise the most common, genetically heterogeneous, fatal neurodegenerative disorders of children. They are characterised by childhood onset, visual failure, epileptic seizures, psychomotor retardation and dementia. CLN3 disease, also known as Batten disease, is caused by autosomal recessive mutations in the CLN3 gene, 80–85% of which are a ~1 kb deletion. Currently no treatments exist, and after much suffering, the disease inevitably results in premature death. The aim of this study was to generate a zebrafish model of CLN3 disease using antisense morpholino injection, and characterise the pathological and functional consequences of Cln3 deficiency, thereby providing a tool for future drug discovery. The model was shown to faithfully recapitulate the pathological signs of CLN3 disease, including reduced survival, neuronal loss, retinopathy, axonopathy, loss of motor function, lysosomal storage of subunit c of mitochondrial ATP synthase, and epileptic seizures, albeit with an earlier onset and faster progression than the human disease. Our study provides proof of principle that the advantages of the zebrafish over other model systems can be utilised to further our understanding of the pathogenesis of CLN3 disease and accelerate drug discovery

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis. © 2013. Published by The Company of Biologists Ltd

Corporate cash hoarding and corporate governance mechanisms : evidence from Borsa Istanbul

This study aims to examine the impact of corporate governance mechanisms on the cash hoarding decision. The study focuses on BIST100 non-financial firms listed on Borsa Istanbul over the period from 2010 to 2014. The study finds that firms with larger size of board of directors are more likely to hoard cash than firms with smaller board size. However, it finds firms with larger size of audit committee are more likely to hold less cash than firms with smaller audit committee size. Besides, it finds that firms with larger percent of independent directors are more likely to hoard more cash than firms with smaller percent of independent directors. It, also, finds that when the CEO of a firm is also the chairman, the firm tends to hoard more cash. Further, the study finds that firms audited by non-big auditor are more likely to hold more cash than firms audited by big auditor. The results suggest that firms with good corporate governance mechanisms (except for percent of independent directors) are less likely to hoard cash