Open Access Peer-reviewed Research Article
A Pharmacological Screening Approach for Discovery of Neuroprotective Compounds in Ischemic Stroke
Published: July 18, 2013
With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-β-hydroxysteroid dehydrogenase-1 (11β-HSD1), gave rise to similarly potent in vivo neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11β-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.
Citation:Beraki S, Litrus L, Soriano L, Monbureau M, To LK, Braithwaite SP, et al. (2013) A Pharmacological Screening Approach for Discovery of Neuroprotective Compounds in Ischemic Stroke. PLoS ONE 8(7): e69233.
Editor:Jinglu Ai, St Michael’s Hospital, University of Toronto, Canada
Received:April 18, 2013; Accepted:June 6, 2013; Published: July 18, 2013
Copyright: © 2013 Beraki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:AGY Therapeutics supported the work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors list includes several individuals who are currently employed by commercial companies (Ms. Lily Litrus at BioSeek division of DiscoverX, Ms. Liza Soriano at Pacific BioDevelopment LLC, Dr. Steven Braithwaite and Dr. Karoly Nikolich at Circuit Therapeutics, Dr. Roman Urfer at Selonterra LLC, and Dr. Donna Oksenberg at Global Blood Therapeutics). Prior to current employment some of the work for the study was completed by the authors while employed at AGY Therapeutics. The team working on this program at AGY Therapeutics consisted of all the coauthors listed above. The current employers played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, and have no financial incentive or gain from publication. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Stroke is the fourth leading cause of adult disability in the United States and a significant public health problem worldwide. Neuroprotective therapies that can be administered after stroke to reduce further neuronal loss are, therefore, a critical area for research and drug development. Tissue plasminogen activator (tPA), currently the only approved therapy, must be administered within 3 hours of stroke onset and carries a risk of inducing cerebral hemorrhage. Novel mechanisms and pharmacological agents are needed to treat patients who suffer a stroke in order to limit neuronal damage and improve clinical outcome. Here we report an approach to screen a library of pharmacologically active compounds in an in vitro model for ischemic injury using primary cortical neurons and hippocampal slices.
Understanding of the mechanisms underlying neuronal death has led to the proposal that several parallel cellular processes including excitotoxicity, ionic imbalance, oxidative stress, and apoptotic–like cell death contribute to delayed ischemic neuronal damage. Despite numerous large clinical trials with compounds targeting these pathways at the individual level, none of these experimental treatments have been successful in generating lead therapeutics for ischemic stroke. This may further suggest that ischemic brain injury following stroke is mediated by activation of several of these complex signaling pathways, and targeting a selective signaling cascade would not be beneficial in protecting the tissue in this disorder. Therefore, approaches that can further define the mechanisms and relevance of pharmacological intervention are necessary to identify compounds of potential benefit.
In this study we used the oxygen glucose deprivation (OGD) model of ischemic neuronal death to identify neuroprotective compounds from a small library. With this approach, we identified Carbenoxolone, a compound known as a gap junction blocker and modulator of 11-β-hydroxysteroid dehydrogenases, as a neuroprotectant. This compound proved to be efficacious in an in vivo model of stroke and further delineation of its mechanism of action identified that inhibition of 11-β-hydroxysteroid dehydrogenase-1 (11β-HSD1) underlies, at least in part, its neuroprotective properties. The role of 11β-HSD1 is to modulate local levels of corticosteroids, acting as an oxoreductase to increase active glucocorticoid levels. Carbenoxolone’s neuroprotective properties were demonstrated in cultured hippocampal neurons, and 11β-HSD1 knockout mice are protected from age related decline in hippocampal function. In addition, Carbenoxolone is neuroprotective when centrally or peripherally administered prior to ischemic injury.
The aim of this study was to discover development candidates by identifying neuroprotective compounds in primary cortical neurons and then confirm their activities in rodent models of stroke. After the initial screen, we focused our profiling on Carbenoxolone. Future efforts will extend our findings in further validating the importance of 11β-HSD1 in neuroprotection and prevention of functional loss in ischemic brain injury.
All experiments were in accordance with protocols approved by AGY’s Animal Care and Use Committee and were performed based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Sufficient actions were considered for reducing pain or discomfort of subjects during the experiments.
All experimental procedures were approved by AGY’s Animal Care and Use Committee. Animal handling was performed in accordance with guidelines of National Institute of Health. Male Wistar rats were supplied by Harlan Laboratories (Harlan Inc., CA) at a body weight of 300–330 grams and approximately 9–10 weeks of age. The Library of Pharmacologically Active Compounds was purchased from Prestwick Chemical (The Prestwick Chemical Library, Illkirch, France) and all other chemicals were purchased from SigmaAldrich. BVT-2733 (3-chloro-2-methyl-N-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethyl) thiazol-2-yl) benzenesulfonamide hydrochloride) was synthesized by a contract research organization.
Rat hippocampal cultures were generated using techniques for culturing brain slices originally described in Stoppini et al with modifications in Cronberg et al. Briefly, the hippocampi of male rats were dissected and immersed in ice-cold HBSS, cut into 250-µm-thick sections using a tissue chopper and plated, one slice per insert, onto Millicell culture inserts (0.4 µm Millicell-CM, 12 mm in diameter, Millipore Corp., Bedford, MA). Cultures were maintained in a humidified atmosphere at 35°C in a CO2 incubator (Thermo-Forma Scientific, Marietta, MA) for 3 weeks before experiments. The culture medium, with osmolarity 330 mosM, consisted of 50% MEM (Eagles with Earl’s balanced salt solution), 25% heat inactivated horse serum, 18% HBSS and 2% B27 and was supplemented with 4 mM l-glutamine and 50 units of penicillin–streptomycin/ml. d-glucose was added to a final concentration of 20 mM. B27 was omitted after the first week o
