Cardiac glycosides provide neuroprotection against ischemic stroke: Discovery by a brain slice-based compound screening platform

Abstract

We report here the results of a chemical genetic screen using small molecules with known pharmacologies coupled with a cortical brain slice-based model for ischemic stroke. We identified a smallmolecule compound not previously appreciated to have neuroprotective action in ischemic stroke, the cardiac glycoside neriifolin, and demonstrated that its properties in the brain slice assay included delayed therapeutic potential exceeding 6 h. Neriifolin is structurally related to the digitalis class of cardiac glycosides, and its putative target is the Na ؉ ͞K ؉ -ATPase. Other cardiac glycoside compounds tested also showed neuroprotective activity, although with lower apparent potencies. In subsequent whole-animal studies, we found that neriifolin provided significant neuroprotection in a neonatal model of hypoxia͞ischemia and in a middle cerebral artery occlusion model of transient focal ischemia. The neuroprotective potential of Na ؉ ͞K ؉ -ATPase is of particular interest because of its known ''druggability''; indeed, Food and Drug Administration-approved, small-molecule compounds such as digitoxin and digoxin have been in clinical usage for congestive heart failure and arrhythmias for several decades. Thus, an existing cardiac glycoside or closely related compound could provide an accelerated path toward clinical trial testing for ischemic stroke. Our findings underscore the important role that hypothesis-neutral, high-content, tissue-based screens can play in the identification of new candidate drugs and drug targets for the treatment of diseases for which validated therapeutic pathways are not currently available.biotechnology ͉ drug discovery ͉ high-content screening ͉ translational medicine

The Nitrone Disodium 2,4-Sulphophenyl-N-Tert-Butylnitrone is Without Cytoprotective Effect on Sodium Nitroprusside-Induced Cell Death in N1E-115 Neuroblastoma Cells in vitro

“…We saw no effect of NXY-059 on tyrosine nitration at concentrations well above the maximal concentration of 200 mmol/L used by Maples et al (2001). A recent paper (Wang et al, 2006) did claim that low concentrations of NXY-059 were cytoprotective in a brain slice preparation, but the nitrone studied was in fact PBN (DC Lo, personal communication). Our study is therefore the first test for a cytoprotective effect of NXY-059 on neuronal-type cells in vitro.…”

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+-ATPase

“…Recently several compounds showing inhibitory potency on Na + ,K + -ATPase, such as cardiac glycosides, ginsenosides, and magnesium lithospermate B (the major water soluble ingredient in the dried roots of medicinal plant Salvia miltiorrhiza), were demonstrated to provide neuroprotection against ischemic stroke in a cortical brain slicebased compound screening platform [6,37,38] . Similarly, oleanolic acid, showing inhibitory potency on Na + ,K + -ATPase in this study, has also been demonstrated to display neuroprotective effect against focal cerebral ischemic injury [39] .…”

High Throughput Screening for Neurodegeneration and Complex Disease Phenotypes

“…Though these small-molecules can reverse phenotypes of several complex neurodegenerative disorders in cell culture and some invertebrate animal models, prioritization and testing in more validated mouse models is a serious challenge. More complex HCS assay and testing in primary cell culture models or invertebrate animal models can provide a reasonable means to prioritize these hits [54,58,64]. However, activity in a small range of secondary assays, though useful for prioritizing such active compounds, is by no means a proof that inactive compounds do not merit further study.…”

“…We have recently described a HCS screen for ischemic stroke based on the creation of a "sentinel" population of pyramidal neurons in cortical brain slice explants via biolistic transfection of Green Fluorescent Protein [58]. Subsequent to oxygen-glucose deprivation (OGD), these GFP-expressing sentinel neurons provide a living index of the extent and time course of the ischemic damage produced.…”