The principle aim of this research is to develop selective and potent human 12/15-LOX inhibitors, which will probe the role of inflammation in stroke, leading to drug candidates for this devastating human disease. Recent failures in testing of neuroprotective agents have highlighted the need for new approaches to acute stroke treatment. Among the novel targets, 12/15-lipoxygenase (12/15-LOX or 15-LOX-1) stands out for numerous reasons. 12/15-LOX expression is increased in the peri-infarct region following middle cerebral artery occlusion (MCAO) in mice, both in neurons and in endothelial cells, which contributes to ischemic brain injury by increasing neuronal cell death, vascular leakage, and edema formation. ALOX15 gene (i.e. 12/15-LOX) knock out mice are protected against these detrimental effects and human 12/15-LOX expression is increased in the ischemic penumbra of ischemic stroke patients, with a cellular pattern very similar to that found in mice. Furthermore, increased levels of the 12/15-LOX metabolite 12-HETE have been found in patients with stroke caused by subarachnoid hemorrhage, suggesting 12/15-LOX may be a target for subarachnoid hemorrhage as well. Targeting this one enzyme may thus provide pleiotropic benefits. The broad objective of the current proposal is to advance one or more candidate molecules toward therapeutic development against stroke, utilizing our assays to identify novel selective inhibitors for the human 12/15-LOX. We have already performed a successful 12/15-LOX high-throughput (HTP) screen in collaboration with the NIH which found over ~1000 potent inhibitors. Of these ~1000 compounds, 50 were re-screened and six potent 12/15-LOX inhibitors were discovered. One of these six inhibitors was re-synthesized and shown to be potent and selective against human 12/15-LOX in vitro, effective in mouse HT-22 neuronal cells and in a mouse stroke animal model, confirming it as a """"""""validated hit"""""""". Our research plan is guided by the following three specific aims. First, we propose to design new molecules based on our """"""""validated hit"""""""", which will have increased potency against human 12/15-LOX. Second, we shall confirm the optimized """"""""validated hit's"""""""" potency in a neuronal cell line, optimize ADME properties, maintain LOX isozyme selectivity and determine its mechanism of inhibition. Third, once we have optimized our inhibitor scaffold, we will evaluate its protective potency in rodent models of stroke. We will utilize these in vitro inhibitors in our transient focal ischemia mouse model and determine if individual inhibitors reduce infarct size, as well as ascertain if our inhibitors redue the biochemical production of 12-HETE, as a direct marker of 12/15-LOX inhibition. Once this is achieved, we will test these compounds in a model of permanent focal ischemia through iron-induced blood clotting, and a clotting ischemia model with tPA-induced reperfusion, where we test the compounds as adjuvants to tPA treatment. Considering that our """"""""validated hit"""""""" manifests a 30% reduction in infarct size, we are confident these studies will discover a 12/15- LOX inhibitor as an effective therapeutic against stroke damage.
The goal of this research is to discover potent and selective 12/15-lipoxygenase inhibitors to prevent stroke injury. Lipoxygenases have been increasingly implicated in stroke damage, thus by developing inhibitors and optimizing their chemical properties, we hope to develop novel stroke therapeutics.
|Green, Abigail R; Barbour, Shannon; Horn, Thomas et al. (2016) Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential. Biochemistry 55:2832-40|
|Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas et al. (2016) Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency. Bioorg Med Chem 24:5380-5387|
|Mashhadi, Zahra; Newcomer, Marcia E; Brash, Alan R (2016) The Thr-His Connection on the Distal Heme of Catalase-Related Hemoproteins: A Hallmark of Reaction with Fatty Acid Hydroperoxides. Chembiochem 17:2000-2006|
|Armstrong, Michelle M; Freedman, Cody J; Jung, Joo Eun et al. (2016) A potent and selective inhibitor targeting human and murine 12/15-LOX. Bioorg Med Chem 24:1183-90|
|Newcomer, Marcia E; Brash, Alan R (2015) The structural basis for specificity in lipoxygenase catalysis. Protein Sci 24:298-309|
|van Leyen, Klaus; Holman, Theodore R; Maloney, David J (2014) The potential of 12/15-lipoxygenase inhibitors in stroke therapy. Future Med Chem 6:1853-5|