Discovery of Potent 12-Lipoxygenase Inhibitors of Platelet Activation

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DESCRIPTION (provided by applicant): The principle aim of this research is to develop selective/potent human 12-LOX inhibitors and probe the biochemistry of inflammation in platelet activation, leading to drug candidates for cardiovascular disease. Recent failures in testing of anti-coagulant agents have highlighted the need for new approaches to acute blood coagulation treatment. Among the novel targets, 12-lipoxygenase (12-LOX) stands out for numerous reasons: 1) It is highly expressed in platelets relative to other cells in circulation, 2) we have previously shown 12-LOX to be important for both hemostasis and thrombosis, and 3) 12-LOX is expressed in the platelets of both human and mice. The broad objective of the current proposal is to advance one or more candidate molecules toward therapeutic development against platelet activation/aggregation, utilizing our assays to identify novel selective inhibitor for the human 12-LOX. We have already performed a successful 12-LOX high-throughput (HTP) screen in collaboration with the NIH, which found over ~1000 potential hits. Of these ~1000 compounds, seven were found to be potent 12-LOX inhibitors. Two of these seven inhibitors were re- synthesized and shown to be potent and selective against human 12-LOX in vitro and effective in numerous human platelet cell assays, confirming them as "validated hits". Our research plan is guided by the following four specific aims. First, we propose to optimize our two "validated hits" and discover additional chemotypes, which have increased potency against human 12-LOX. Second, we shall optimize the ADME/PK properties of our "hits", maintain LOX isozyme selectivity and determine their mechanism of inhibition. Third, once we have optimized our various inhibitor chemotypes, we shall evaluate their protective potency in a variety of platelet activation screens. We will utilize these in vitro inhibitors in our platelet models and determine if individual inhibitors reduce aggregation, as well as ascertain if our inhibitors reduc the biochemical production of 12- HETE, as a direct marker of 12-LOX inhibition. Fourth, we will evaluate the in vivo efficacy of these 12-LOX inhibitors toward inhibition of thrombosis in mice while retaining normal levels of hemostasis. We will further confirm the activity of the 12-LOX inhibitors in vivo through assessment of 12-HETE production and ex vivo platelet function from mice treated with 12-LOX inhibitors. These inhibitors will additionally be tested for their abilityto prevent heparin-induced thrombocytopenia (HIT), as recent work suggests HIT is regulated in a 12- LOX-dependent manner. We are confident these studies will discover a 12-LOX inhibitor that is potentially an effective therapeutic against platelet aggregation in a number of pathological settings including athero- thrombosis, venous thrombosis, and HIT, and help us to better understand the biology of cardiovascular disease, a pathophysiological condition which worsens with age.