Armstrong Michelle M, Freedman Cody J, Jung Joo Eun, Zheng Yi, Kalyanaraman Chakrapani, Jacobson Matthew P, Simeonov Anton, Maloney David J, van Leyen Klaus, Jadhav Ajit, Holman Theodore R
Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95060, United States.
Neuroprotection Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, United States.
Bioorg Med Chem. 2016 Mar 15;24(6):1183-90. doi: 10.1016/j.bmc.2016.01.042. Epub 2016 Jan 21.
Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4±0.5 μM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0±0.08 μM and a Kiu of 6.0±3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.
人网织红细胞12/15-脂氧合酶（h12/15-LOX）是一种脂质氧化酶，在没有磷脂酶的情况下，它可以直接氧化脂质膜，从而直接攻击细胞器，如线粒体。中风后，h12/15-LOX的这种细胞毒性活性在神经元和内皮细胞中上调，并被认为与神经元细胞死亡和血脑屏障渗漏均有关。在体外选择性靶向重组h12/15-LOX以及在体内对小鼠直系同源物具有活性的抑制剂的发现，可能会为中风治疗提供一种新的治疗策略。在此，我们报告了在高通量筛选（HTS）中发现的一类新的抑制剂，它们对重组h12/15-LOX和细胞小鼠12/15-脂氧合酶（m12/15-LOX）具有选择性且强效。母体分子MLS000099089（化合物99089）在体外对h12/15-LOX的IC50效力为3.4±0.5μM，在小鼠神经元细胞系HT-22中体内IC50效力约为10μM。当在20μM抑制剂浓度下进行测试时，化合物99089对h5-LOX和COX-2的选择性大于30倍，对h15-LOX-2的选择性为15倍，对h12-LOX的选择性为10倍。稳态抑制动力学表明，99089对h12/15-LOX的抑制模式是一种混合型抑制剂，Kic为1.0±0.08μM，Kiu为6.0±3.3μM。这些数据表明，99089及其相关衍生物可能因其在体外选择性靶向h12/15-LOX和在体内靶向m12/15-LOX的能力，而成为抗中风治疗药物开发的起点。
