Selective Inhibitors of Pro-Ferroptotic Lipoxygenases - Next Generation Radiomitigators

尽管有意或意外的辐射照射构成严重威胁，但辐射后损伤的分子机制仍然存在着一些问题。 全身照射（TBI）并不完全清楚，因此放射性方法仍然不足。我们 氧化心磷脂（CL）作为TBI细胞凋亡所需阶段的发现已经导致 作为抗细胞凋亡放射性抑制剂的靶向GS-nitroxides和咪唑取代的脂肪酸。同样地， 通过由15-羟基-磷脂酰乙醇胺（HOO-PE）产生的氢过氧化-磷脂酰乙醇胺（HOO-PE）来解读TBI铁凋亡信号传导。 脂氧合酶（15 LOX）与磷脂酰乙醇胺结合蛋白1（15-LOX/PEBP 1）的复合物指导我们 作为新型放射性抑制剂的新型抑制剂。我们通过细菌病原体证明了“盗窃-铁下垂”， 铜绿假单胞菌利用其15-LOX（pLoxA）（12）触发宿主（上皮）细胞的铁凋亡，表明 TBI诱导的非无菌性炎症也可以作为治疗靶点。我们的核心假设是辐射 触发反应涉及几种类型的程序性坏死性死亡，特别是胃肠道上皮细胞的铁凋亡 以及主要的先天免疫细胞，中性粒细胞和巨噬细胞，随着时间的推移而进化，并驱动坏死性炎症 vs无菌和非无菌炎症期间的促消退凋亡反应， 功能障碍和死亡率。这为TBI机制的主要新理解奠定了基础，并导致 一种新的协调放射治疗策略，针对主要细胞死亡的时间和机制特异性靶向 途径。我们的假设将被测试如下：目的1：通过采用氧化还原脂质组学鉴定特异性 在上皮细胞、中性粒细胞和巨噬细胞中主要死亡程序的含氧磷脂信号， 在无菌和非无菌坏死性炎症的不同阶段，辐照小鼠的GI。这些新发现的TBI 脂质生物标志物将涉及：i）细胞死亡的特异性蛋白标志物，ii）主要的促炎和抗炎脂质 介质，iii）细胞因子，和iv）破坏上皮屏障和免疫抑制。目的2：探索分子 极化为M1（N1）和M2（N2）的巨噬细胞和中性粒细胞对铁凋亡的敏感性/抵抗机制 在促炎/抗炎条件下的反应状态和15 LOX和iNOS/NO·系统的表达， TBI诱导体内铁凋亡。我们将使用15只LOX和iNOS KO小鼠，并定量评估 P.铜绿假单胞菌及其pLoxA在小鼠肠铁缺乏和TBI放射敏感性中的作用。目标3：设计新的选择性 哺乳动物15 LOX以及铜绿假单胞菌的原核pLoxA的促铁蛋白酶催化活性的抑制剂， 在TBI辐射疾病的无菌和非无菌阶段，测试它们作为抗铁毒性辐射调节剂。总体而言，这 该项目是基于一个全新的概念，放弃了寻找一个单一的“银弹”radiomitigator， 包括几种缓解剂的协调组合，及时控制TBI引发的异常反应， 依赖机制的方式。

In spite of the serious threats of intentional or accidental exposures to radiation, molecular mechanisms of injury after total body irradiation (TBI) are not completely clear, hence radiomitigative approaches remain insufficient. Our discoveries of oxygenated cardiolipins (CLs) as required stages of TBI apoptosis have already resulted in mitochondria-targeted GS-nitroxides and imidazole-substituted fatty acids as anti-apoptotic radiomitigators. Similarly, deciphering the TBI ferroptotic signaling by hydroperoxy-phosphatidylethanolamines (HOO-PEs) produced by 15- lipoxygenases (15LOX) complexes with phosphatidylethanolamine-binding protein 1 (15-LOX/PEBP1) guides us to new inhibitors as novel radiomitigators. Our demonstration of “theft- ferroptosis” by a bacterial pathogen, Pseudomonas aeruginosa, utilizing its 15-LOX (pLoxA) (12) to trigger ferroptosis of the host (epithelial) cells indicates that TBI induced non-sterile inflammation may be also therapeutically targeted. Our central hypothesis is that radiation triggered responses engage several types of programmed necrotic death, particularly ferroptosis, in GI epithelial cells and the major innate immune cells, neutrophils and macrophages, evolving over time and driving necro-inflammatory vs pro-resolving apoptotic responses during sterile and non-sterile inflammation and culminating in multiple organ dysfunction and mortality. This sets the stage for a principally new understanding of the TBI mechanisms and leads to a new harmonized radiomitigation strategy of time- and mechanism-specific targeting of the leading cell death pathways. Our hypothesis will be tested as follows: Aim 1: By employing Redox Lipidomics identify specific oxygenated phospholipid signatures of the major death programs in epithelial cells, neutrophils and macrophages of the GI of irradiated mice at different stages of sterile and non-sterile necro-inflammation. These newly discovered TBI lipid biomarkers will be related to: i) specific protein markers of cell death, ii) major pro- and anti-inflammatory lipid mediators, iii) cytokines, and iv) breach of the epithelial barrier and immunosuppression. Aim 2: Explore molecular mechanisms of sensitivity/resistance to ferroptosis of macrophages and neutrophils polarized to M1 (N1) and M2 (N2) states in response to pro-/anti-inflammatory conditions and expression of 15LOX and iNOS/NO• system in vitro and TBI induced ferroptosis in vivo. We will employ 15LOX and iNOS KO mice and also quantitatively assess the role of P. aeruginosa and its pLoxA in gut ferroptosis and radiosensitivity of mice to TBI. Aim 3: Design new selective inhibitors of pro-ferroptotic catalytic activity of mammalian 15LOX as well as prokaryotic pLoxA of P. aeruginosa and test them as anti-ferroptotic radiomitigators during sterile and non-sterile stages of TBI radiation disease. Overall, this project is based on an entirely new concept that abandons the search for a single “silver-bullet” radiomitigator and includes a harmonized combination of several mitigators controlling TBI triggered aberrant reactions in time- and mechanism-dependent manner.