Selenocyanate as a novel treatment of cystic fibrosis lung disease

项目摘要 感染介导的炎症过程驱动进行性肺病的机制仍然是 难以捉摸。新的初步数据支持一种进化选择性代谢途径，这可能解释了 肺防御病原体的定植而在此过程中不伤害自身。我们发现 含有硫氧还蛋白还原酶（Sec-TrxR）的哺乳动物硒代半胱氨酸，一种在肺中发现的酶，可以 使卤代过氧化物酶产生的次硫氰酸盐（HOSCN）解毒并将其再循环回-SCN。我们假设 原核细菌如大肠杆菌中含有半胱氨酸的硫氧还蛋白还原酶（Cys-TrxR）。coli和P. 铜绿假单胞菌不能解毒HOSCN由于酶结构的差异，特别是缺乏C-末端 硒代半胱氨酸残基只存在于高等真核生物中。囊性纤维化（CF）是由于囊性纤维化的突变。 编码顶端膜阴离子的纤维化跨膜传导调节因子（CFTR）基因 转运包括-SCN在内的多种阴离子的转运蛋白。CF受试者患有慢性肺 导致与这种遗传性疾病相关的发病率和死亡率的感染。CFTR蛋白 将-SCN转运到气道表面液中，我们假设这是异常宿主的一个关键特征， 与CF肺病相关的防御。-SCN可以直接与HOCl反应产生HOSCN，其可以是 被宿主代谢而不是病原体。我们提供了初步的数据支持的能力，哺乳动物 Sec-TrxR解毒HOSCN和原核Cys-TrxR不能解毒HOSCN。我们已经发表 关于CFTR在维持气道液体-SCN水平中的重要性的体外和体内数据以及初步的 研究表明，吸入SCN可显著改善铜绿假单胞菌的发病率并减少肺部炎症。 肺部感染小鼠模型。新的数据表明，SCN类似物，硒氰酸盐（-SeCN），是更有效的 抗微生物比SCN，但共享所有的选择性解毒的好处-SCN。工作假设是 在CF中，肺不能最佳地利用-SCN，导致卤代过氧化物酶产生更多的次氯酸盐（HOCl）， 没有选择性地解毒，并产生更多的肺损伤和炎症。具体目标1将决定 含Sec的TrxR抵抗HOSCN/HOSeCN氧化失活的化学机制， 含Cys的TrxR被HOSCN/HOSeCN灭活。具体目标2将检查CFTR和Sec-TrxR 在-SCN/HOSCN介导的肺宿主防御细菌感染的重要性中的作用。具体目标3 检查-SeCN作为使用野生型和CFTR治疗肺部炎症和感染的潜在疗法 KO和ENaC Tg小鼠模型。该方案的主要创新点在于：1）我们发现了选择性的 通过哺乳动物Sec-TrxR对HOSCN/HOSeCN进行解毒，使肺防御病原体 不伤害自身; 2）HOSCN/HOSeCN对原核Cys-TrxR的新靶向作用; 3）-SCN/-SeCN可 改善中性粒细胞和巨噬细胞宿主防御功能; 4）改善肺部感染模型， 雾化-SeCN;和5）将-SeCN疗法引入临床的原理研究的证明。

PROJECT SUMMARY The mechanism(s) by which infection-mediated inflammatory processes drive progressive lung disease remains elusive. Novel preliminary data supports an evolutionary selective metabolism pathway that may explain how the lung defends against pathogen colonization without harming itself in the process. We have discovered that mammalian selenocysteine containing thioredoxin reductase (Sec-TrxR), an enzyme found in the lung, can detoxify the haloperoxidase produced hypothiocyanate (HOSCN) and recycle it back to -SCN. We hypothesize that cysteine-containing thioredoxin reductase (Cys-TrxR) from prokaryotic bacteria such as E. coli and P. aeruginosa cannot detoxify HOSCN due to differences in enzyme structure, especially the lack of a C-terminal selenocysteine residue found only in higher eukaryotes. Cystic fibrosis (CF) is due to mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene that encodes for an apical membrane anion transporter protein that transports a number of anion species including -SCN. CF subjects have chronic lung infections that contribute to morbidity and mortality associated with this genetic disease. The CFTR protein transports -SCN into the airway surface fluid and we hypothesize that this is a critical feature of the aberrant host defense associated with CF lung disease. -SCN can directly react with HOCl producing HOSCN which can be metabolized by the host but not the pathogen. We provide preliminary data supporting the ability of mammalian Sec-TrxR to detoxify HOSCN and the inability of prokaryotic Cys-TrxR to detoxify HOSCN. We have published in vitro and in vivo data on the importance of CFTR in maintaining airway fluid -SCN levels and preliminary studies that inhaled -SCN dramatically improves morbidity and decreases lung inflammation in a P. aeruginosa lung infection mouse models. Novel data is presented that a SCN analog, selenocyanate (–SeCN), is more potent antimicrobial than SCN, but shares all the selective detoxification benefits of -SCN. The working hypothesis is that in CF the lung cannot use -SCN optimally resulting in haloperoxidases make more hypochlorite (HOCl) which is not selectively detoxified and produces more lung damage and inflammation. Specific aim 1 will determine the chemical mechanism by which Sec-containing TrxR resists inactivation by HOSCN/HOSeCN oxidation and Cys-containing TrxR are inactivated by HOSCN/HOSeCN. Specific aim 2 will examine CFTR’s and Sec-TrxR’s role in the importance of –SCN/HOSCN -mediated lung host defense against bacterial infection. Specific aim 3 Examine -SeCN as a potential therapy for treatment of lung inflammation and infection using wild type and CFTR KO and -ENaC Tg mouse models. The major innovations in this proposal are: 1) our novel discovery of selective detoxification of HOSCN/HOSeCN by mammalian Sec-TrxR that allows the lung to defend against pathogens without harming self; 2) novel targeting of prokaryotic Cys-TrxR by HOSCN/HOSeCN; 3) –SCN/-SeCN may improve neutrophil and macrophage host defense functions; 4) improvement in lung infection models with nebulized -SeCN; and 5) proof of principle studies to bring -SeCN therapy into the clinic.