Therapeutic Intervention of Lewisite-Mediated Cutaneous Blistering-Inflammation

描述（由申请人提供）：路易氏剂已被确定为可能用于化学战的潜在威胁化学品。局部暴露于路易氏剂导致皮肤起泡和炎症，这可能是严重和痛苦的。在本申请中，我们将测试这样的假设：由于与紧密连接和水/甘油运输相关的蛋白质被破坏，路易斯石通过渗透皮肤而破坏皮肤屏障功能。这些作用通过激活hippo信号传导途径、FAK激酶、泛素连接酶、蛋白酶如组织蛋白酶和其他溶菌酶蛋白酶介导。急性炎症是通过激活砷依赖性活性氧（ROS）产生触发的未折叠蛋白反应（UPR）信号和激活DNA损伤反应信号介导的。这些复杂的信号通路之间的串扰导致疼痛性水疱和炎症的发病机制。通过小分子阻断这些分子靶标可以阻断这些效应。目的1通过研究路易氏剂对表皮紧密连接蛋白claudins、occludin、zonula occludens（ZO）等的影响，探讨路易氏剂对皮肤屏障功能破坏和水疱的影响。此外，蛋白质参与水/甘油运输的调节，水通道蛋白，将进行研究。路易氏剂对急性皮肤炎症反应的影响也将通过确定炎症反应的动力学和在其峰值时评估增生和炎症细胞浸润来研究。然后，我们将检测促炎介质，IL-6，干扰素-β，前列腺素E2、ROS、RNS（包括NO）等，使用来自路易氏剂处理的Ptch 1 +/-/SKH-1无毛小鼠的皮肤活组织检查。我们将确定路易氏体调节的UPR信号是否调节这些促炎作用。在目标2中，我们将筛选饮食和合成测试试剂对描述屏障功能、起泡和炎症的生物标志物的有效性。基于其阻断参与屏障功能/起泡/炎症的分子靶点的已知潜力，从一系列膳食和合成化学品中选择的两种最有效的药物将在此目的中进一步评价。在确定治疗窗后，将评估这些选定药物的作用动力学、逆转路易氏体诱导的分子变化的能力和潜在的皮肤炎症/水疱。因此，该提议将采用一种新的小鼠模型来解开路易氏剂的分子机制，以开发基于机制的解毒剂/治疗。通过提交前瞻性申请（R 01或U 01），小分子候选先导化合物可以很容易地用于进一步的小鼠研究，并最终基于其已知的毒性特征和用于其他疾病的临床试验。拟议研究的结果很可能对在大规模人口接触战争威胁化学品的情况下保护人类健康产生重大影响。

DESCRIPTION (provided by applicant): Lewisite has been identified as a potential threat chemical which could be used in chemical warfare. Topical exposure to lewisite results in cutaneous blistering and inflammation, which may be severe and painful. In this application, we will test the hypothesis that lewisite, by penetrating skin, ruptures the cutaneous barrier functions as a consequence of disruption of proteins associated with tight junctions and water/glycerin transport. These effects are mediated via activation of hippo signaling pathway, FAK kinases, ubiquitin ligases, proteases such as Cathepsin, and other lysozymal proteases. The acute inflammation is mediated through the activation of unfolded protein response (UPR) signaling triggered by arsenic-dependent reactive oxygen species (ROS) production and activation of DNA damage response signaling. Crosstalk between these intricate signaling pathways results in the pathogenesis of painful blisters and inflammation. Blocking these molecular targets by small molecules may intercept these effects. Aim 1 will investigate the effects of lewisite on disruption of skin barrier function and blistering by studying its effects o tight junction proteins such as claudins, occludin, zonula occludens (ZO), etc. in epidermis. In addition, proteins involved in the regulation of water/glycerin transport, the aquaporins, will be studied. The effects of lewisite on acute cutaneous inflammatory response will also be investigated by determining the kinetics of inflammatory responses and, at their peak, assessing hyperplasia and inflammatory cell infiltration. Then, we will assay the pro-inflammatory mediators, IL-6, interferon-?, prostaglandin E2, ROS, RNS (including NO) etc. using skin biopsies from lewisite- treated Ptch1+/-/SKH-1 hairless mice. We will determine whether lewisite-modulated UPR signaling regulates these pro-inflammatory effects. In Aim 2, we will screen the effectiveness of dietary and synthetic test agents on biomarkers depicting barrier function, blistering, and inflammation. The two most efficacious agents chosen from a series of dietary and synthetic chemicals based on their known potential to block molecular targets involved in barrier function/blistering/inflammation will be evaluated further in this aim. After defining the therapeutic window, kinetics of action, ability to reverse lewisite-induced molecular changes and underlying cutaneous inflammation/blistering by these select agents will be assessed. Thus, this proposal will employ a novel murine model to unravel the molecular mechanism of lewisite in order to develop mechanism-based antidotes/therapy. Small molecule candidate lead-compounds can easily be taken to further murine studies by submitting a prospective application (R01 or U01) and finally to clinical trials based on their known toxicity profile and use for other conditions. The outcome of the proposed research is likely to have a significant impact on human health protection in the event of mass population exposure to war threat chemicals.