Therapeutic Intervention of Lewisite-Mediated Cutaneous Blistering-Inflammation

描述(申请人提供)：刘易斯已被确定为一种潜在的威胁化学品，可用于化学战。局部暴露于路易氏剂会导致皮肤起泡和发炎，这可能是严重和疼痛的。在这项应用中，我们将检验这一假设，即路易氏剂通过穿透皮肤，破坏与紧密连接和水/甘油运输相关的蛋白质，从而破坏皮肤屏障功能。这些作用是通过激活河马信号通路、FAK激酶、泛素连接酶、组织蛋白酶等蛋白酶和其他溶菌酶来实现的。急性炎症是通过激活未折叠蛋白反应(UPR)信号和激活DNA损伤反应信号来实现的。这些错综复杂的信号通路之间的串扰导致了疼痛水泡和炎症的发病机制。用小分子阻断这些分子靶标可能会截获这些效应。目的1通过研究路易威特对表皮紧密连接蛋白Claudins、occludin、zonula occludens(ZO)等的影响，探讨其对皮肤屏障功能的破坏和水泡形成的影响。此外，还将研究与水/甘油运输调节有关的蛋白质--水通道蛋白。路易威特对急性皮肤炎症反应的影响也将通过测定炎症反应的动力学以及在其高峰期评估增生和炎症细胞的渗透来进行研究。然后，我们将检测促炎症介质，IL-6，干扰素-β，前列腺素E2，ROS，RNS(包括NO)等，使用路易斯汀治疗ptch1/-/SKH-1无毛小鼠的皮肤活检。我们将确定路易威特调节的UPR信号是否调节这些促炎效应。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、水泡和炎症的生物标记物的有效性。从一系列饮食和合成化学品中选择的两种最有效的药物，基于它们已知的阻止屏障功能/起泡/炎症的分子靶标的潜力，将在这一目标中进一步评估。确定治疗窗口后，将评估这些选定药物的作用动力学、逆转路易威特诱导的分子变化的能力以及潜在的皮肤炎症/水泡。因此，本提案将采用一种新的小鼠模型来解开路易威特的分子机制，以开发基于机制的解毒剂/治疗方法。通过提交预期的申请(R01或U01)，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，并最终根据它们已知的毒性特征和用于其他条件进行临床试验。如果人们大量接触战争威胁化学品，拟议的研究结果可能会对人类健康保护产生重大影响。

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.