Controlling Radiation Injury by TLR5 Agonists

描述（由申请人提供）：电离辐射（IR）对哺乳动物的毒性主要是由于对最辐射敏感的组织，胃肠道（GI）和造血系统（HP）的损伤。已经开发了许多方法和化合物来保护或恢复HP，包括IR暴露后的免疫功能。相比之下，胃肠道放射综合征的治疗仍然很差，导致大量死亡率和幸存者的生活质量下降。初步研究表明，细菌鞭毛蛋白作为一种NF-？toll样受体5 （TLR5）的B激活激动剂是一种强大的放射保护剂和放射缓释剂，分别在致死性IR之前或之后给予，能够提高生存率。值得注意的是，鞭毛蛋白的抗辐射作用与保护胃肠道和HP系统有关。通过删除鞭毛蛋白中对TLR5激活非必需的部分，产生了一种具有降低免疫原性的药理学优化的TLR5激动剂（CBLB502）。CBLB502对小鼠和非人类灵长类动物有效且无毒，正在开发成为一种稳定的、自给药的辐射解毒剂，预计将用于生物防御应用。本文主要研究CBLB502的分子和细胞作用机制。目的1将涉及鉴定CBLB502对辐照小鼠小肠保护和愈合作用的细胞介质，特别关注骨髓源性细胞的作用。目标2将致力于分析CBLB502诱导的分子信号通路，这对其辐射防护和辐射缓解特性至关重要。假设驱动和无偏功能筛选方法的结合将用于确定CBLB502分子靶标。最后，Aim 3将集中评估CBLB502对其他放射敏感组织（包括肝、肾和肺）中IR的短期和长期病理后果的影响。纤维化和癌症发展的频率和严重程度将在小鼠模型中进行评估。该项目的完成将为一类新兴的辐射解毒剂提供关键信息，这将使人们更好地了解它们作为针对急性辐射综合征的人类治疗药物的潜力。当今世界可能发生的核事故和恐怖袭击意味着由于系统暴露于放射性物质而造成大量辐射伤害和伤亡。全身照射（TBI）的毒性与急性辐射综合征（ARS）的诱导有关，主要涉及对高度辐射敏感的胃肠道（GI）和造血系统的损害。虽然已经开发了许多方法和化合物来保护或恢复创伤性脑损伤后的HP功能，但胃肠道辐射综合征仍然难以治疗，导致严重的死亡率和幸存者的生活质量下降。理想的辐射对策既能保护GI系统，又能保护HP系统，既能进行辐射防护（预防），又能减轻辐射伤害。此外，生物防御应用需要一种易于自我施用的药物，具有较长的保质期，不需要特殊的处理或储存条件。我们最近发现了一类新的制剂，满足上述所有要求，最显著的是对胃肠道辐射综合征和HP综合征有效。我们的先导化合物，命名为CBLB502，是一种强大的辐射保护剂和缓解辐射损伤，适用于肌肉注射。它是一种从细菌蛋白鞭毛蛋白中提取的多肽，作为toll样受体5 （TLR5）的激动剂，在肠和骨髓祖细胞的放射敏感细胞中表达，与配体结合后，激活几种由NF-？B信号。在此，我们提出了一项计划，全面表征CBLB502抗辐射活性的分子和细胞机制，特别是针对GI辐射综合征。我们还将研究CBLB502对IR在其他组织中的病理后果的影响，包括辐射致癌性。拟议的实验将为先前观察到的CBLB502的辐射防护和减缓作用提供基本的机制见解。

DESCRIPTION (provided by applicant): The toxicity of ionizing radiation (IR) in mammals is largely due to damage to the most radiosensitive tissues, the gastrointestinal (GI) tract and the hematopoietic (HP) system. A number of approaches and compounds have been developed to protect or restore HP, including immune, function following IR exposure. In contrast, GI radiation syndrome remains poorly treatable resulting in significant mortality and reduced quality of life for survivors. In preliminary studies it was demonstrated that the bacterial flagellin protein, acting as an NF-?B activating agonist of Toll-like receptor 5 (TLR5), is a powerful radioprotectant and radiomitigator capable of improving survival when given either before or after lethal IR, respectively. Notably, the anti-radiation effects of flagellin are associated with protection of the GI tract as well as the HP system. A pharmacologically optimized TLR5 agonist with reduced immunogenicity (CBLB502) was generated by deleting portions of flagellin that are non-essential for TLR5 activation. CBLB502 is effective and non-toxic in mice and non-human primates and is being developed into a stable, self-administrable radiation antidote projected for biodefense applications. This proposal is focused on deciphering the molecular and cellular mechanisms of action of CBLB502. Aim 1 will involve identification of cellular mediators of CBLB502's effect on protection and healing of small intestine in irradiated mice with particular attention on the role of bone marrow-derived cells. Aim 2 will be devoted to analysis of molecular signaling pathways induced by CBLB502 that are critical for its radioprotection and radiomitigation properties. A combination of hypothesis-driven and unbiased functional screening approaches will be used to determine CBLB502 molecular targets. Finally, Aim 3 will concentrate on assessing the effects of CBLB502 on the short- and long-term pathological consequences of IR in other radiosensitive tissues, including liver, kidney and lung. The frequency and severity of fibrosis and cancer development will be assessed in a mouse model. Completion of this program will provide critical information about an emerging class of radiation antidotes that will allow improved understanding of their potential as human therapeutics targeting acute radiation syndrome. The potential scenarios for nuclear accidents and terrorist attacks in today's world imply mass radiation injury casualties affected by systemic exposure to radioactive materials. The toxicity of total body irradiation (TBI) is associated with induction of acute radiation syndrome (ARS) primarily involving damage to the highly radiosensitive gastrointestinal (GI) tract and hematopoietic (HP) system. While a number of approaches and compounds have been developed to protect or restore HP function following TBI, GI radiation syndrome remains poorly treatable, resulting in significant mortality and reduced quality of life for survivors. An ideal radiation countermeasure would protect both the GI and HP systems and would be capable of both radioprotection (prophylaxis) and mitigation of radiation injury. Moreover, biodefense applications require a drug that is easily self-administered, has a long shelf life and does not require special handling or storage conditions. We have recently discovered a new class of agents that satisfy all of the above requirements, most notably being effective against GI radiation syndrome as well as HP syndrome. Our lead compound, named CBLB502, is a powerful radioprotectant and mitigator of radiation injury suitable for intramuscular injections. It is a polypeptide derived from bacterial protein flagellin that acts as an agonist of Toll-like receptor 5 (TLR5), which is expressed in radiosensitive cells of the intestine and bone marrow progenitors and, upon binding with the ligand, activates several pro-survival pathways, mediated by NF-?B signaling. Here we propose a program to comprehensively characterize the molecular and cellular mechanisms underlying the anti-radiation activity of CBLB502, particularly focusing on GI radiation syndrome. We will also investigate the effect of CBLB502 on the pathological consequences of IR in other tissues, including radiation carcinogenicity. The proposed experiments will provide essential mechanistic insight into the previously observed radio-protective and -mitigative effects of CBLB502.