Mechanistic probes to study the immune response in periodontal disease

摘要 42%的美国成年人患有牙周病，其特点是细菌引起的炎症 骨质流失。传统的和新兴的牙周炎治疗方法通常不针对宿主 免疫反应，这是组织损伤的主要来源。染色质的脱甲基化活性 重塑酶赖氨酸特异性去甲基酶1(KDM1A)位于转录激活标志组蛋白3赖氨酸 4(H3K4)导致促炎细胞因子转录减少。相比之下，染色质的重塑 酶赖氨酸特异性去甲基酶4B(KDM4B)特异性去甲基化转录失活标志 组蛋白3三甲基赖氨酸9(H3K9me3)，导致促炎细胞因子表达上调 (图)。有趣的是，这两种酶之间的串扰导致了一个平衡的系统，其中赖氨酸9 KDM4B的低甲基化是KDM1a对赖氨酸4进行低甲基化的先决条件。这项研究 本提案中概述的计划将利用这种串扰来设计用于 帕金森病的表观遗传学基础研究。这项研究的中心假设是KDM1A的促进 通过引入特定的KDM4B或KDM4E抑制剂的活性将通过减少表达 病变组织中的PICS和减少破骨细胞的形成。这样确定的抑制剂将作为化学物质有用 探讨帕金森病炎症和骨丢失的生化基础。我们将检验这一假设 通过完成以下具体目标：具体目标1：我们将采用基于结构的设计 发现新的KDM4B或4E抑制剂作为化学工具用于阐明其机制的技术 帕金森病的潜在炎症和骨丢失；特定目标2：我们将定义 KDM4B/4E过度表达导致牙周炎和骨质流失；具体目标3：我们将 使用两种帕金森病模型评估新的和已知的KDM4B/4E抑制剂在体内的免疫调节活性。 我们的初步结果表明，KDM4B和4E蛋白在牙周组织中的体内含量更丰富 且抑制KDM4B可显著降低PIC产量和 病变周围脂多糖预处理的组织中破骨细胞的形成。新的和现有的缓蚀剂 将用于进一步验证KDM4B作为PD的治疗靶点，以及计算化学对接 将使用与物理化合物筛选配对的实验来关联化合物结构和 疾病进展标志物的变化。选定的KDM4B抑制剂将在体外和在 体内药效和毒性。这项研究将对表观遗传机制提供更有力的理解。 对牙周病进展起重要作用，验证KDM4B作为牙周炎的药物靶点， 并开发出具有局部免疫调节佐剂治疗潜力的化学探针 帕金森病的治疗。该项目将打破牙周学和药物设计领域之间的壁垒，并 将促进关于翻译口腔健康科学迫切需要的方面的知识的发展。

Abstract Periodontal diseases affect 42% of adult Americans and are characterized by bacterial-driven inflammatory bone loss. Traditional and emerging treatments for periodontitis management do not typically target the host immune response, which is the major source of tissue damage. The demethylation activity of the chromatin remodeling enzyme lysine-specific demethylase 1 (KDM1A) at the transcription activating mark histone 3 lysine 4 (H3K4) leads to a decrease in pro-inflammatory cytokine transcription. By contrast, the chromatin remodeling enzyme lysine specific demethylase 4B (KDM4B) specifically demethylates the transcription deactivating mark histone 3 trimethyllysine 9 (H3K9me3), leading to up regulated expression of pro-inflammatory cytokines (PICs). Interestingly, cross talk between these two enzymes leads to a balanced system wherein lysine 9 hypomethylation by KDM4B serves as a prerequisite to lysine 4 hypomethylation by KDM1A. The research plan outlined in this proposal will exploit this crosstalk for the design of new chemical probes for use in the study of the epigenetic basis for PD. The the central hypothesis of this study is that promotion of KDM1A activity by introduction of a specific KDM4B or KDM4E inhibitor will alleviate PD by reducing the expression of PICs in diseased tissue and reducing osteoclast formation. Inhibitors so identified will be useful as chemical probes to study the biochemical basis of inflammation and bone loss in PD. We will test this hypothesis through completion of the following Specific Aims: Specific Aim 1: We will use structure-based design techniques to discover novel inhibitors of KDM4B or 4E for use as chemical tools to elucidate the mechanism underlying inflammation and bone loss in PD; Specific Aim 2: We will define the cellular mechanism by which KDM4B/4E over expression contributes to periodontal inflammation and bone loss; Specific Aim 3: We will evaluate novel and known KDM4B/4E inhibitors for immunomodulatory activity in vivo using two models of PD. Our preliminary results demonstrate that KDM4B and 4E protein is more abundant in vivo in periodontally diseased connective tissue, and that inhibition of KDM4B results in significant decreases in PIC production and osteoclastogenesis in tissue pre-treated with a periopathogenic lipopolysaccharide. New and existing inhibitors will be used to further validate KDM4B as a therapeutic target in PD, and computational chemistry docking experiments paired with physical compound screens will be employed to correlate compound structure and changes in disease progression markers. Selected KDM4B inhibitors will be interrogated both in vitro and in vivo for efficacy and toxicity. This study will provide a more robust understanding of epigenetic mechanisms that play a significant role periodontal disease progression, validate KDM4B as a drug target for periodontitis, and result in development of chemical probes with therapeutic potential for local immunomodulatory adjuvant treatment of PD. This project will break down barriers between the fields of periodontics and drug design and will foster the development of knowledge on a critically needed aspect of translational oral health science.