Mechanistic probes to study the immune response in periodontal disease

摘要 牙周病影响42%的成年美国人，其特征是细菌驱动的炎症性疾病。 骨质流失传统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。染色质的去甲基化活性 重构酶赖氨酸特异性脱甲基酶1（KDM 1A）在转录激活标记组蛋白3赖氨酸 4（H3 K4）导致促炎细胞因子转录的减少。相比之下， 赖氨酸特异性脱甲基酶4 B（KDM 4 B）特异性地使转录失活标记脱甲基 组蛋白3三甲基赖氨酸9（H3 K9 me 3），导致促炎细胞因子表达上调 （太平洋岛屿国家）。有趣的是，这两种酶之间的串扰导致平衡系统，其中赖氨酸9 KDM 4 B的低甲基化是KDM 1A的赖氨酸4低甲基化的先决条件。研究 本提案中概述的计划将利用这种串扰来设计新的化学探针， PD的表观遗传学基础研究。本研究的中心假设是KDM 1A的促进作用 通过引入特异性KDM 4 B或KDM 4 E抑制剂的活性将通过减少 病变组织中的PIC和减少破骨细胞的形成。如此鉴定的抑制剂将可用作化学制剂。 探讨PD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：我们将使用基于结构的设计 发现KDM 4 B或4 E的新型抑制剂的技术，用作阐明机制的化学工具 PD中潜在的炎症和骨丢失;具体目标2：我们将定义细胞机制， KDM 4 B/4 E过表达有助于牙周炎症和骨丢失;具体目标3：我们将 使用两种PD模型评价新型和已知KDM 4 B/4 E抑制剂的体内免疫调节活性。 我们的初步研究结果表明，KDM 4 B和4 E蛋白在牙周组织中的含量更高， 疾病的结缔组织，并且KDM 4 B的抑制导致PIC产生的显著减少， 在用病原体周围脂多糖预处理的组织中的破骨细胞生成。新的和现有的抑制剂 将用于进一步验证KDM 4 B作为PD治疗靶点，以及计算化学对接 将采用与物理化合物筛选配对的实验来关联化合物结构， 疾病进展标志物的变化。选择的KDM 4 B抑制剂将在体外和体内进行研究。 体内的功效和毒性。这项研究将提供一个更强大的理解表观遗传机制 在牙周病进展中起重要作用，验证KDM 4 B作为牙周炎药物靶点， 并导致具有局部免疫调节佐剂治疗潜力的化学探针的开发 治疗PD。该项目将打破牙周病学和药物设计领域之间的障碍， 将促进知识的发展转化口腔健康科学的一个急需的方面。

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.