TLR/IL-1R signaling intermediaries and a target-specific therapeutic for arthriti

描述(申请人提供)：本研究的目的是了解Toll样受体(TLR)/白介素1受体(IL-1R)信号通路中蛋白激酶D1(PKD1)的确切功能，并通过干扰TLR/IL-1R信号转导，开发一种有效和安全的方法来抑制关节炎炎症的恶性循环。 使用纳米体基的局部递送抑制性多肽。类风湿关节炎(RA)是一种自身免疫性疾病，表现为慢性炎症、关节肿胀、自身反应性T淋巴细胞发育以及关节软骨和骨骼的破坏。虽然引发和维持炎症的因素还不是很清楚，但人们认为RA可能部分是通过TLRs发出信号而启动的，TLRs有助于发展一种自我持续反应，导致慢性炎症和致病的自身反应性Th1/Th17细胞的发展。因此，破坏大多数TLRs使用的信号通路可能提供一个机会，在不可逆转的关节损伤发生之前，在发育的最早阶段停止RA过程。初步研究表明，全身性抑制PKD1的激活实质上可以改善小鼠模型的关节炎。此外，PKD1在RA滑膜细胞中被结构性激活，并负责这些细胞表达促炎介质。这些发现支持我们的假设，即PKD1是关节促炎反应发展的关键因素，并可能成为治疗干预的有效分子靶点。我们已经证明，在依赖于MyD88的TLR/IL-1R配体的刺激下，PKD1(一种丝氨酸/苏氨酸激酶)被招募到同源受体复合体中并在其中激活。在TLR/IL-1R信号通路中，PKD1的激活位于IRAK4和IRAK1的下游，而TRAF6泛素化的上游。然而，允许组装受体复合体和在TLR/IL-1R信号转导中发挥作用的PKD1的特定结构域尚未确定。我们的目标是确定PKD1激活的分子机制和在TLR/IL-1R信号转导中的作用，识别PKD1的特定结构域，允许组装IRAK4/IRAK1/PKD1/TRAF6复合体，用于开发一种新的方法，以TLR/IL-1R途径特异性的方式抑制PKD1的激活，并利用创新的靶向特异性药物传递系统选择性地将治疗剂(阻断TLR/IL-1R信号转导)输送到炎症关节。具体地说，我们将1)确定PKD1在TLR/IL-1R信号转导中的激活和作用机制；2)开发能够向炎症关节输送治疗剂量的基于多肽的PKD1激活抑制物的纳米体；以及3)检验以下假设： 一种针对炎性巨噬细胞/单核细胞的PKD1激活的特异性抑制剂将有效地改变动物模型中的炎症环境，预防关节炎。这一结果可以转化为一种创新的治疗方法，以比目前的方案更少的副作用有效地控制类风湿关节炎的进展。

DESCRIPTION (provided by applicant): The objectives of this study are to understand the precise function of protein kinase D1 (PKD1) in the Toll- like receptor (TLR)/interleukin-1 receptor (IL-1R) signaling pathway and to develop an effective and safe way to suppress the vicious cycle of inflammation in arthritic joints by interfering with TLR/IL-1R signal transduction using nanosome-based local delivery of inhibitory peptides. Rheumatoid arthritis (RA) is an autoimmune disease manifested by chronic inflammation, swollen joints, development of auto-reactive T-lymphocytes, and destruction of cartilage and bone in joints. While the factors that initiate and perpetuate inflammation are not well understood, it is thought that RA may be initiated in part by signaling through TLRs that contribute to the development of a self-perpetuating reaction leading to chronic inflammation and development of pathogenic auto-reactive Th1/Th17 cells. Therefore, disruption of the signaling pathway used by most TLRs may provide an opportunity to halt the RA process in the earliest stages of development before irreversible joint damage occurs. Preliminary studies show that systemic suppression of PKD1 activation substantially ameliorates arthritis in murine models. Also, that PKD1 is constitutively activated in RA synoviocytes and is responsible for expression of proinflammatory mediators by those cells. These findings support our hypothesis that PKD1 is a critical factor for development of proinflammatory reactions in the joint and might be an effective molecular target for therapeutic intervention. We have shown that following stimulation with MyD88-dependent TLR/IL-1R ligands, PKD1 (a serine/threonine kinase) is recruited to and activated within the cognate receptor complex. Activation of PKD1 in TLR/IL-1R signaling is down-stream of IRAK4 and IRAK1, but up-stream of TRAF6 ubiquitination. However, the specific domains of PKD1 that allow assembly of the receptor complex and the function of PKD1 in TLR/IL-1R signal transduction have not been established. Our goals are to define the molecular mechanism of PKD1 activation and action in TLR/IL-1R signaling, to identify the specific domains of PKD1 that allow for assembly of the IRAK4/IRAK1/PKD1/TRAF6 complex that can be applied to the development of a novel approach to inhibit PKD1 activation in a TLR/IL-1R pathway-specific manner, and to selectively deliver a therapeutic agent (that blocks TLR/IL-1R signal transduction) to the inflamed arthritic joints using an innovative target-specific drug delivery system. Specifically we will 1) determine the activation and action mechanisms of PKD1 in TLR/IL-1R signaling; 2) develop nanosomes that can deliver a therapeutic dose of peptide-based inhibitors of PKD1 activation to inflamed joints; and 3) test the hypothesis that delivery of a specific inhibitor of PKD1 activation targeted to inflammatory macrophages/monocytes will effectively alter the inflammatory milieu and prevent arthritis in an animal model. The results could be translated into an innovative therapeutic approach to effectively control RA progression with fewer side effects than current regimens.