Development of Imaging Probes for Risk Assessment of Alzheimer's Disease using Phage Display

项目摘要 家长资助金摘要： 蛋白水解酶是人类基因组中最大、最具特性的酶家族之一。 此外，还有许多人类健康状况，如癌症，与 蛋白水解酶活性和功能。因此，允许单个蛋白酶活性的特定分子探针 在体内疾病发展的过程中被成像将在我们对 蛋白质降解事件，有助于疾病病理，同时也为早期疾病提供了直接的方法 监测和对治疗的反应。在过去的十年里，已经产生了许多不同类别的分子探测器 可用于成像应用。也许这些试剂中最有效的一种是活性- 基于探针(ABP)。然而，ABPS的广泛应用通常受到以下需要的限制： 使用合成化学来优化探针，并且探针通常对给定的靶标酶缺乏绝对的特异性。 该提案将专注于建立一种创新技术，使快速设计的ABP具有 对任何给定的关注目标的蛋白酶具有特殊的专一性。这将涉及到应用噬菌体展示 一种筛选与蛋白水解酶反应相连的化学受限双环肽的不同文库的方法 电泳法反复筛选高效和选择性的共价结合元件。我们建议 利用两种基质细胞衍生的蛋白酶靶标--组织蛋白酶建立和验证噬菌体筛选方法 S(CAT S)和成纤维细胞激活蛋白(FAP)参与了肿瘤发生的关键环节。这些新的探测器 然后将在癌症小鼠模型中进行成像应用验证。在这方面开发的技术 该方案的提出不仅为蛋白水解酶ABP的开发提供了一种新的通用方法，而且还将生产出 在肿瘤影像方面具有潜在临床应用前景的探头。 补充项目摘要： 阿尔茨海默病(AD)是一种以炎症和细胞为特征的进行性神经退行性疾病 导致认知功能丧失的脑内死亡。尽管一些遗传链接已经被 已建立的和许多与疾病病理有关的细胞事件已被建立，很少是 已知这种致衰性疾病的关键风险因素和非遗传诱因。最近的发现之一是 与阿尔茨海默病发病相关的主要蛋白质，b-淀粉样蛋白，可以起到抗菌肽的作用。 研究确定传染病病原体作为可能的致病原因的作用。为此，一些国家提出了 最近的努力已经确定了致病细菌牙龈卟啉单胞菌的存在 阿尔茨海默病患者死后脑的90%以上。这种病原体通常是在口腔中发现的。 参与慢性牙周炎(CP)的进展，但也可在 身体包括冠状动脉、胎盘和肝脏。牙龈假单胞菌产生和分泌几种蛋白酶 被称为牙龈痛的物质是细菌有效定植所必需的毒力因子。这些 已经在AD患者的大脑中发现了酶，它们的水平与疾病密切相关 标记物，如tau和泛素。在体外、体内和口服中也发现姜痛具有神经毒性。 感染牙龈假单胞菌的小鼠会导致大脑暴露，并增加淀粉样斑块的积累。 最近，一家名为Cortex yme的公司开发了一种赖氨酸特异性牙龈痛(KGP)的小分子抑制剂，并 表明它能有效地治疗小鼠脑内牙龈假单胞菌感染，导致对 海马神经元。这些结果导致了AD患者的I期试验(NCT03418688)，该试验显示 积极的保护价值。目前的第二/第三阶段试验(NCT03823404)正在进行中，结果预计在年中 到2021年底。由于这些最新的发现，目前对非侵入性成像的需求尚未得到满足 这些方法将使进一步研究大脑中牙龈痛活动与AD风险之间的联系以及 以确定哪些患者可能从牙龈痛抑制剂的治疗中受益。因此，在这个 作为补充，我们计划使用目前在家长拨款中开发的噬菌体展示技术来识别 针对牙周疼痛的高度选择性共价结合环肽。噬菌体的主要好处是 展示方法是所得到的环肽显示出高度特异的靶向结合，这是 有效的显像剂。选择性共价抑制剂可以转化为含有荧光的化合物 以及用于成像应用的PET放射性示踪剂。增刊的主要目的如下：目的1-- 筛选赖氨酸特异性(Kgp)和精氨酸特异性牙龈痛(RgpA和RgpA)的环肽噬菌体文库 RgpB)以鉴定选择性共价结合序列。目标2将序列从噬菌体筛选转换为 针对每个牙痛靶点的选择性共价结合环肽探针。目标3证明了荧光 探针可以在细胞和感染牙龈假单胞菌的小鼠模型中标记牙龈痛。在完成 在一年的补充中，我们将至少有一个可以标记18F的探针用于小鼠的PET成像， 进展到IND前研究，并最终进入人类临床研究。

Project Summary Parent Grant Summary: Proteases represent one of the largest and most well characterized families of enzymes in the human genome. Furthermore, there are many human health conditions such as cancer that are associated with alterations in protease activity and function. Therefore, specific molecular probes that allow individual protease activities to be imaged during disease progression in vivo would both be transformative in our understanding of the roles of proteolytic events that contribute to disease pathology while also providing a direct methods for early disease monitoring and response to therapy. The past decade has produced many diverse classes of molecular probes that can be used for imaging applications. Perhaps one of the most powerful of these reagents is the activity- based probe (ABP). However, the broad application of ABPs is typically limited by the need to painstakingly optimize probes using synthetic chemistry and often probes lack absolute specificity for a given target enzyme. This proposal will focus on establishing an innovative technology that will allow rapid design of ABPs with exceptional specificity for any given protease target of interest. This will involve application of a phage display method to screen diverse libraries of chemically constrained bi-cyclic peptides linked to a protease reactive electrophile to iteratively screen for covalent binding elements with high potency and selectivity. We propose to establish and validate the phage screening method using two stromal-cell derived protease targets, cathepsin S (cat S) and fibroblast activation protein (FAP), involved in key aspects of tumorigenesis. These new probes will then be validated for imaging applications in mouse models of cancer. The technology developed in this proposal will result in not only a new general method for protease ABP development but also will produce probes with potential future clinical applications in cancer imaging. Supplement Project Summary: Alzheimer’s Disease (AD) is a progressive neurodegenerative disease characterized by inflammation and cell death in the brain that leads to loss of cognitive function. Although a number of genetic links have been established and many cellular events associated with disease pathology have been established, very little is known about key risk factors and non-genetic triggers of this debilitating disease. Recent findings that one of the primary proteins linked to AD pathogenesis, b-amyloid, can function as an anti-microbial peptide motivated studies to identify a role for an infectious agent as a possible cause for the disease. To this end, a number of recent efforts have identified the presence of the pathogenic bacteria, Porphyromonas gingivalis, in greater than 90% of postmortem brains of patients with AD. This pathogen is typically found in the mouth where it is involved in the progression of chronic periodontitis (CP) but can also be found in other locations within the body including coronary arteries, placenta and liver. P. gingivalis produces and secretes several proteases called gingipains that act as virulence factors necessary for effective colonization by the bacteria. These enzymes have been identified in the brains of AD patients and their levels strongly correlate with disease markers such as tau and ubiquitin. Gingipains were also found to be neurotoxic in vitro and in vivo and oral infection of mice with P. gingivalis leads to brain exposure and increased accumulation of amyloid plaques. Recently, a company, Cortexyme, developed a small molecule inhibitor of lysine-specific gingipain (Kgp) and showed that it can effectively treat P. gingivalis infection in the brain in mice, leading to protection of hippocampal neurons. These results led to a phase I trial (NCT03418688) in patients with AD which showed positive protective value. A current phase II/III trial (NCT03823404) is underway with results expected in mid to late 2021. As a result of these recent findings, there is a current unmet need for non-invasive imaging methods that will enable further studies of the link between gingipain activity in the brain and AD risk as well as to identify patients who would likely benefit from treatment with gingipain inhibitors. Therefore, in this supplement, we plan use the phage display technology currently being developed in the parent grant to identify highly selective covalent binding cyclic peptides that target the gingipains. The major benefit of the phage display approach is that the resulting cyclic peptides show highly specific target binding necessary for an effective imaging agent. Selective covalent inhibitors can be converted to compounds containing a fluorescent and PET radiotracer for imaging applications. The primary aims for the supplement are as follows: Aim 1- screen cyclic peptide phage libraries against lysine-specific (Kgp) and arginine-specific gingipains (RgpA and RgpB) to identify selective covalent binding sequences. Aim 2 Convert sequences from phage screening into selective covalent binding cyclic peptide probes for each gingipain target. Aim 3 Demonstrate that fluorescent probes can label gingipains in cells and in a mouse model of P. gingivalis infection. At the completion of the one-year supplement, we will have at least one probe that can be labeled with 18F for PET imaging in mice, advancement into pre-IND studies and eventually clinical studies in humans.