Small molecular weight targeted killing agents against a periodontal pathogen

描述（由申请人提供）：牙周病提供了一个机会，以了解从免疫稳态导致慢性炎症的过程。从生物膜聚生体中消除所选微生物的能力将提供一种重要工具，用于确定推定病原体在导致慢性炎症的事件进展中的作用以及细菌减轻这种破坏性免疫反应的潜力。更广泛地说，肠道微生物群落在维持许多粘膜系统的健康方面起着不可或缺的作用，并且需要选择性杀灭剂来有效治疗这些非无菌界面处的粘膜感染。我们成功地针对生物膜的牙周病原菌放线菌伴聚合菌（Aa）与光敏剂，并获得光激活的杀伤。我们使用单克隆抗体（mAb）作为靶向部分。单克隆抗体大小的生物分子进入生物膜的运输受到显著阻碍。目前没有针对Aa的小分子量靶向杀伤剂，并且没有产生针对任何给定细菌病原体的小分子量选择性靶向部分的通用方法。我们的目标是填补这两个空白。小分子量试剂不仅具有快速转运到牙周袋和Aa生物膜中的优点，而且不太可能是抗原性的，并且更适合于常规合成和应用。我们将采用两种方法生产针对Aa的靶向杀伤剂：1）噬菌体展示（具体目标1）。我们将使用已建立的方案来筛选对三种靶点具有高亲和力的肽：Aa生物膜、Aa LPS和Aa外膜蛋白。我们将构建与光敏剂（PS）缀合的高亲和力肽的同源和异源二聚体对。2)抗体CDR二聚体（具体目的2）。使用抗Aa mAb的集合作为起点，我们将产生由互补决定区（CDR）组成的小分子量靶向肽。对Aa生物膜表现出高亲和力的CDR将通过亲水性间隔物偶联并缀合至PS。我们的具体目标的成功完成将产生一个工具，可用于探测在动物和人类研究中的牙周疾病进展的Aa的作用，并提供了一种方法，用于生产小分子量的针对各种牙周病原体的靶向杀伤剂。

DESCRIPTION (provided by applicant): Periodontal disease provides an opportunity to understand the processes that lead from immune homeostasis to chronic inflammation. The ability to eliminate a selected microbe from a biofilm consortium would provide an essential tool for determining the role of putative pathogens in the progression of events that lead to chronic inflammation and the potential of commensals to mitigate this destructive immune response. More broadly, commensal microbial communities play an integral role in maintaining the health of a number of mucosal systems and selective killing agents will be needed for effective treatment of mucosal infections at these non- sterile interfaces. We successfully targeted biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) with a photosensitizer and obtained light activated killing. We used a monoclonal antibody (mAb) as the targeting moiety. Transport of biomolecules the size of mAb into biofilms is significantly hindered. There are currently no small molecular weight targeted killing agents for Aa, and there are no general methods for producing small molecular weight selective targeting moieties against any given bacterial pathogen. Our objective is to fill both these gaps. Small molecular weight agents will not only have the advantage of rapid transport into periodontal pockets and Aa biofilm, but are less likely to be antigenic and will be more appropriate for routine synthesis and application. We will pursue two approaches for producing targeted killing agents against Aa: 1) Phage display (Specific aim 1). We will use established protocols for screening peptides with high affinity for three targets: Aa biofilm, Aa LPS and an Aa outer membrane protein. We will construct homo and hetero dimer pairs of high affinity peptides conjugated to a photosensitizer (PS). 2) Antibody CDR dimers (Specific aim 2). Using a collection of anti-Aa mAb as a starting point we will produce small molecular weight targeting peptides consisting of complementarity determining regions (CDRs). CDRs exhibiting high affinity to Aa biofilm will be coupled via a hydrophilic spacer and conjugated to a PS. Successful completion of our specific aims will produce a tool that can be used to probe the role of Aa in periodontal disease progression in both animal and human studies and provide an approach for producing small molecular weight targeted killing agents against a variety of periopathogens.