Highly specific anti-toxin therapies for severe bacterial gut infections

 性状（申请方提供）：滋贺毒素1和2（Stx 1/2）是产滋贺毒素大肠杆菌的主要毒力因子。大肠杆菌（STEC）是困扰发达国家和发展中国家的主要食品和水媒病原体。据估计，美国每年发生265，000例STEC感染。除了严重和经常出血的腹泻，危及生命的并发症溶血性尿毒综合征（HUS）发生在2-7%的受害者（主要是儿童）。除支持性治疗外，没有STEC疫苗或治疗方法可用。不使用抗生素，因为它们会增加毒素的产生和HUS的风险。STEC感染的严重性，爆发的倾向和缺乏有效的治疗使STEC成为潜在的生物恐怖制剂和公共卫生优先事项。 在这里，我们描述了一种创新的策略，发现D-肽抑制剂的Stx 1/2，以打击STEC感染。D-肽是天然L-肽的镜像，不能被蛋白酶消化，因此具有长的体内半衰期和低免疫原性的潜力。与小分子相比，它们可以很容易地以高效力和特异性破坏蛋白质界面，并且生产成本比抗体低得多。D-肽是肠道和/或体循环中Stx 1/2中和的理想候选物。 Navigen的药物发现平台采用对映体筛选技术（镜像噬菌体展示）与蛋白质设计相结合。我们已经通过鉴定HIV、RSV和埃博拉病毒的D肽抑制剂成功验证了这一平台技术。我们的抗HIV D-肽是第一个被发现的有效和特异性D-肽抑制剂，并处于先进的临床前试验中。它结合HIV三聚体表面糖蛋白gp 41上功能关键和保守的疏水“口袋”。该D肽的三聚体形式结合所有三个gp 41口袋，提供强的亲合力增强。 Stx 1/2各自由单个酶活性A亚基和形成五聚体环的五个受体结合B亚基组成。每个B五聚体亚基都含有一个类似于我们的病毒靶标的易受攻击的口袋。Stx 1/2 B亚基口袋是我们接下来将我们的专业知识应用于D-肽药物设计的极好靶点，因为1）我们成功地将类似口袋靶向功能关键界面，2）五聚体Stx 1/2靶点，其激发了具有强亲合力的五聚体D-肽的设计，和3）D-肽在肠道中稳定性和活性而不干扰天然植物群的可能性。此外，来自志贺氏菌的Stx 1和Stx的B亚基是相同的，使得抗Stx 1 B D-肽具有双重治疗用途。这些益处引起了GI传染病临床医生对该项目的强烈热情，他们了解抗Stx 1/2 D肽在临床上的巨大潜在影响。 在这项为期两年的资助中，我们建议发现，结构表征和优化五聚体D肽抑制剂，以高效中和Stx 1/2。该项目的成功将推出一类新的抑制剂，用于对抗对全球健康至关重要的病原菌。

 DESCRIPTION (provided by applicant): Shiga toxins 1 and 2 (Stx1/2) are the primary virulence factors of Shiga toxin-producing E. coli (STEC), which are major food-, and waterborne pathogens afflicting both developed and developing countries. An estimated 265,000 STEC infections occur annually in the US. In addition to severe and often bloody diarrhea, the life-threatening complication hemolytic uremic syndrome (HUS) occurs in 2-7% of victims (primarily children). No STEC vaccines or therapies beyond supportive care are available. Antibiotics are not used since they can increase toxin production and risk of HUS. The severity of STEC infection, propensity for outbreaks, and lack of effective treatments make STEC concerning potential bioterror agents and a public health priority. Here, we describe an innovative strategy to discover D-peptide inhibitors of Stx1/2 to combat STEC infection. D-peptides, the mirror images of natural L-peptides, cannot be digested by proteases and, therefore, have the potential for long in vivo half-lives and low immunogenicity. They can readily disrupt protein interfaces with high potency and specificity compared to small molecules and are much less expensive to produce than antibodies. D-peptides are ideal candidates for Stx1/2 neutralization in the gut and/or systemic circulation. Navigen's drug discovery platform employs an enantiomeric screening technology (mirror-image phage display) coupled with protein design. We have successfully validated this platform technology by identifying D- peptide inhibitors of HIV, RSV, and Ebola. Our anti-HIV D-peptide was the first potent and specific D-peptide inhibitor to be discovered and is in advanced preclinical trials. It binds a functionally critical and conserved hydrophobic "pocket" on HIV's trimeric surface glycoprotein, gp41. A trimeric version of this D-peptide binds to all three gp41 pockets, providing a strong avidity boost. Stx1/2 is each composed of a single enzymatically active A subunit and five receptor-binding B subunits that form a pentameric ring. Each of the B pentamer subunits contains a vulnerable pocket analogous to those of our viral targets. The Stx1/2 B subunit pockets are excellent targets for us to next apply our expertise in D- peptide drug design given 1) our success in targeting analogous pockets at functionally critical interfaces, 2) the pentameric Stx1/2 target, which inspires design of pentameric D-peptides with strong avidity, and 3) the likelihood of D-peptide stability and activity in the gut without disturbing native flora. Furthermore, the B subunits of Stx1 and Stx from Shigella dysenteriae are identical, enabling dual therapeutic use for an anti- Stx1B D-peptide. These benefits have generated strong enthusiasm for this project from GI infectious disease clinicians, who understand the dramatic potential impact an anti-Stx1/2 D-peptide would have in the clinic. In this two-year grant, we propose to discover, structurally characterize, and optimize pentameric D-peptide inhibitors that will neutralize Stx1/2 with high potency. Success in this project will launch a new class of inhibitors against pathogenic bacteria important to global health.