Brewing anti-toxin drugs using probiotic yeast

利用益生菌酵母酿造抗毒素药物

• 批准号: 10687670
• 负责人: Nathan C. Crook
• 金额: $ 133.37万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10687670
• 关键词: Animal Model; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Attenuated; Bacteria; Bacterial Infections; Bacterial Toxins; Biomanufacturing; Cell surface; Cells; Clostridium difficile; Coupled; Disease; Disease Progression; Drug usage; Engineered Probiotics; Engineering; Epithelial Cells; Family; Gene Deletion; Genome; Glucose; Growth; Health Promotion; Human; Individual; Infection; Lead; Location; Mass Spectrum Analysis; Modeling; Peptide Library; Peptides; Pharmaceutical Preparations; Play; Predisposition; Prevalence; Probiotics; Proteins; Recurrence; Ribose; Saccharomyces cerevisiae; Site; Therapeutic; Toxin; Virulence; Work; Yeasts; antibiotic resistant infections; antitoxin; commensal bacteria; commensal microbes; delivery vehicle; glycosyltransferase; in vitro Assay; inhibitor; novel therapeutics; pathogen; pathogenic bacteria; prevent; sugar; synergism; usability

Abstract Bacterial infections of the gut afflict millions of individuals worldwide. While treatment with antibiotics is currently highly effective, the increasing prevalence of antibiotic resistance is making these infections more difficult to treat. Furthermore, antibiotics can damage an individual’s health-promoting commensal bacteria, making them susceptible to C. difficile infections, which can be recurrent in 20% of cases. New drugs are therefore needed which can synergize with or prolong the utility of antibiotics. Bacteria commonly express toxins during infection, which play key roles in virulence by damaging host epithelial cells. In support of their importance, pathogen virulence is attenuated or eliminated entirely when their toxin genes are deleted. These toxins act through a variety of mechanisms, but one large and important family are the glycosyltransferase toxins, which cause cytopathic effects by attaching sugars (commonly glucose or ribose) to key locations on host proteins. A promising strategy, synergistic with antibiotics, is to neutralize the toxins, as this would halt the progression of disease and avoid off-target effects on commensal microbes. Unfortunately, toxin-neutralizing drugs do not exist for many bacterial pathogens. For the anti-toxin therapies that do exist, they can be prohibitively expensive, or target mutable regions of the toxins. In this work, we propose to develop peptides that neutralize the highly conserved enzymatic activity of bacterial toxins. To do so, we will exploit the observation that baker’s yeast (S. cerevisiae) is susceptible to these toxins. Because S. cerevisiae is so easy to engineer, it is therefore possible to screen massive peptide libraries and identify potent toxin inhibitors that rescue yeast growth. In fact, we have performed a pilot screen and have already identified a lead peptide inhibitor of C. difficile TcdB. We will first expand this screen to identify peptide inhibitors of 5 additional bacterial toxins. The potency of these inhibitors will be investigated in cell-based models of toxin activity, and the inhibitory mechanism of promising leads will be identified using in vitro assays, coupled with mass spectrometry. Finally, these leads will be encoded in the genome of probiotic yeast, enabling continuous biomanufacturing of these drugs at the site of disease. Probiotic yeast will also be engineered to display toxin binders on its cell surface, thereby sequestering additional toxin and preventing toxin contact with human cells. The efficacy of the peptides and yeast delivery vectors will be evaluated in animal models. Taken together, this work develops a generalizable platform for discovery, characterization, and delivery of anti-toxin therapeutics that has the potential to prolong the usability of existing antibacterial drugs.

抽象的 肠道细菌感染困扰着全世界数百万人。虽然目前使用抗生素治疗 非常有效，抗生素耐药性的日益普遍使这些感染更加难以治愈 对待。此外，抗生素会损害个体促进健康的共生细菌，使它们 易受艰难梭菌感染，20% 的病例可能会复发。因此需要新药 它可以与抗生素协同作用或延长抗生素的效用。细菌在感染过程中通常会表达毒素， 通过破坏宿主上皮细胞在毒力中发挥关键作用。为了支持其重要性，病原体 当它们的毒素基因被删除时，毒力就会减弱或完全消除。这些毒素通过 机制多种多样，但糖基转移酶毒素是一个重要的大家族，它会导致 通过将糖（通常是葡萄糖或核糖）附着到宿主蛋白质的关键位置来产生细胞病变效应。一个 与抗生素协同作用的有前途的策略是中和毒素，因为这将阻止疾病的进展 疾病并避免对共生微生物的脱靶影响。不幸的是，毒素中和药物并不存在 对于许多细菌病原体。对于现有的抗毒素疗法来说，它们可能非常昂贵，或者 针对毒素的可变区域。在这项工作中，我们建议开发能够中和高度 细菌毒素的保守酶活性。为此，我们将利用面包酵母（S. cerevisiae）对这些毒素敏感。由于酿酒酵母非常容易设计，因此有可能 筛选大量肽库并鉴定可挽救酵母生长的有效毒素抑制剂。事实上，我们有 进行了初步筛选，并已鉴定出艰难梭菌 TcdB 的先导肽抑制剂。我们首先会 扩展此筛选以识别另外 5 种细菌毒素的肽抑制剂。这些抑制剂的效力 将在基于细胞的毒素活性模型中进行研究，有希望的先导化合物的抑制机制将 使用体外测定并结合质谱法进行鉴定。最后，这些线索将被编码在 益生菌酵母的基因组，能够在疾病部位连续生物制造这些药物。益生菌 酵母也将被改造为在其细胞表面展示毒素结合剂，从而隔离额外的毒素 并防止毒素与人体细胞接触。肽和酵母递送载体的功效将是 在动物模型中进行评估。总而言之，这项工作开发了一个通用的发现平台， 抗毒素疗法的表征和交付，有可能延长现有药物的可用性 抗菌药物。