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.

摘要 肠道细菌感染困扰着全球数百万人。虽然目前使用抗生素治疗 高效，抗生素耐药性的日益流行使这些感染更难 请客。此外，抗生素会损害个人的健康促进共生细菌，使它们 容易感染艰难梭菌，在20%的病例中可能会复发。因此，需要新的药物 它可以与抗生素协同或延长抗生素的使用时间。细菌通常在感染期间表达毒素， 它们通过破坏宿主上皮细胞在毒力中发挥关键作用。为了支持它们的重要性，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。这些毒素通过一种 机制多种多样，但有一个重要的大家族是糖基转移酶毒素，它能引起 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而产生的细胞病变效应。一个 有希望的策略，与抗生素的协同作用，是中和毒素，因为这将阻止 并避免对共生微生物产生偏离目标的影响。不幸的是，毒素中和药物并不存在。 对许多细菌病原体来说。对于确实存在的抗毒素疗法来说，它们可能昂贵得令人望而却步，或者 锁定毒素的可变区。在这项工作中，我们建议开发能够中和高度 保存细菌毒素的酶活性。为此，我们将利用观察到的面包师酵母(S. 酿酒酵母)对这些毒素敏感。因为酿酒酵母很容易改造，所以这是可能的。 筛选大量的多肽文库，并确定有效的毒素抑制剂，以挽救酵母的生长。事实上，我们有 进行了中试筛选，并已经确定了艰难梭菌TcdB的先导肽抑制剂。我们将首先 扩展此屏幕以识别其他5种细菌毒素的多肽抑制物。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，有希望的线索的抑制机制将 使用体外分析结合质谱仪进行鉴定。最后，这些线索将被编码到 益生菌酵母的基因组，使这些药物能够在发病部位连续生物制造。益生菌 酵母还将被改造成在其细胞表面显示毒素结合剂，从而隔离额外的毒素 并防止毒素与人体细胞接触。多肽和酵母递送载体的功效将是 在动物模型中进行评估。综上所述，这项工作开发了一个可推广的发现平台， 表征和交付抗毒素疗法，有可能延长现有的可用性 抗菌药物。