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; 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 肠道的细菌感染全世界数百万个人。目前用抗生素治疗 高效，抗生素耐药性的患病率增加使这些感染更加困难 对待。此外，抗生素会损害个人促进健康的共生细菌 容易受到艰难梭菌感染的影响，在20％的病例中可能会复发。因此需要新药 细菌通常在感染过程中表达毒素， 通过损害宿主上皮细胞，在病毒中起关键作用。为了支持其重要性，病原体 当毒素基因被删除时，病毒被完全减弱或消除。这些毒素通过 多种机制，但一个大而重要的家族是糖基转移酶毒素，这会导致 通过将糖（通常是葡萄糖或核糖）连接到宿主蛋白上的关键位置来通过将糖（通常是葡萄糖或核糖）连接起来。一个 有希望的策略，与抗生素协同作用是中和毒素，因为这将停止的进展 疾病并避免对共生微生物的脱靶影响。不幸的是，毒素中和药不存在 对于许多细菌病原体。对于确实存在的抗毒素疗法，可以禁止，昂贵或 靶标的毒素可变区域。在这项工作中，我们建议开发中和高度中和的肽 细菌毒素的保守酶活性。为此，我们将利用贝克酵母的观察（S。 酿酒酵母容易受到这些毒素的影响。因为S. cerevisiae非常容易设计，因此可能 筛选大量肽库并确定挽救酵母菌生长的潜在毒素抑制剂。实际上，我们有 进行了试点筛查，并且已经确定了艰难梭菌TCDB的铅肽抑制剂。我们将首先 扩展此屏幕以鉴定5种其他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，有希望的铅的抑制机制将 可以使用体外评估与质谱法相结合。最后，这些线索将在 益生菌酵母的基因组，可以在疾病部位对这些药物进行连续的生物制造。益生菌 酵母也将经过设计以在其细胞表面显示毒素结合剂，从而隔离其他毒素 并防止毒素与人类细胞接触。 Petides和酵母交付向量的效率将是 在动物模型中进行了评估。综上所述，这项工作开发了一个可概括的发现平台， 抗毒素理论的表征和传递，有可能延长现有的可用性 抗菌药物。