Development of non-antibiotic therapeutics for Clostridium difficile infection (CDI)

艰难梭菌感染（CDI）非抗生素疗法的开发

• 批准号: 9464655
• 负责人: Matthew Bogyo
• 金额: $ 39.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9464655
• 关键词: Active Sites; American; Anaerobic Bacteria; Animal Model; Antibiotic Resistance; Antibiotics; Antibodies; Antitoxins; Bacteria; Bacterial Toxins; Binding; Biochemical; Biological Assay; Caspase; Cells; Chemicals; Clinical; Clinical Data; Clinical Research; Clostridium difficile; Colon; Coupled; Data; Development; Diarrhea; Disease; Epithelial Cells; Eukaryota; Event; Family; Fluorescence Polarization; Formulation; Goals; Hamsters; Health; Healthcare Systems; Human; Incidence; Infection; Inflammation; Injection of therapeutic agent; Intestines; Lead; Length; Mediating; Mediator of activation protein; Metronidazole; Modeling; Mus; Pathology; Peptide Hydrolases; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Phase; Phytic Acid; Property; Prophylactic treatment; Protease Domain; Proteins; Recurrence; Reporting; Risk; Selenium; Severity of illness; Signaling Molecule; Solid; Solubility; Structure; Symptoms; Targeted Toxins; Tertiary Protein Structure; Testing; Therapeutic; Tissues; Toxic effect; Toxin; Validation; Virulence Factors; Virulent; Work; analog; bacterial resistance; base; clinical development; cost; cross reactivity; design; ebselen; high throughput screening; improved; in vivo; infectious disease treatment; inhibitor/antagonist; microbial; mouse model; nanomolar; next generation; novel; novel therapeutic intervention; novel therapeutics; pathogen; pressure; prevent; response; small molecule; small molecule inhibitor; treatment strategy; uptake

Project Summary C. difficile infection (CDI) is triggered by use of broad-spectrum antibiotics which disrupts the natural microbial flora of the gut and allows the Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of the disease requires new strategies for optimal treatment. Furthermore, decreased response rates to metronidazole, high recurrence rates with the use multiple antiboitics, and emergence of multiple antibiotic resistant bacteria demonstrate the urgent need to develop new therapies. A paradigm shift in the general treatment of infectious diseases, including CDI, is necessary to prevent an increase in antibiotic resistance and alternatives to antibiotics should be considered.  The goal of this project is to develop a novel non-antibiotic therapy against C. difficile based on the inhibition of the major bacterial virulence factors that mediate the disease. These toxins are post-translationally activated inside intestinal epithelial cells via allosteric activation of their cysteine protease domain (CPD) by the eukaryote-specific small molecule inositol-hexakisphosphate (InsP6). We have developed an activity-based probe that can report on toxin CPD activation. Using this probe, we conducted a high-throughput fluorescence polarization screen to identify novel small-molecule inhibitors of the toxins. The screen revealed a significant number of novel lead compounds as well as an existing phase II clinical drug with nanomolar activity against the toxin. We have used the clinical drug to demonstrate the therapeutic value of blocking toxin function, using both toxigenic and infection models in mice. These studies have confirmed that inhibition of the CPD blocks pathogen-mediated toxicity in host tissues. However, its poor pharmacological properties coupled with its broad target selectivity makes it less than ideal for clinical development for CDI. Therefore, this project will focus in the first 3 years of the project to identify viable lead compounds with improved potency, selectivity, solubility and cellular uptake. We will focus on the clinical lead compound as well as several novel chemical entities (NCEs) identified in our HTS efforts. We will identify three chemically distinct lead molecules and then perform validation in mouse and hamster models of CDI. We will advance one of these molecules (keeping the others as backups) into formulation studies, with the end goal being to identify a single lead molecule and formulation strategy that can move into IND enabling studies after completion of this project.

项目概要 艰难梭菌感染 (CDI) 是由使用破坏天然微生物的广谱抗生素引发的 肠道菌群并允许革兰氏阳性厌氧病原体繁殖。发病率增加和 该疾病的严重程度需要新的最佳治疗策略。此外，响应率下降 甲硝唑、使用多种抗生素的高复发率以及多种抗生素的出现 耐药细菌表明迫切需要开发新疗法。总体范式转变 治疗传染病（包括 CDI）对于防止抗生素耐药性增加和 应考虑抗生素的替代品。  该项目的目标是开发一种新型非抗生素 基于抑制介导艰难梭菌的主要细菌毒力因子的治疗 疾病。这些毒素通过变构激活在肠上皮细胞内进行翻译后激活 通过真核生物特异性小分子肌醇六磷酸对其半胱氨酸蛋白酶结构域 (CPD) 进行改造 （InsP6）。我们开发了一种基于活动的探针，可以报告毒素 CPD 激活情况。使用这个探头， 我们进行了高通量荧光偏振筛选来鉴定新型小分子抑制剂 毒素。筛选显示了大量新型先导化合物以及现有的 II 期化合物 具有纳摩尔抗毒素活性的临床药物。我们已经用临床药物证明了 使用小鼠产毒和感染模型阻断毒素功能的治疗价值。这些研究 已经证实，抑制 CPD 可以阻断宿主组织中病原体介导的毒性。然而其贫 药理学特性加上其广泛的靶点选择性使其不太适合临床 CDI 的发展。因此，该项目将在项目的前 3 年重点寻找可行的线索 化合物具有改进的效力、选择性、溶解度和细胞摄取。我们将专注于临床领先 化合物以及我们在 HTS 工作中发现的几种新型化学实体 (NCE)。我们将确定三个 化学上不同的先导分子，然后在 CDI 的小鼠和仓鼠模型中进行验证。我们将 将其中一种分子（保留其他分子作为备份）推进配方研究，最终目标 确定可进入 IND 的单一先导分子和配方策略，以便在之后进行研究 该项目的完成。

项目成果

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

• DOI: 10.1371/journal.ppat.1008852
• 发表时间: 2020-09
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Bilverstone TW;Garland M;Cave RJ;Kelly ML;Tholen M;Bouley DM;Kaye P;Minton NP;Bogyo M;Kuehne SA;Melnyk RA
• 通讯作者: Melnyk RA

Pre-Trained Deep Convolutional Neural Network for Clostridioides Difficile Bacteria Cytotoxicity Classification Based on Fluorescence Images.

• DOI: 10.3390/s20236713
• 发表时间: 2020-11-24
• 期刊: Sensors (Basel, Switzerland)
• 作者: Brodzicki A;Jaworek-Korjakowska J;Kleczek P;Garland M;Bogyo M
• 通讯作者: Bogyo M