Unlocking the cidal activity of echinocandins against Aspergillus fumigatus

解锁棘白菌素对烟曲霉的杀灭活性

• 批准号: 10378147
• 负责人: Jarrod R. Fortwendel
• 金额: $ 46.42万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10378147
• 关键词: Anabolism; Antifungal Agents; Aspergillosis; Aspergillus; Aspergillus fumigatus; Biological Assay; CRISPR/Cas technology; Cell Wall; Clinical; Combined Modality Therapy; Complex; Coupled; Cyclic AMP-Dependent Protein Kinases; Data; Development; Effectiveness; Engineering; Exhibits; Future; Generations; Genes; Genetic; Genome; Germination; Goals; Hyphae; Immune system; Immunocompromised Host; In Vitro; Incidence; Individual; Industrial fungicide; Infection; Injury; Intervention; Libraries; Mechanics; Mediator of activation protein; Modeling; Molds; Mutation; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphotransferases; Polyenes; Predisposition; Protein Kinase; Reporting; Reproduction spores; Research; Residual state; Resistance; Septate; Stress; Structure; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Tissues; Toxic effect; Treatment Efficacy; Triazoles; Work; biological adaptation to stress; breakthrough infection; deletion library; fungus; high-throughput drug screening; improved; in vivo; mortality; mouse model; mutant; novel; programs; response; therapeutically effective; transcription factor; transcriptomics; treatment effect

Invasive aspergillosis (IA), caused mainly by A. fumigatus, is the most prevalent invasive mold infection of immunocompromised individuals and is associated with mortality rates of 35-90%. Only three classes of anti- Aspergillus drugs exist. The global rise of resistance to the triazole class and the unacceptably high patient toxicity of the polyene class limits the use of two of these. The remaining class, the echinocandins, are generally considered effective therapeutic agents as their target is fungus-specific (cell wall biosynthesis) and they exhibit low toxicity. However, the echinocandins are not fungicidal for Aspergillus species and a paradoxical effect of treatment, characterized by decreased drug effectiveness with increasing drug concentrations, has been described both in vivo and in vitro. Likely due to these issues, high incidence breakthrough infections during echinocandin therapy have been reported. Therefore, echinocandin use for IA is also limited. The discovery of mechanisms essential for echinocandin stress adaptation and survival is expected to improve therapeutic efficacy with these important compounds by identifying fungal targets for future combination therapies. To delineate novel mechanisms orchestrating echinocandin stress responses, we recently completed the generation of a protein kinase disruption mutant library in a wild type genetic background. This library was constructed using CRISPR/Cas9 gene-editing to disrupt each of the 142 putative protein kinases encoded by the A. fumigatus genome. A total of 118 non-essential, unique gene disruptions were achieved and subsequently employed for in vitro echinocandin susceptibility assays. Our preliminary assays identified a total of 12 protein kinase disruption mutants displaying 4- to >32-fold decreased minimum effective concentrations (MEC) compared to the parental strain. We have discovered that two of these mutations, residing in the previously uncharacterized SepL and SidB kinases, impart fungicidal anti-Aspergillus activity to multiple echinocandins. These protein kinases are predicted to be core components of the Septation Initiation Network (SIN), a three- kinase cascade that is necessary for septation in fungi. Our exciting preliminary data show that blocking septation via loss of any single SIN kinase causes widespread hyphal damage and loss of viability in response to echinocandin treatment. In addition, employing the sepL disruption mutant, we have found that echinocandin therapy improves survival and eliminates residual tissue burden in a mouse model of IA. As septa are important for the limitation of mechanical injury to hyphae, and the A. fumigatus SIN is completely uncharacterized, exploration of this network is expected to reveal novel effectors of echinocandin stress survival. Our aims are to identify core SIN components required for unlocking echinocandin cidal activity (Aim 1), define temporal requirements for enhancement of echinocandin activity resulting from septation blockade (Aim 2), and to delineate the SIN pathway-dependent septum construction machinery (Aim 3). Our work will identify the SIN components with the highest potential for benefit when targeted in combination with echinocandin therapy.

侵袭性曲霉病（IA）主要由A.烟曲霉，是最普遍的侵入性霉菌感染， 免疫功能低下的个体，并与35- 90%的死亡率相关。只有三类反- 曲霉菌药物存在。全球对三唑类药物耐药性的上升和患者对三唑类药物的高耐药率 多烯类的毒性限制了其中两种的使用。剩下的一类，棘白菌素， 被认为是有效的治疗剂，因为它们的靶标是真菌特异性的（细胞壁生物合成），并且它们表现出 低毒性。然而，棘白菌素对曲霉属物种不具有杀真菌作用，并且棘白菌素对曲霉属物种具有矛盾的作用。 治疗的特点是随着药物浓度的增加，药物有效性降低， 在体内和体外都有描述。很可能由于这些问题， 棘白菌素疗法已有报道。因此，棘白菌素用于IA也是有限的。的发现 棘白菌素应激适应和存活所必需的机制有望改善治疗效果。 通过鉴定用于未来联合治疗的真菌靶点，研究这些重要化合物的有效性。到 描述了棘白菌素应激反应的新机制，我们最近完成了 在野生型遗传背景中产生蛋白激酶破坏突变体库。该文库 使用CRISPR/Cas9基因编辑构建，以破坏由CRISPR/Cas9编码的142种推定蛋白激酶中的每一种。 助理烟曲霉基因组总共实现了118个非必需的独特基因破坏， 用于体外棘白菌素敏感性测定。我们的初步分析共鉴定了12种蛋白质 显示最低有效浓度（MEC）降低4至>32倍的激酶破坏突变体 与亲本菌株相比。我们发现，其中两个突变，居住在以前的 未表征的SepL和SidB激酶赋予多种棘白菌素杀真菌抗曲霉活性。 这些蛋白激酶被预测为分隔起始网络（SIN）的核心组成部分， 真菌中分隔所必需激酶级联。我们令人兴奋的初步数据表明， 通过任何单一SIN激酶的损失，引起广泛的菌丝损伤和活力损失， 棘白菌素治疗。此外，利用sepL破坏突变体，我们发现棘白菌素 在IA的小鼠模型中，治疗提高了存活率并消除了残余组织负荷。因为隔膜很重要 限制菌丝的机械损伤;烟曲霉SIN是完全没有特征的， 对该网络的探索有望揭示棘白菌素应激存活的新效应物。我们的目标是 鉴定解锁棘白菌素杀灭活性所需的核心SIN组分（目标1）， 增强分隔阻断引起的棘白菌素活性的要求（目的2），以及 描述SIN通路依赖的隔膜施工机械（目标3）。我们的工作将确定SIN 当靶向与棘白菌素治疗组合时，具有最高获益潜力的组分。