Overcoming Emerging Aspergillus fumigatus Azole Resistance Via Protease Inhibition

项目摘要。由丝状真菌烟曲霉及其相关种属引起的感染 尽管有现代抗真菌药物治疗，但仍与显著的发病率和死亡率相关。许多 导致不良治疗结果的因素包括真菌在感染部位的生理状态， 感染和三唑类耐药菌株的全球出现。一个主要的监管机制， 真菌的疾病进展和抗三唑类药物活性是蛋白水解激活转录 调节子SrbA。SrbA在体内的激活是真菌毒力和内源性三唑类药物的绝对必要条件 抗性，作为SrbA调节因子的无效突变体，例如真菌特异性激活丝氨酸蛋白酶 RbdB和E3泛素连接酶（DSC）在侵袭性曲霉病（IA）的动物模型中是无毒的， 三唑敏感性显著增加。响应RFA-AI-17-036的本提案旨在 鉴定抑制SrbA活化的小分子并将其开发成高级治疗候选物 对三唑耐药丝状真菌具有广谱活性。SrbA的有效抑制剂- 依赖性信号传导通路将被开发用于临床，作为与药物组合的连续治疗。 用于治疗IA的三唑类抗真菌剂。预期这种连续疗法将提供几种 与三唑单一疗法相比的优势，包括在缺氧条件下的生长抑制和增加的 三唑类药物在药物敏感性和耐药性感染中的抗真菌活性。作为SrbA 途径在大多数人类真菌病原体中是保守的，其中一些是固有的唑类耐药，我们 预期除了由A.烟熏。我们的方法利用了 已充分表征的蛋白酶和连接酶抑制剂化学文库的可用性， 在许多疾病的设置，与Microbiotix公司的专业知识，和达特茅斯的克拉默实验室。的 本申请的R21阶段将利用基于细胞的高通量筛选确定的靶向小分子 文库，以鉴定和确认SrbA调节蛋白酶和/或连接酶抑制剂，并验证其抗真菌性 活性、途径特异性和早期命中和先导物的哺乳动物毒性。在R33阶段，经验证的命中率 将进行化学优化，验证，定义，确定作用机制，最后 在已建立的鼠模型中进行体内药理学和毒理学分析以及抗真菌功效 侵袭性曲霉病

PROJECT SUMMARY. Infections caused by the filamentous fungus Aspergillus fumigatus and related species are associated with significant morbidity and mortality despite contemporary antifungal drug therapies. Many factors contribute to poor treatment outcomes including the physiological state of the fungus at the site of infection and the global emergence of triazole drug resistant strains. One major regulatory mechanism used by the fungus to progress disease and resist triazole drug activity is proteolytic activation of the transcriptional regulator, SrbA. Activation of SrbA in vivo is absolutely required for fungal virulence and intrinsic triazole drug resistance, as null mutants of SrbA regulatory factors such as the fungal specific activating serine protease RbdB and E3 ubiquitin ligases (DSCs) are avirulent in animal models of invasive aspergillosis (IA) and have significant increases in triazole susceptibility. The objective of this proposal in response to RFA-AI-17-036 is to identify small molecules that inhibit SrbA activation and develop them into advanced therapeutic candidates with broad-spectrum activity against triazole resistant filamentous fungi. Potent inhibitors of the SrbA- dependent signaling pathway will be developed for clinical use as an adjunctive therapy in combination with a triazole antifungal agent that is used to treat IA. The adjunctive therapy is expected to provide several advantages over triazole monotherapy, including growth inhibition in hypoxic conditions and increased antifungal activity of the triazole drug in both drug susceptible and drug resistant infections. As the SrbA pathway is conserved among most human fungal pathogens, some of which are inherently azole resistant, we anticipate broad spectrum clinical utility beyond infections caused by A. fumigatus. Our approach leverages the availability of well characterized protease and ligase inhibitor chemical libraries, both known druggable targets in many disease settings, with the expertise of Microbiotix Inc. and the Cramer Laboratory at Dartmouth. The R21 phase of this application will utilize high-throughput cell based screens of defined targeted small molecule libraries to identify and confirm SrbA regulatory protease and/or ligase inhibitors and validate their antifungal activity, pathway specificity, and mammalian toxicity of early hits and leads. In the R33 phase, validated hits will be chemically optimized, validated, defined pharmacologically, determine mechanism of action, and finally proceed to in vivo pharmacologic and toxicology profiling and antifungal efficacy in established murine models of invasive aspergillosis.