Overcoming Emerging Aspergillus fumigatus Azole Resistance Via Protease Inhibition

项目总结。由丝状真菌烟曲霉菌及相关物种引起的感染 尽管有现代的抗真菌药物治疗，但仍与显著的发病率和死亡率有关。许多 导致治疗效果不佳的因素包括真菌在感染部位的生理状态 感染和全球出现的三氮唑耐药菌株。使用的一种主要监管机制 真菌对疾病的进展和对三氮唑类药物的抗药性是蛋白水解酶的转录激活 监管者，sba。SrbA在体内的激活是真菌毒力和内在三氮唑药物所必需的 抗性，作为真菌特异性激活丝氨酸蛋白酶等sbA调节因子的零突变 RbdB和E3泛素连接酶(DSCs)在侵袭性曲霉病(IA)动物模型中是无毒的 三氮唑类药物敏感性显著增加。本提案针对RFA-AI-17-036的目标是 识别抑制srbA激活的小分子并将其发展为先进的候选治疗方案 对三唑耐药丝状真菌具有广谱活性。有效的srbA抑制剂- 依赖的信号通路将被开发为临床上使用的辅助疗法，与 三氮唑类抗真菌药，用于治疗IA。这种辅助疗法有望提供几种 与三氮唑单一疗法相比的优势，包括在低氧条件下抑制生长和增加 三唑类药物在药物敏感和耐药感染中的抗真菌活性。作为塞族共和军 途径在大多数人类真菌病原体中是保守的，其中一些是天生的对唑类抗药性，我们 预期除烟曲霉菌引起的感染外，还具有广泛的临床应用价值。我们的方法利用 具有良好特性的蛋白酶和连接酶抑制剂化学文库的可用性，这两个化学文库都是已知的药物靶标 在许多疾病环境中，利用MicroBiotix Inc.和达特茅斯的Cramer实验室的专业知识。这个 该应用的R21阶段将利用基于高通量细胞的已定义靶向小分子的筛选 鉴定和确认srbA调节酶和/或连接酶抑制剂并验证其抗真菌作用的文库 早期HITs和Lead的活性、途径特异性和哺乳动物毒性。在R33阶段，验证的命中率 将进行化学优化、验证、药理学定义、确定作用机制，并最终 在已建立的小鼠模型中进行体内药理和毒理学分析及抗真菌效果 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.