Kamath Manali M, Adams Emily M, Lightfoot Jorge D, Wells Becca L, Fuller Kevin K
Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
bioRxiv. 2024 Aug 8:2024.08.08.607189. doi: 10.1101/2024.08.08.607189.
The fungal unfolded protein response consists of a two-component relay in which the ER-bound sensor, IreA, splices and activates the mRNA of the transcription factor, HacA. Previously, we demonstrated that is essential for virulence in a murine model of fungal keratitis (FK), suggesting the pathway could serve as a therapeutic target. Here we investigate the antifungal properties of known inhibitors of the mammalian Ire1 protein both and in a treatment model of FK.
The antifungal activity of Ire1 inhibitors was tested against conidia of several isolates by a microbroth dilution assay and against fungal biofilm by XTT reduction. The influence of 4μ8C on mRNA splicing in was assessed through gel electrophoresis and qRT-PCR of UPR regulatory genes. The toxicity and antifungal profile of 4μ8C in the cornea was assessed by applying drops to uninfected or -infected corneas 3 times daily starting 4 hours post-inoculation. Corneas were evaluated daily through slit-lamp imaging and optical coherence tomography, or at endpoint through histology or fungal burden quantification via colony forming units.
Among six Ire1 inhibitors screened, the endonuclease inhibitor 4μ8C displayed the strongest antifungal profile with an apparent fungicidal action. The compound both blocked conidial germination and hyphal metabolism of Af293 in the same concentration range that blocked splicing and UPR gene induction (60-120 μM). Topical treatment of sham-inoculated corneas with 0.5 and 2.5 mM 4μ8C did not impact corneal clarity, but did transiently inhibit epithelialization of corneal ulcers. Relative to vehicle-treated Af293-infected corneas, treatment with 0.5 and 2.5 mM drug resulted in a 50% and >90% reduction in fungal load, respectively, the latter of which corresponded to an absence of clinical signs of infection or corneal pathology.
The data suggest that 4μ8C displays antifungal activity against through the specific inhibition of IreA. Topical application of the compound to the murine cornea can furthermore block the establishment of infection, suggesting this class of drugs can be developed as novel antifungals that improve visual outcomes in FK patients.
真菌未折叠蛋白反应由一个双组分信号转导途径组成，其中内质网结合传感器IreA对转录因子HacA的mRNA进行剪接并激活。此前，我们证明该途径对于真菌性角膜炎（FK）小鼠模型中的毒力至关重要，这表明该途径可作为治疗靶点。在此，我们研究了已知的哺乳动物Ire1蛋白抑制剂在体外和FK治疗模型中的抗真菌特性。
通过微量肉汤稀释法测试Ire1抑制剂对几种曲霉菌株分生孢子的抗真菌活性，并通过XTT还原法测试其对真菌生物膜的活性。通过凝胶电泳和UPR调节基因的qRT-PCR评估4μ8C对曲霉菌中HacA mRNA剪接的影响。在接种后4小时开始，每天3次向未感染或感染曲霉菌的角膜滴注药物，评估4μ8C在角膜中的毒性和抗真菌谱。每天通过裂隙灯成像和光学相干断层扫描评估角膜，或在实验终点通过组织学或通过菌落形成单位进行真菌载量定量评估。
在筛选的六种Ire1抑制剂中，核酸内切酶抑制剂4μ8C表现出最强的抗真菌活性，具有明显的杀菌作用。该化合物在阻断HacA剪接和UPR基因诱导的相同浓度范围内（60 - 120μM），既能阻止烟曲霉Af293分生孢子的萌发，又能抑制其菌丝代谢。用0.5和2.5 mM 4μ8C对假接种的角膜进行局部治疗，不影响角膜清晰度，但会短暂抑制角膜溃疡的上皮化。相对于载体处理的Af293感染角膜，用0.5和2.5 mM药物治疗分别使真菌载量降低了50%和>90%，后者对应于无感染的临床体征或角膜病变。
体外数据表明，4μ8C通过特异性抑制IreA对烟曲霉具有抗真菌活性。将该化合物局部应用于小鼠角膜还可以阻止感染的建立，表明这类药物可以开发成为改善FK患者视力的新型抗真菌药物。
