Argentilactone molecular targets in Paracoccidioides brasiliensis identified by chemoproteomics

Paracoccidioidomycosis (PCM) is the cause of several deaths from systemic mycoses. The etiological agents of PCM belong to the Paracoccidioides genus, which is restricted to Latin America. The infection is acquired through inhalation of conidia that primarily lodges in the lungs and may disseminate to other organs/tissues. Treatment of PCM is commonly achieved via the administration of antifungals such as amphotericin B, co-trimoxazole, and itraconazole. The antifungal toxicity and side effects, in addition to the long treatment time, have driven research for new bioactive compounds. Argentilactone, a compound isolated from the Brazilian savanna plant Hyptis ovaliofolia, has been suggested to be a potent antifungal, inhibiting the dimorphism of P. brasiliensis and enzymatic activity of isocitrate lyase, a key enzyme of the glyoxylate cycle. Furthermore, argentilactone has no cytotoxicity and genotoxicity in fibroblast cells at concentrations that inhibit fungal growth. This work was developed due to the importance of elucidating the putative mode of action of argentilactone. The chemoproteomics approach, by affinity chromatography, is the methodology used to explore the interactions between P. brasiliensis proteins and argentilactone. A total of 109 proteins was identified and classified functionally, with those related to amino acid metabolism, energy, and detoxification being the most representative. The interactome of argentilactone binding proteins was predicted. Argentilactone inhibited the enzymatic activity of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase. Furthermore, argentilactone induced the production of reactive oxygen species. In addition, our data were compared to previously obtained proteomics and transcriptional data. Altogether, our results reveal argentilactone as a promising antifungal.

副球孢子菌病（Paracoccidioidomycosis, PCM）是引发多例系统性真菌病（systemic mycoses）致死病例的病因。该病的致病原（etiological agents）隶属于副球孢子菌属（Paracoccidioides genus），该属真菌仅分布于拉丁美洲。人体可通过吸入分生孢子（inhalation of conidia）感染该病，孢子主要定植于肺部，还可播散至其他器官或组织。临床治疗副球孢子菌病通常采用两性霉素B（amphotericin B）、复方新诺明（co-trimoxazole）及伊曲康唑（itraconazole）等抗真菌药物（antifungals）。但抗真菌药物的毒性与副作用，加之治疗周期漫长，推动了新型生物活性化合物（bioactive compounds）的研发。
从巴西稀树草原植物（Brazilian savanna plant）长苞香科科（Hyptis ovaliofolia）中分离得到的银内酯（argentilactone）被认为是一种强效抗真菌成分，可抑制巴西副球孢子菌（P. brasiliensis）的二态性（dimorphism），以及乙醛酸循环（glyoxylate cycle）关键酶异柠檬酸裂解酶（isocitrate lyase）的活性。此外，在抑制真菌生长的浓度下，银内酯对成纤维细胞（fibroblast cells）无细胞毒性（cytotoxicity）与遗传毒性（genotoxicity）。
本研究旨在阐明银内酯潜在的作用机制。研究采用亲和色谱法（affinity chromatography）结合化学蛋白质组学（chemoproteomics）的方法，探究巴西副球孢子菌蛋白与银内酯之间的相互作用。最终共鉴定出109种蛋白质并完成功能分类，其中与氨基酸代谢、能量代谢及解毒相关的蛋白质占比最高。本研究还预测了银内酯结合蛋白的相互作用组（interactome）。实验结果显示，银内酯可抑制苹果酸脱氢酶（malate dehydrogenase）、柠檬酸合酶（citrate synthase）及丙酮酸脱氢酶（pyruvate dehydrogenase）的酶活性，同时可诱导活性氧（reactive oxygen species）的产生。此外，本研究将所得数据与既往发表的蛋白质组学（proteomics）及转录组数据（transcriptional data）进行了对比。综上，本研究结果证实银内酯是一种极具潜力的抗真菌候选药物。