血小板生成素受體致效劑eltrombopag之新穎抗隱球菌活性

Abstract

全世界預估約有12億人口遭受真菌性感染，新型隱球菌(Cryptococcus neoformans)和格特隱球菌(Cryptococcus gattii)可造成人類隱球菌症(cryptococcosis)，是一種每年可造成約60萬人口死亡的人體伺機性病原。然而，治療策略卻十分侷限，因為傳統藥物開發的時程緩慢，加上抗藥性菌株有逐年增加的趨勢，因此，研發創新、有效且低毒性的抗真菌藥物為論文研究之目標。透過快速且大量篩選1,018種美國食品藥物管理局核准之藥物，我們從中發現一種過去用來治療自體免疫性血小板減少性紫斑症的藥物eltrombopag在最低抑制濃度0.125至 0.5 μg/mL下具有抑制隱球菌、光滑念珠菌及皮癬菌的效果。進一步實驗結果發現，eltrombopag與鈣調磷酸酶抑制劑 FK506具有協同作用(synergistic effect)。另外也發現此藥物可以顯著干擾隱球菌重要的致病因子，例如生物膜的形成、莢膜的形成、黑色素的合成與生長於37 ºC。掃描式電子顯微鏡的結果顯示隱球菌細胞表面出現許多破碎殘體，推測施用eltrombopag可能會破壞部分細胞表面結構，此結果可能與eltrombopag為抑菌而非殺菌的特性有關係。透過RNA測序(RNA sequencing)及基因功能分類分析(Gene ontology analysis)，結果顯示受藥物處理的大部分基因群和脂質生合成、跨膜運輸及細胞膜的組成有很大的關聯，即時反轉錄聚合酶鏈式反應的結果也證實受藥物處理後的樣本，其基因表現與對照組的有顯著性差異。為了證明eltormbopag確實對隱球菌之細胞膜造成受損，我們使用propidium iodide (PI)染色方法來評估受eltrombopag處理過後的細胞膜對PI染劑的滲透性。動物模式的結果顯示1 mg/kg/day eltrombopag可以顯著降低隱球菌在小鼠腦部的菌落數，但在存活率的數據上並無顯著差異。本研究是第一篇發現eltrombopag具有抗隱球菌活性的藥物，上述研究成果認為eltrombopag除了原本對紫斑病的治療，另外也具有潛力並開發應用於臨床上治療隱球菌症。

About 1.2 billion people worldwide are estimated to suffer from fungal infections. Cryptococcus neoformans and Cryptococcus gattii are the major opportunistic pathogens that cause cryptococcosis and responsible for approximately 600,000 deaths annually on a global scale. However, treatments remain challenging due to limited therapeutic options, rapidly emerging drug-resistant isolates and time-consuming research and development of new drugs. In this study, our objective is to discover compounds with low toxicity but exhibit novel antifungal activity. We identified eltrombopag, a medication that had been approved for the treatment of immune thrombocytopenia (ITP), via screening a compound library containing 1,018 FDA-approved drugs. Eltrombopag is firstly demonstrated to exhibit antifungal activity against fungal pathogens, such as Cryptococcus species with minimum inhibitory concentration (MIC) at 0.125 μg/mL, Candidia glabrata at 0.25 μg/mL, and Trichophyton rubrum at 0.5 μg/mL. Meanwhile, eltrombopag showed synergistic effect with a calcineurin inhibitor FK506 against Cryptococcus species. Furthermore, we showed that eltrombopag affected cryptococcal virulence factors such as biofilm formation, capsule formation, melanin production and growth ability at 37 ºC. Assisted by scanning electron microscopy, we observed impaired cryptococcal cell surface after eltrombopag treatment which may be associated with the fungistatic effect of eltrombopag against Cryptococcus species. RNA sequencing experiments revealed that many genes involved in lipid biogenesis, membrane components and transmembrane transporter were regulated by eltrombopag and confirmed by real time RT-PCR. To validate that the function of cell membrane was impaired, we assessed membrane permeability by propidium iodide staining. In the murine model of cryptococcosis, the results of fungal burden revealed that 1 mg/kg/day eltrombopag could significantly reduce cryptococcal colony formation units (CFU) in the brains, but the studies of survival rate did not show any difference in all treatments. Our data suggests that eltrombopag may be a promising antifungal agent against cryptococcal infections in addition to its original treatment of ITP.