台灣本土真菌Neosartorya fischeri之二次代謝物avenaciolides於抑制抗甲氧西林金黃色葡萄球菌之機轉

Abstract

在現代醫學中，抗生素的發展及其應用在許多成功的感染治療中一直扮演著舉足輕重的角色。然而，隨著幾十年來抗生素的濫用，使抗藥性菌株不斷地增長，進而使現今醫學可運用的抗生素漸漸趨少。舉例來說，在革蘭氏陽性抗藥菌株中，抗甲氧西林金黃色葡萄球菌(MRSA)位於院內交叉感染率及死亡率之首。在此抗生素後世代，新藥的尋找及發展成為了現今刻不容緩的課題。 在本論文的研究中，我們發現一株來自於花蓮縣的本土真菌「Neosartorya fischeri」會分泌一種二級代謝產物並有效的抑制抗甲氧西林金黃色葡萄球菌的生長。本篇研究利用以活性為導向的策略，純化且分離出一系列的燕麥麯黴素衍生物。在穿透式電子顯微鏡研究中，發現其中有三個燕麥麯黴素衍生物藉由抑制細菌細胞壁合成來達成其抑菌之活性。藉由「結構-活性」關係中，我們發現在燕麥麯黴素衍生物上的a，b-共振不飽和羰基是抑菌活性中不可或缺的單元。因此，在經過進一步的文獻搜索以及核磁共振磷谱和質譜的分析中，證實了燕麥麯黴素抑制細胞壁合成的第一步酵素「乙醯氨基葡萄糖轉移酶」。雖然，目前臨床中以磷黴素為乙醯氨基葡萄糖轉移酶之唯一抑制劑；但近年來，抗磷黴素突變菌株卻不斷地被發現及報導。其最嚴重的抗磷黴素機轉，為乙醯氨基葡萄糖轉移酶之催化中心的胺基酸突變(半胱氨酸突變成天門冬氨酸)。 在此研究中，一號與二號燕麥麯黴素衍生物不僅針對野生性也針對抗磷黴素(半胱氨酸突變成天門冬氨酸)之乙醯氨基葡萄糖轉移酶有抑制活性。並在經由分子模擬計算，我們推論出二號化合物競爭性地阻擾酵素受質中間體的產生。最後，在所有衍生物中，二號燕麥麯黴素被證實具為有潛力之先驅化合物，可作為未來發展且抑制抗甲氧西林金黃色葡萄球菌及抗磷黴素之乙醯氨基葡萄糖轉移酶突變的指標性藥物。

Antibiotics, the major breakthrough of modern medicine, play a pivotal role in many successful medical practices. However, the emergence of resistant traits against multiple classes of antibiotics has progressively narrowed the available treatment options for some pathogens for decades. Among the Gram-positive drug resistant microbes, methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for nosocomial and community-acquired bacterial infections around the world. Since we are now living in the post-antibiotics era in which the crisis of antimicrobial resistance is worse than ever before, discovering alternative antibiotics is urgent to counteract the ever increasing phenomenon. In this study, we report four avenaciolide derivatives (1-4) isolated from Neosartorya fischeri, an indigenous fungus from Hualien, Taiwan, three of which had significant antimicrobial activity against MRSA. Based on the TEM results, the morphology of avenaciolide-treated cells was protoplast-like, which indicated that cell wall biosynthesis was interrupted. Comparing the structures and MICs of 1-4, the the a,b-unsaturated carbonyl group seems to be an indispensable moiety for antimicrobial activity. Based on a structural similarity survey of other inhibitors with the same moiety, we revealed that MurA was the drug target. This conclusion was validated by 31P NMR spectroscopy and MS/MS analysis. Although fosfomycin, which is the only clinically used MurA-targeted antibiotic, is ineffective for treating bacteria harboring the catalytically important Cys-to-Asp mutation, avenaciolides 1 and 2 inhibited not only wild type but also fosfomycin-resistant MurA in an unprecedented way. Molecular simulation revealed that 2 competitively perturbs the formation of the tetrahedral intermediate in MurA. Our findings demonstrated that 2 is a potent inhibitor of MRSA and fosfomycin-resistant MurA, laying the foundation for the development of new scaffolds for MurA-targeted antibiotics.