MKT-077选择性清除外排泵高表达唑类耐药真菌的作用机理及其抗真菌靶点发现

Fungal infection has become one of the most common clinical infections. Despite the use of antifungal therapies, the battle against candidiasis has remained largely unsuccessful because of a limited number of antifungal drugs available and the occurrence of antifungal-resistance. Therefore, it is of great significance to develop novel antifungal agents and effective strategies to overcome the evolved-resistance. Collateral sensitivity means that a mutation that confers resistance to one drug can increases the sensitivity to other types of drugs and this phenomenon is commonly observed in microorganisms and tumors. Given this property, we screened a compound library including natural products and drug molecules through using a co-culture model of azole-sensitive and azole-resistant fungi, and found that MKT-077 selectively combated drug-resistant fungi caused by overexpression of MDR1. Co-localization experiments showed MKT-077 was accumulated in the fungal mitochondria, suggesting that it may exert antifungal effects through disrupting the mitochondrial function. This study aims at revealing the selective mechanism of MKT-077 by comparing the differences between MDR1-activated strain and its parent strain, and investigating the mode of action of MKT-077 in affecting mitochondrial function through transcriptome analysis and various target-discovering methods. Besides, we will optimize its structure by computer-assisted drug screening and explore its antifungal activity in vivo. The purpose of this study is to lay a foundation for the development of new antifungal drugs.

真菌感染是临床常见的感染性疾病，抗真菌药物种类少以及耐药的多发严重制约着真菌感染的治疗，因而寻找新型、特别是针对耐药菌的抗真菌药物具有重要意义。微生物和肿瘤均存在旁系敏感现象，即某种耐药突变会增加对其它类型药物的敏感性。我们利用该特性，采用荧光标记的敏感菌和耐药菌共培养模型对天然分子和药物分子库进行筛选，发现具有抗肿瘤活性的花菁染料类分子MKT-077对由外排泵MDR1高表达引起的唑类耐药菌具有选择性清除作用，其最小抑菌浓度为唑类敏感株的1/8；研究发现MKT-077聚集于真菌线粒体，提示其可能通过损伤线粒体功能发挥抗真菌作用。本课题拟寻找MDR1高表达株与其母体菌株的差异揭示MKT-077的选择性作用机制；通过转录组学、合成连接生物素标记的探针等方法探索药物作用靶点；通过计算机辅助药物筛选进行结构优化，并进一步研究MKT-077及衍生物的体内抗真菌活性，为开发新型抗真菌药物奠定研究基础。

念珠菌导致的系统性真菌感染的发生率和死亡率逐年升高，其中白色念珠菌作为临床最常见的机会致病菌，在机体免疫系统受损时侵入血液和组织，进一步造成重症念珠菌感染。尽管目前已经有较为合理的抗真菌感染治疗方案，但由于抗真菌药物种类有限以及真菌耐药的广泛出现，临床真菌感染形势依然不容乐观。因而寻找新型、特别是针对耐药菌的抗真菌药物具有重要意义。微生物和肿瘤均存在旁系敏感现象，即某种耐药突变会增加对其它类型药物的敏感性。申请人利用该特性，建立了一个药物筛选模型并筛选得到一系列能够选择性清除多药耐药外排泵编码基因MDR1高表达唑类耐药白色念珠菌的化合物，选取其中具有抗耐药选择性指数高的花菁类分子MKT-077进行深入研究。MKT-077对由外排泵MDR1高表达引起的唑类耐药菌具有选择性清除作用，其最小抑菌浓度为唑类敏感株的1/4；且选择性与该类小分子在MDR1高表达菌株中累积增加有关。进一步通过benomyl诱导实验和MKT-077耐药子发现MDR1表达与MKT-077的胞内累积成正相关，因此申请人推测Mdr1介导MKT-077的摄取。利用酵母异源表达系统发现白色念珠菌、热带念珠菌、近平滑念珠菌的Mdr1均能介导MKT-077进入胞内。进一步实验发现MKT-077可以与benomyl联用清除其他因素引起的唑类耐药菌；与两性霉素B联用清除成熟被膜，具有较好的临床应用价值。通过对MKT-077进行结构修饰获得了仍具有选择性清除活性且抗菌活性更佳的MD-1。采用蜡螟评价模型发现MKT-077对Mdr1高表达菌株感染蜡螟模型具有较好治疗效果，为开发抗真菌药物奠定了研究基础。该发现不仅为克服外排泵Mdr1引起的耐药提供了潜在的先导分子，还颠覆了传统的外排泵功能认识。