新型抗耐药真菌化合物的作用靶点及分子机制研究

The incidence of invasive fungal infections is increasing year by year, mortality remains high. Natural and acquired drug resistance of fungi brings enormous difficulties for the treatment of fungal infections. No new drug acting at novel target has been developed against drug-resistant fungi over the last decade. Based on the new strategy of synergistic effect, we have found multiple natural small molecules possess strong activity against drug-resistant fungi. The preliminary structure-activity relationship (SAR) study suggested that these compounds have similar pharmacophore. But until now, their target and molecular mechanisms have not been determined. In the present project, we first plan to continue the SAR and structural optimization study, for screening the compound with higher activity and specificity, and ensuring the ligand can be immobilized affinity gel with target recognition capability. Second, we want to investigate the cellular and molecular mechanisms of the active compounds against drug-resistant fungi. Third, we will screen the interaction protein in fungi with the above active compounds by SILAC-based quantitative proteomics technology and affinity adsorption technology, and then combine bioinformatics technologies to analyze the target of the active compounds against drug-resistant fungi. In the end, to validate the target of the novel compounds, we will knockout the target gene in fungi cell and compare the phenotype between gene deletion strain and wild type strain after incubation with active compound, combine association analysis between biological function of the target protein and mechanism of the active compounds against fungi. We aim to not only identify the target and clarify the antifungal mechanism of the active compound, but also provide theoretical and experimental basis for new effective antifungal agent development.

侵袭性真菌感染的发生率逐年上升，病死率居高不下，真菌天然耐药和获得性耐药为真菌感染治疗带来巨大困难，且近十多年来未发现基于新靶点的抗耐药真菌新药。本课题组从协同抗耐药真菌的新策略出发，发现了多个天然小分子化合物具有很强的抗耐药真菌活性，构效关系研究发现了它们共有的药效基团，但作用靶点和机制尚不明确。本项目拟对活性化合物深入进行构效关系及结构优化研究，以发现具有更高活性、特异性的化合物和能够固载于亲和凝胶上并具有靶点识别能力的配体；深入研究活性化合物抗耐药真菌的细胞和分子机制；采用基于SILAC的定量蛋白质组学技术结合亲和吸附技术研究活性化合物与真菌细胞内特异相互作用蛋白和作用靶点；采用基因敲除技术构建靶蛋白基因缺失菌，比较基因缺失菌和野生菌与活性化合物作用后的表型差异，关联分析靶蛋白的生物学功能及化合物作用机制，对其作用靶点进行确证，为研究新型高效的抗耐药真菌药物提供理论和实验依据。

侵袭性真菌感染的发生率逐年上升，病死率居高不下。真菌天然耐药和获得性耐药为真菌感染的治疗带来巨大困难，且近十多年来未发现基于新靶点的抗耐药真菌新药。本课题从协同抗耐药真菌的新策略出发，设计合成并测试了多个系列的化合物，发现氮唑类化合物NT-2在体内有强于氟康唑和伏立康唑的抗耐药白念和新生隐球菌活性，并实现了成果转化；发现氨基吡啶类化合物GPI-89能抑制真菌菌丝形成和生长繁殖，体内（4mg/kg、 8mg/kg、16mg/kg）能剂量依赖性协同氟康唑抗耐药白念珠菌感染；发现新化合物3e有更强于原型物的协同抗耐药真菌活性。采用SILAC技术结合亲和层析技术研究小分子探针TSJ-9、BBR3、BE-2与白念珠菌的相互作用蛋白，发现蛋白Hsp90、Eno1、Hsp70、Tal1、Cat2、Fba1等可能与小檗碱、黄芩素等活性化合物抗耐药真菌的作用有关。我们表达纯化了Hsp90(片段)和Eno1、Cat2、Fba1的全长蛋白，采用BiaCore技术检测了上述蛋白与BBR及BE 的亲和力，结果发现Hsp90、Eno1与BBR及BE有一定的结合。进一步利用杂交瘤技术制备并筛选了抗真菌Hsp90的单抗，经过大量的筛选，发现了体内能协同氟康唑抗耐药真菌并实现了成果转化的单抗，BiaCore法测定其与Hsp90结合的Kd值达到纳摩儿级水平。围绕Eno1我们研究其结构功能，建立了检测Eno1-PLG结合、Eno1烯醇化酶及Eno1谷氨酰胺转氨酶活性模型，发现小分子化合物TGI和抗Eno1单抗（KD值达10-9 M）动物体内能够协同氟康唑发挥抗真菌感染作用。研究发现Flo8基因缺失菌预免疫对后续的小鼠真菌感染具有显著的保护作用，该保护作用是胸腺依赖性的。进一步研究发现，发现Flo8缺失菌细胞壁甘露糖通过促进胸腺产生IL-10，起抗胸腺淋巴细胞凋亡，保护机体抗真菌免疫的作用，有潜在的治疗学意义。