抗感染土壤源放线菌产抗MDROs新颖大环内酯的结构及突变选择窗研究

Multidrug-resistant organisms (MDROs) are resistant to a variety of structural types of antibiotics, and the research and development of new anti-MDROs drugs is particularly urgent. In view of the anti-MDROs potential and good druggability of macrolides, macrolide-producing strains ZZ027 and ZZ021 were screened out from sixty-one actinomycetes strains isolated from anti-infectious soil. Preliminary studies showed that eighteen macrocyclic lactones including at least six new ones were contained in the broth of two strains, and these macrocyclic lactones showed anti-MDROs activity including anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) and anti-vancomycin-resistant Enterococcus (anti-VRE). To clarify their chemical structures and determine their anti-MDROs activities and difficulty degree of MDROs resistant to them, these macrocyclic lactones will be isolated and prepared by all kinds of separation and purification techniques such as column chromatography and crystallization, and which will be respectively guided by the HPLC-UV characteristics of macrocyclic lactones produced by two strains. Their chemical structures will be elucidated on the basis of physic-chemical properties, physic-chemical constants and spectroscopic evidences determined. Further, their mutant selection windows against MDROs will be researched with MRSA and VRE used as indicator bacteria. Anti-MDROs lead compounds with novel structure, remarkable activity and narrow mutant selection window will be expected to obtain through this research, and a certain foundation will be established for the research of new anti-MDROs drug with independent intellectual property rights.

多重耐药菌（MDROs）对多种结构类型的抗生素耐药，新型抗多重耐药菌药物的研发显得尤为迫切。鉴于大环内酯抗多重耐药菌的潜力及良好成药性，课题组从61株抗感染土壤源放线菌中筛得大环内酯产生菌ZZ027和ZZ021，前期研究表明：两菌株发酵液中含有18个抗多重耐药菌MRSA和VRE的大环内酯，且至少有6个为新大环内酯。为阐明这些大环内酯的化学结构，明确其抗多重耐药菌活性及多重耐药菌对其产生耐药的难易程度，该课题将分别以两菌株所产大环内酯的HPLC-UV特征为导向，选用各种柱色谱和结晶等分离纯化技术，对这些大环内酯进行分离制备，并通过其理化性质、理化常数和波谱的测定与综合解析，确定各大环内酯的化学结构，然后以MRSA和VRE为指示菌，对其抗多重耐药菌的突变选择窗进行研究。以期获得结构新颖、活性显著且突变选择窗小的抗多重耐药菌先导化合物，为拥有自主知识产权的抗多重耐药菌新药研究打下一定基础。

近年来，多重耐药菌(MDROs)感染遍及全球各地，感染率呈持续上升趋势，并对多种结构类型的临床抗生素相继出现耐药，其速度已超过目前新抗生素的研发速度，新型抗多重耐药菌药物的研发显得尤为迫切。鉴于此，该课题对前期发现具有抗多重耐药菌活性的土壤放线菌菌株所产的目标产物（大环内酯）进行研究，共计分离鉴定12个具有广谱抗菌（包括抗多重耐药菌MRSA等）活性的大环内酯化合物，其中包括7个新的阿扎霉素F类似物，同时对该菌株产阿扎霉素F类化合物的发酵进行了优化，制得阿扎霉素F纯组分325.7 g。结合阿扎霉素F类似物结构研究时的新发现，制备了抗MRSA活性显著增强的7个新阿扎霉素F衍生物，其中抗MRSA活性最强的为去丙二酸单酰基阿扎霉素F5a、F4a和F3a，对测试MRSA菌株的最低抑菌浓度为0.25-0.50 μg/mL，为具有抗MRSA活性的阿扎霉素F类似物的化学和生物学研究打下了基础，并提供了活性更强、水溶性更好和稳定性较佳的新型抗MRSA苗头化合物，给可能的抗MRSA新药研发打下了坚实基础。由此，课题组以代表性化合物阿扎霉素F5a、F4a、F3a、去丙二酸单酰基阿扎霉素F5a、F4a和F3a为主要研究对象，对阿扎霉素F抗MRSA的活性、消除生物膜作用和突变选择窗进行了深入研究。结果表明：阿扎霉素F或去丙二酸单酰基阿扎霉素F与氢醌类化合物（如维生素K3）具有显著的协同抗MRSA活性，阿扎霉素F具有消除MRSA生物膜作用，阿扎霉素F5a具有较窄的突变选择性窗、且与维生素K3联用时其抗MRSA的突变选择窗可被关闭。由此显示阿扎霉素F在抗MRSA新药研发方面的良好前景，值得深入研究。另外，课题组还分得1个具有γ-氨基丁酸转氨酶抑制活性的新化合物5-氨甲基-2-亚氨基咪唑烷-4-羧酸，其对γ-氨基丁酸转氨酶的IC50为60μM （阳性对照药Vigabatrin的IC50为36μM），由此为γ-氨基丁酸衍生物在治疗γ-氨基丁酸缺乏而引起脑功能失常方面的疾病添加了新的结构亚类，并为其在此方面的研究打下了基础。

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