Development of the Screening System for Inhibitors of Bacterial Drug Exporters

细菌类药品出口抑制剂筛选系统的研制

• 批准号: 07557150
• 负责人: YAMAGUCHI Akihito
• 金额: $ 4.22万
• 依托单位: Osaka University (1996-1997)Chiba University (1995)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557150/
• 关键词: inhibitor; multidrug exporter; NorA; MexAB; TetK; TetB; screening; multidrug resistance; 緑膿菌; 黄色ぶどう球菌; テトラサイクリン耐性; 薬剤排出蛋白; 黄色ブドウ球菌; 耐性菌

A lot of bacterial drug exporters have recently been found in pathogens confering the mutidrug resistance. The development of an inhibitor for these multidrug exporters are very useful for chemotherapy. In this study, we developed the system for screening inhibitors for bacterial drug exporters and checked more than hundred thousand compounds. At first, norA and mexA/B-expressing S.aureus and P.aeruginosa strains, respectively, were constructed using these gene-defective strains as host cells. The resistance levels of these exporter gene-expresssing strains and their control host strains against quinolone of beta-lactam antibiotics were systematically determined in the presence or absence of various screening compounds. As a result, some inhibitor candidates were found, however, the probability for the appearance of the inhibitors was unexpectedly low, indicating that it is difficult to overcome the bacterial multidrug resistance based on the multidrug exporters. Similar results were independently obtained by Microcide Co. in USA.In the separate experiments, we investigated the inhibitors for a tetracycline exporter, Tet(K), in S.Aureus. An indane derivative was revealed to be a Tet (K)-specific inhibitor.

近年来，在引起多重耐药的病原菌中发现了大量的细菌性药物输出菌。针对这些多药输出者的抑制剂的开发对于化疗是非常有用的。在这项研究中，我们开发了用于筛选细菌药物出口抑制剂的系统，并检查了数十万种化合物。首先，使用这些基因缺陷菌株作为宿主细胞，分别构建了表达诺拉和mexA/B的金黄色葡萄球菌和铜绿假单胞菌菌株。在存在或不存在各种筛选化合物的情况下，系统地测定这些输出基因表达菌株及其对照宿主菌株对喹诺酮类或β-内酰胺类抗生素的耐药性水平。结果，发现了一些抑制剂候选物，然而，抑制剂出现的概率出乎意料地低，表明难以克服基于多药输出体的细菌多药耐药性。在单独的实验中，我们研究了金黄色葡萄球菌中四环素输出蛋白泰特（K）的抑制剂。茚满衍生物被证明是泰特（K）特异性抑制剂。

项目成果

Someya, Y.et al: "Mercaptide Formed between the Residue Cys70 and Hg^<2+> or Co^<2+> Behaves as a Functional Positively-Charged Side Chain Operative in the Arg70→Cys Mutant of the Metal-Tetracycline/H^+ Antiporter of Escherichia coli." Biochemistry. 35. 9

Someya, Y. 等人：“残基 Cys70 和 Hg^<2+> 或 Co^<2+> 之间形成的硫醇在金属四环素的 Arg70→Cys 突变体中充当功能性带正电荷的侧链操作物/大肠杆菌的 H^+ 逆向转运蛋白。”生物化学。 35. 9

Kimura, T., Inagaki, Y., Sawai, T.and Yamaguchi, A.: "Substrate-Induced Acceleration of N-Ethylmaleimide Reaction with the Cys-65 Mutant of the Transposon Tn10-Encoded Metal-Tetracycline/H^+ Antiporter Depends on the Interaction of Asp-66 with the Substra

Kimura, T.、Inagaki, Y.、Sawai, T. 和 Yamaguchi, A.：“底物诱导的 N-乙基马来酰亚胺反应与转座子 Tn10 编码的金属四环素/H^ 逆向转运蛋白的 Cys-65 突变体的加速取决于

Kimura, T., Nakatani, M., Kawabe, T.and Yamaguchi, A.: "Roles of Conserved Arginine Residues in the Metal-Tetracycline/H^+ Antiporter of Escherichia coli." Biochemistry. 37, (in press). (1998)

Kimura, T.、Nakatani, M.、Kawabe, T. 和 Yamaguchi, A.：“保守精氨酸残基在大肠杆菌金属四环素/H^ 逆向转运蛋白中的作用”。

T.Kimura: "Asp-285 of the Metal-Tetracycline/H^+ Antiporter of Escherichia coli is Essential for Substrate Binding" FEBS Letters. 388. 50-52 (1996)

T.Kimura：“大肠杆菌金属四环素/H^ 逆向转运蛋白的 Asp-285 对于底物结合至关重要”FEBS Letters。

Kimura,T.et al.: "Cysteine-Scanning Mutagenesis around Transmembrane Segment III of Tn10-Encoded Metal-Tetracycline/H^+ Antiporter." J.Biol.Chem.273. 1-5 (1998)

Kimura,T.et al.：“Tn10 编码的金属四环素/H^ 反转运蛋白跨膜片段 III 周围的半胱氨酸扫描诱变。”