绿脓杆菌SecA ATPase抑制剂筛选模型的建立和应用

The Establishment and Application of Inhibitors Screening Model Targeting to Pseudomonas aeruginosa SecA ATPase

摘要:

Sec途径(分泌途径, Secretion pathway)是蛋白质转运的主要途径。其中, SecA ATPase是蛋白质转运途径中的“动力泵”, 它通过ATP的水解循环驱使蛋白质前体穿过细菌内膜。SecA蛋白在细菌中是独有且不可缺少的。克隆和高效表达绿脓杆菌PasecAN75蛋白(绿脓杆菌SecA蛋白N端645个氨基酸残基组成的片段, 大小约75 kD)并优化其ATPase酶活测定体系, 在此基础上建立了更为灵敏的SecA蛋白ATPase活性抑制剂的筛选模型。运用该模型从化合物库的3220个样品中筛选得到可抑制绿脓杆菌SecA ATP酶的活性阳性化合物4个, 从7196个微生物发酵液中得到66个阳性样品, 筛选阳性率为0.67% (以抑制率大于30%为筛选阳性标准)。而后通过已建立的细胞水平筛选模型对其抗菌活性进行验证。研究结果表明3个化合物样品和6个发酵液样品在酶水平和细胞水平对绿脓杆菌SecA ATPase均有较好的抑制作用, 值得进一步研究。

Abstract:

The most of secreted proteins are exported by Sec translocase (Secretion pathway). SecA ATPase is the preprotein translocase nanomotor that undergoes membrane insertion and deinsertion to drive preprotein across the bacterial inner membrane, which is unique and indispensable to bacteria. It should be presumed that the compound which inhibits the activity of SecA ATPase probably can be used as the candidate of bactericide. Pseudomonas aeruginosa secA gene product, whose amino acid sequence displays PaSecAN75 (The N-terminal first 645 amino acids of Pseudomonas aeruginosa SecA), was cloned, overexpressed and purified in order to establish enzyme inhibitor screening model. After establishment and optimization of screening assay, a primary screening was performed with compounds library and microbial fermentation collection in our institute. 4 out of 3220 compouds and 66 out of 7196 microbial fermentation samples were identified as primary hit. To confirm the hits from the primary screening, a cell-based SecA activity assay was used to assess their inhibitory effect in vivo. Results showed that 3 compounds and 6 microbial fermentation samples inhibited enzymic activity of SecA in vitro and impaired its function in vivo.