Crowther Gregory J, Quadri S Arshiya, Shannon-Alferes Benjamin J, Van Voorhis Wesley C, Rosen Henry
University of Washington, Seattle, WA, USA.
J Biomol Screen. 2012 Apr;17(4):535-41. doi: 10.1177/1087057111431606. Epub 2012 Jan 10.
More than 20% of bacterial proteins are noncytoplasmic, and most of these pass through the SecYEG channel en route to the periplasm, cell membrane, or surrounding environment. The Sec pathway, encompassing SecYEG and several associated proteins (SecA, SecB, YidC, SecDFYajC), is of interest as a potential drug target because it is distinct from targets of current drugs, is essential for bacterial growth, and exhibits dissimilarities in eukaryotes and bacteria that increase the likelihood of selectively inhibiting the microbial pathway. As a step toward validating the pathway as a drug target, we have adapted a mechanism-based whole-cell assay in a manner suitable for high-throughput screening (HTS). The assay uses an engineered strain of Escherichia coli that accumulates beta-galactosidase (β-gal) in its cytoplasm if translocation through SecYEG is blocked. The assay should facilitate rapid identification of compounds that specifically block the Sec pathway because widely, toxic compounds and nonspecific protein synthesis inhibitors prevent β-gal production and thus do not register as hits. Testing of current antibiotics confirmed that they do not generally act through the Sec pathway. A mini-screen of 800 compounds indicated the assay's readiness for larger screening projects.
超过20%的细菌蛋白是非胞质蛋白，其中大多数在转运至周质、细胞膜或周围环境的过程中会穿过SecYEG通道。Sec途径由SecYEG和几种相关蛋白（SecA、SecB、YidC、SecDFYajC）组成，作为一个潜在的药物靶点备受关注，因为它与现有药物的靶点不同，对细菌生长至关重要，并且在真核生物和细菌中表现出差异，这增加了选择性抑制微生物途径的可能性。作为将该途径验证为药物靶点的第一步，我们采用了一种基于机制的全细胞检测方法，使其适用于高通量筛选（HTS）。该检测方法使用一种工程改造的大肠杆菌菌株，如果通过SecYEG的转运受阻，该菌株会在其细胞质中积累β-半乳糖苷酶（β-gal）。该检测方法应有助于快速鉴定特异性阻断Sec途径的化合物，因为广泛的有毒化合物和非特异性蛋白质合成抑制剂会阻止β-gal的产生，因此不会被记录为阳性结果。对现有抗生素的测试证实，它们通常不是通过Sec途径起作用的。对800种化合物的小型筛选表明该检测方法已准备好用于更大规模的筛选项目。
