Department of Cell Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, 0310 Oslo, Norway.
J Antimicrob Chemother. 2010 May;65(5):924-30. doi: 10.1093/jac/dkq071. Epub 2010 Mar 12.
In order to search for novel antibacterial compounds we used a previously developed screening strain designed specifically to discover inhibitors of the bacterial initiator protein, DnaA. This strain (SF53) is not viable at 30 degrees C due to overinitiation. Therefore, compounds that are able to restore growth to SF53 cells are likely to cause either partial or complete inhibition of DnaA function. In this study we used SF53 cells to screen the Library of Pharmacologically Active Compounds (LOPAC).
An SF53 screen of LOPAC in 384-well plates was performed. The effects of compounds identified as positive were studied further by growth assays specific for replication proteins as well as an in vitro assay of the activity of purified DNA gyrase.
One of the compounds that tested positive in this screening was the benzazepine derivate (+/-)-6-chloro-PB hydrobromide (S143). We found that the substance did not target DnaA directly, but that it most probably reduces overinitiation by inhibiting DNA gyrase. Benzazepines have not previously been reported as gyrase inhibitors.
These findings indicate that a screening with SF53 will be able to identify compounds that also target other replication proteins (in addition to DnaA). Screening of LOPAC with SF53 cells led to the discovery of a novel DNA gyrase inhibitor.
为了寻找新的抗菌化合物，我们使用了一种先前开发的筛选菌株，该菌株专门设计用于发现细菌起始蛋白 DnaA 的抑制剂。由于过度起始，这种菌株（SF53）在 30°C 时无法存活。因此，能够使 SF53 细胞恢复生长的化合物很可能导致 DnaA 功能的部分或完全抑制。在这项研究中，我们使用 SF53 细胞筛选了药理学活性化合物库（LOPAC）。
在 384 孔板中对 LOPAC 进行了 SF53 筛选。通过针对复制蛋白的生长测定以及体外纯化 DNA 拓扑异构酶活性测定，进一步研究了鉴定为阳性的化合物的作用。
在该筛选中测试为阳性的一种化合物是苯并氮杂环庚烷衍生物（±）-6-氯-PB 氢溴酸盐（S143）。我们发现该物质不是直接靶向 DnaA，而是很可能通过抑制 DNA 拓扑异构酶来减少过度起始。苯并氮杂环庚烷以前没有被报道为拓扑异构酶抑制剂。
这些发现表明，使用 SF53 进行筛选将能够识别出除 DnaA 之外还靶向其他复制蛋白的化合物。使用 SF53 细胞对 LOPAC 进行筛选导致发现了一种新型的 DNA 拓扑异构酶抑制剂。
