Identification of the target and mode of action for the prokaryotic nucleotide excision repair inhibitor ATBC.

In bacteria, nucleotide excision repair (NER) plays a major role in repairing DNA damage from a wide variety of sources. Therefore, its inhibition offers potential to develop a new antibacterial in combination with adjuvants, such as UV light. To date, only one known chemical inhibitor of NER is 2-(5-amino-1,3,4-thiadiazol-2-yl)benzo(f)chromen-3-one (ATBC) exists and targets Mycobacterium tuberculosis NER. To enable the design of future drugs, we need to understand its mechanism of action. To determine the mechanism of action, we used in silico structure-based prediction, which identified the ATP-binding pocket of Escherichia coli UvrA as a probable target. Growth studies in E. coli showed it was nontoxic alone, but able to impair growth when combined with DNA-damaging agents, and as we predicted, it reduced by an approximately 70% UvrA’s ATPase rate. Since UvrA’s ATPase activity is necessary for effective DNA binding, we used single-molecule microscopy to directly observe DNA association. We measured an approximately sevenfold reduction in UvrA molecules binding to a single molecule of dsDNA suspended between optically trapped beads. These data provide a clear mechanism of action for ATBC, and show that targeting UvrA’s ATPase pocket is effective and ATBC provides an excellent framework for the derivation of more soluble inhibitors that can be tested for activity.

在细菌中，核苷酸切除修复（NER）在修复来自多种来源的DNA损伤方面起着重要作用。因此，抑制它有可能与佐剂（如紫外线）联合开发一种新的抗菌剂。到目前为止，已知的NER化学抑制剂只有2 - (5 - 氨基 - 1,3,4 - 噻二唑 - 2 - 基)苯并(f)色烯 - 3 - 酮（ATBC），它靶向结核分枝杆菌的NER。为了能够设计未来的药物，我们需要了解其作用机制。为了确定作用机制，我们使用了基于计算机结构的预测，该预测确定大肠杆菌UvrA的ATP结合口袋可能是一个靶点。在大肠杆菌中的生长研究表明，它单独使用时无毒，但当与DNA损伤剂联合使用时能够损害生长，并且正如我们所预测的，它使UvrA的ATP酶活性降低了约70%。由于UvrA的ATP酶活性对于有效的DNA结合是必需的，我们使用单分子显微镜直接观察DNA结合情况。我们测量到结合在光镊微珠之间悬浮的单分子双链DNA的UvrA分子数量减少了约7倍。这些数据为ATBC提供了一个明确的作用机制，并表明靶向UvrA的ATP酶口袋是有效的，而且ATBC为推导更多可溶的、可用于活性测试的抑制剂提供了一个极好的框架。