Alexander Evan M, Kreitler Dale F, Guidolin Valeria, Hurben Alexander K, Drake Eric, Villalta Peter W, Balbo Silvia, Gulick Andrew M, Aldrich Courtney C
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Department of Structural Biology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14203, United States.
ACS Infect Dis. 2020 Jul 10;6(7):1976-1997. doi: 10.1021/acsinfecdis.0c00326. Epub 2020 Jun 24.
Tilimycin is an enterotoxin produced by the opportunistic pathogen that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell (IC = 29 ± 4 μM) through the inhibition of NpsA ( = 29 ± 4 nM).
替利霉素是由一种机会致病菌产生的肠毒素，该菌会引发抗生素相关性出血性结肠炎（AAHC）。这种吡咯并苯二氮卓（PBD）天然产物是通过由三种蛋白质NpsA、ThdA和NpsB组成的双模块非核糖体肽合成酶（NRPS）途径合成的。我们描述了完全重组的NRPS系统的功能和结构特征，并报告了所有天然底物和辅因子的稳态动力学分析以及NpsA和ThdA的结构特征。通过在替利霉素暴露于真核细胞后检测推定的N-2鸟嘌呤烷基化，使用DNA加合物组学技术证实了替利霉素的作用机制，提供了细胞中形成的PBD-DNA加合物的首次结构表征。最后，我们报告了通过抑制NpsA（IC = 29 ± 4 nM）来阻断全细胞中替利霉素生物合成的小分子抑制剂的合理设计（IC = 29 ± 4 μM）。
