Center for Life Nano Science@Sapienza, Italian Institute of Technology, Rome, Italy.
Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Laboratory affiliated to Pasteur Italia-Fondazione Cenci Bolognetti, Rome, Italy.
J Antimicrob Chemother. 2020 Sep 1;75(9):2564-2572. doi: 10.1093/jac/dkaa200.
Colistin is a last-resort treatment option for many MDR Gram-negative bacteria. The covalent addition of l-aminoarabinose to the lipid A moiety of LPS is the main colistin resistance mechanism in the human pathogen Pseudomonas aeruginosa.
Identification (by in silico screening of a chemical library) of potential inhibitors of ArnT, which catalyses the last committed step of lipid A aminoarabinosylation, and their validation in vitro as colistin adjuvants.
The available ArnT crystal structure was used for a docking-based virtual screening of an in-house library of natural products. The resulting putative ArnT inhibitors were tested in growth inhibition assays using a reference colistin-resistant P. aeruginosa strain. The most promising compound was further characterized for its range of activity, specificity and cytotoxicity. Additionally, the effect of the compound on lipid A aminoarabinosylation was verified by MS analyses of lipid A.
A putative ArnT inhibitor (BBN149) was discovered by molecular docking and demonstrated to specifically potentiate colistin activity in colistin-resistant P. aeruginosa isolates, without relevant effect on colistin-susceptible strains. BBN149 also showed adjuvant activity against colistin-resistant Klebsiella pneumoniae and low toxicity to bronchial epithelial cells. Lipid A aminoarabinosylation was reduced in BBN149-treated cells, although only partially.
This study demonstrates that in silico screening targeting ArnT can successfully identify inhibitors of colistin resistance and provides a promising lead compound for the development of colistin adjuvants for the treatment of MDR bacterial infections.
多黏菌素是治疗许多耐多药革兰氏阴性菌的最后手段。脂多糖的脂质 A 部分与 l-阿拉伯糖共价结合是铜绿假单胞菌等人类病原体产生多黏菌素耐药的主要机制。
通过对化学文库进行计算机筛选，鉴定出催化脂质 A 氨基阿拉伯糖基化的最后一步的 ArnT 的潜在抑制剂，并在体外验证其作为多黏菌素佐剂的作用。
利用现有的 ArnT 晶体结构，对内部天然产物文库进行基于对接的虚拟筛选。使用参考的多黏菌素耐药铜绿假单胞菌菌株，在生长抑制试验中测试得到的潜在 ArnT 抑制剂。对最有前途的化合物进行了更广泛的活性、特异性和细胞毒性特征分析。此外，通过对脂质 A 的 MS 分析验证了该化合物对脂质 A 氨基阿拉伯糖基化的影响。
通过分子对接发现了一种潜在的 ArnT 抑制剂（BBN149），并证明其可特异性增强多黏菌素在多黏菌素耐药铜绿假单胞菌分离株中的活性，而对多黏菌素敏感株无相关作用。BBN149 对多黏菌素耐药的肺炎克雷伯菌也具有佐剂活性，且对支气管上皮细胞的毒性较低。BBN149 处理的细胞中脂质 A 的氨基阿拉伯糖基化减少，但仅部分减少。
本研究表明，针对 ArnT 的计算机筛选可以成功鉴定出多黏菌素耐药抑制剂，并为开发治疗多重耐药细菌感染的多黏菌素佐剂提供了有前景的先导化合物。
