College of Pharmaceutical Science & Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technologygrid.469325.f, Hangzhou, China.
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
Microbiol Spectr. 2022 Aug 31;10(4):e0276421. doi: 10.1128/spectrum.02764-21. Epub 2022 Jul 20.
One of the challenges associated with the treatment of Pseudomonas aeruginosa infections is the high prevalence of multidrug resistance (MDR). Since conventional antibiotics are ineffective at treating such bacterial infections, innovative antibiotics acting upon novel targets or via mechanisms are urgently required. In this study, we identified a quorum sensing inhibitor (QSI), norharmane, that uniquely shows weak antibacterial activity but strongly inhibits pyocyanin production and biofilm formation of MDR P. aeruginosa. Biophysical experiments and molecular docking studies showed that norharmane competes with anthraniloyl-AMP for anthranilyl-CoA synthetase PqsA of P. aeruginosa at the ligand-binding pocket, which is not exploited by current inhibitors, thereby altering transcription regulatory activity. Moreover, norharmane exhibits synergy with polymyxin B. This synergism exhibits a high killing rate, low probability of resistance selection, and minimal cytotoxicity. Notably, norharmane can effectively boost polymyxin B activity against MDR P. aeruginosa-associated infections in animal models. Together, our findings provide novel insight critical to the design of improved PqsA inhibitors, and an effective combination strategy to overcome multiantibiotic bacterial resistance using conventional antibiotics and QSIs. Pseudomonas aeruginosa is a dominant hospital-acquired bacterial pathogen typically found in immunocompromised individuals. It is particularly dangerous for patients with chronic lung diseases and was identified as a serious threat for patients in the 2019 Antimicrobial Resistance Threats report (https://www.cdc.gov/drugresistance/biggest-threats.html). In this study, we used activity-based high-throughput screening to identify norharmane, a potent and selective inhibitor of P. aeruginosa PqsA, which is a well-conserved master quorum sensing (QS) regulator in multidrug resistant (MDR) P. aeruginosa. This compound competitively binds anthranilyl-CoA synthetase PqsA at the anthraniloyl-AMP binding domain, which has not been exploited by known inhibitors. Remarkably, norharmane can significantly block the production of the virulence factor, pyocyanin (87%), and biofilm formation (80%) in MDR P. aeruginosa. Furthermore, norharmane is capable of augmenting polymyxin B activity against MDR P. aeruginosa in cell cultures and animal models. Taken together, these results suggest that norharmane may be an effective adjuvant for combating multiantibiotic bacterial resistance.
铜绿假单胞菌感染治疗面临的挑战之一是多药耐药性（MDR）的高流行率。由于传统抗生素对治疗此类细菌感染无效，因此迫切需要针对新靶点或通过新机制发挥作用的创新抗生素。在这项研究中，我们发现一种群体感应抑制剂（QSI），即 norharmane，它具有独特的弱抗菌活性，但能强烈抑制多药耐药铜绿假单胞菌的绿脓菌素产生和生物膜形成。生物物理实验和分子对接研究表明，norharmane与 anthraniloyl-AMP 竞争铜绿假单胞菌的 anthranilyl-CoA 合成酶 PqsA 的配体结合口袋，而当前的抑制剂并未利用这一结合口袋，从而改变了转录调节活性。此外，norharmane 与多粘菌素 B 表现出协同作用。这种协同作用具有高杀伤率、低耐药选择概率和最小细胞毒性的特点。值得注意的是，norharmane 可以有效地增强多粘菌素 B 对多药耐药铜绿假单胞菌相关感染的活性，在动物模型中。总之，我们的研究结果为设计改良 PqsA 抑制剂提供了新的见解，并为利用传统抗生素和 QSIs 克服多抗生素细菌耐药提供了一种有效的联合策略。铜绿假单胞菌是一种主要的医院获得性细菌病原体，通常在免疫功能低下的个体中发现。它对患有慢性肺部疾病的患者尤其危险，并在 2019 年抗生素耐药性威胁报告（https://www.cdc.gov/drugresistance/biggest-threats.html）中被确定为患者的严重威胁。在这项研究中，我们使用基于活性的高通量筛选来鉴定 norharmane，这是一种有效的抑制剂铜绿假单胞菌 PqsA 的抑制剂，它是多药耐药（MDR）铜绿假单胞菌中一种保守的主群体感应（QS）调节剂。这种化合物竞争性地结合 anthranilyl-CoA 合成酶 PqsA 在 anthraniloyl-AMP 结合域，这一结合域尚未被已知的抑制剂利用。值得注意的是，norharmane 能显著抑制多药耐药铜绿假单胞菌毒力因子绿脓菌素（87%）和生物膜形成（80%）的产生。此外，norharmane 能够增强多粘菌素 B 对细胞培养物和动物模型中多药耐药铜绿假单胞菌的活性。总之，这些结果表明 norharmane 可能是一种有效的辅助药物，用于对抗多抗生素细菌耐药性。
