Machado Diana, Fernandes Laura, Costa Sofia S, Cannalire Rolando, Manfroni Giuseppe, Tabarrini Oriana, Couto Isabel, Sabatini Stefano, Viveiros Miguel
Unidade de Microbiologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade NOVA de Lisboa, UNL, Lisboa, Portugal.
Current affiliation: Laboratório de Diagnóstico Molecular Veterinário GeneVet, Algés, Portugal.
PeerJ. 2017 Apr 26;5:e3168. doi: 10.7717/peerj.3168. eCollection 2017.
Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone - PQQ4R), against by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux in reducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of the inner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future adjuvants of conventional therapy against and other Gram-negative bacteria, especially their multidrug resistant forms. Their major limitation is the high toxicity for human cells at the concentrations needed to be effective against bacteria. Their future molecular optimization to improve the efflux inhibitory properties and reduce relative toxicity will optimize their potential for clinical usage against multi-drug resistant bacterial infections due to efflux.
外排泵抑制剂引起了人们极大的兴趣，因为将其用作细菌化疗佐剂可以提高抗生素的细胞内浓度，并有助于对抗抗生素耐药菌的增加。在这项工作中，我们通过研究2-苯基喹啉外排抑制剂（4-(2-(哌嗪-1-基)乙氧基)-2-(4-丙氧基苯基)喹诺酮 - PQQ4R）的外排抑制能力、与抗生素联合使用时的协同活性，并将其与抑制剂苯基-精氨酸-β-萘酰胺（PAβN）和氯丙嗪（CPZ）的效果进行比较，描述了其对[具体细菌名称未给出]的作用模式。结果表明，PQQ4R与已知易被外排的抗生素以浓度依赖性方式协同作用，通过抑制其外排来降低它们的最低抑菌浓度（MIC）。实时荧光测定法表明，亚抑制浓度的PQQ4R促进溴化乙锭在细胞内的积累，抑制其外排，这与PAβN或CPZ类似，PAβN和CPZ是已知的革兰氏阴性菌外排泵抑制剂，但其临床应用因在临床和细菌学有效浓度下的毒性水平而受到限制。时间杀菌研究表明，杀菌浓度的PQQ4R具有快速的抗菌活性，伴随着细胞内ATP水平的快速下降。结果还表明，PQQ4R的作用模式涉及内膜电位的不稳定和ATP产生的损害，最终通过干扰外排系统所需的能量导致外排泵抑制。在杀菌浓度下，膜通透性增加，最终ATP完全耗尽导致细胞死亡。由于外排泵活性介导的耐药性很大程度上取决于质子动力势（PMF），像PQQ4R这样的PMF耗散剂可被视为未来针对[具体细菌名称未给出]和其他革兰氏阴性菌，尤其是其多重耐药形式的传统治疗佐剂。它们的主要局限性是在有效对抗细菌所需的浓度下对人类细胞具有高毒性。它们未来的分子优化以改善外排抑制特性并降低相对毒性，将优化其针对因外排导致的多重耐药细菌感染的临床应用潜力。
