Overcoming antimicrobial resistance of bacteria producing MCRS by metal-based drugs

Abstract

Antimicrobial has been regarded as one of the most successful drugs, and has saved innumerable lives and revolutionized medicine in many respects. However, antimicrobial resistance (AMR) is destroying the bloom and then turns to be the worldwide crisis. Currently, a plasmid-borne transmissible colistin resistance gene (mcr) might be the toughest problem because it facilitates the desensitization of colistin, the last-line antibiotic, against mcr carrying bacteria. Once carrying the drug-resistance gene, the bacteria will desensitize colistin with a MIC from 0.25-1 μg ml-1 (colistin-sensitive) to 4-32 μg ml-1 (colistin-resistant). Up till now, no effective therapy is available for clinical treatment of these bacterial infections. Silver/gold-based drugs such as auranofin (®Ridaura), have long been used in clinic. In this thesis, silver nitrate (and silvadene cream, silver nano-particles) and auranofin (and aurothioglucose, aurothiosulfate, aurothiomalate, gold nano- particles) are repurposed to restore activities of colistin against multiple bacteria carrying mcr-1, as well as other mcr genes, with the FICI of 0.025 (auranofin, mcr-1) or 0.375 (silver nitrate, mcr-1) respectively. Importantly, they can slow down the speed of mcr-1 gene evolution by limiting 70% overexpression of MCR -1. Combinations of these metallo-agents with colistin can also reduce bacteria loads in liver and spleen (over 20-fold) in murine peritonitis model (as well as in Shigella-HepG2 invasion assay) and then significantly increase the survival ratio of mice infected with mcr-1 carrying bacterial pathogens. All mice can be rescued upon administration of 0.5 mg kg-1 colistin and 0.25 mg kg-1 auranofin, prompting their potentials to be further developed. Herein, silver nitrate prefers to serve in the treatment for non-internal infection, whereas auranofin can be used for infection internally. These two classes of drugs could be complementary. Biophysical data revealed that all Zn2+ cofactors of MCR-1 can be replaced by Ag+/Au+ within 60 minutes, which might attribute to their higher affinities than Zn2+ (over 10-fold). The considerable interaction was further observed in vitro and in vivo based on MALDI-TOF-MASS and CETSA assays, which prompt the significance of drug metabolism of silver/gold (I)-based compounds. X-ray structures of metal-bound proteins confirm the replacement of Zn2+ by Ag+/Au+. An unprecedented tetra-nuclear of silver cluster is firstly noted in the active pocket of MCR-1 and bridging critical residues in the area. Other mechanisms are also observed, including interference in the binding of phosphorylethanolamine (pEA) substrate to the enzyme and possible enhancement in the rigidity of several critical residues. All contribute to the inactivation of MCRs enzyme and then re-sensitization of bacteria carrying mcr genes against colistin. The study provides crucial information for combating superbug infections and contributes to a referred basis for development of more effective antimicrobial drugs.