Novel ruthenium metal-based complexes as antimicrobial agents

Ryder, Steven (2023) Novel ruthenium metal-based complexes as antimicrobial agents. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Antimicrobial resistance (AMR) is becoming increasingly prevalent amongst clinically significant bacteria. The World Health Organization (WHO) has declared AMR as one of the greatest public health threats facing humanity. There has been a sharp decline in the number of new clinically approved antibiotics, with most new antibiotics being based on pre-existing antibiotic scaffolds. As a result, there is an urgent need for new novel ways to treat infections caused by AMR bacteria. There has been an increased focus on Ruthenium (Ru) complexes acting as antimicrobial agents. This is in part due to their biological compatibility, multiple oxidation states and bonding configuration allowing for specific geometries that are ideally suited for biological applications. This study evaluated the antimicrobial activity of 12 repurposed Ru complexes. Preliminary screening against a diverse selection of clinically significant bacteria identified Ru complexes 1 (C22H23Cl3N2SRu) and 7 ([Ru(NH3)6]Cl3) as potential lead candidates with the Ru complexes acted as bactericidal agents against S. aureus USA300 JE2 and P. aeruginosa PAO1 respectively. Eukaryotic cytotoxicity testing against HeLa and HEK 293T cell lines demonstrated Ru complex 7 exhibited no significant cytotoxic effects against both cell lines (p>0.05), whilst Ru complex 1 was significantly cytotoxic (p<0.05). S. aureus USA300 JE2 and E. coli EC958 were able to tolerate an 11-fold increase in MIC after long term incrementally increasing concentrations of Ru complex 1. Comparative genome analysis of S. aureus USA300 JE2 showed long term exposure to Ru complex 1 increased the rate of mutagenesis and led to 17 de novo mutations being identified within eight genes. Furthermore, significant gene expression changes in clpP, katA and norA were reported in S. aureus USA300 JE2 after exposure to Ru complex 1, indicating Ru complex 1 affected a wide array of cellular functions. The mechanisms of action for antimicrobials Ru complex 1 and 7 were investigated. Ru complex 1 displayed no significant outer membrane or inner membrane permeabilising effects, whilst Ru complex 7 caused no significant outer membrane permeabilising but did stimulate elevated depolarisation of the inner membrane in a number of bacterial species. Scanning electron microscopy confirm that both complexes appeared not to be directly targeting the outer membrane as no cellular morphological changes were observed. Cellular metal uptake studies using Ru complexes 1 and 7 showed elevated intracellular concentrations in S. aureus USA300 JE2 and P. aeruginosa PAO1 respectively compared to the exposure concentrations. Electrophoretic mobility shift assays (EMSA) and competitive binding assays showed that intracellular concentrations of Ru complexes 1 and 7 had a significant impact on DNA mobility and displacement of SYTO 9 from the SYTO 9/DNA complex. Exposure to Ru complexes 1 and 7 caused elevated but not significant levels of reactive oxygen species generation (ROS) in S. aureus USA300 JE2, P. aeruginosa PAO1 and E. coli EC958 The results of the thesis demonstrate the potential to use mononuclear Ru complexes as antimicrobial agents. Notably, the potent antimicrobial activity of Ru complex 7 against P. aeruginosa PAO1, coupled with low levels of cytotoxicity make this an ideal candidate for further in vivo investigation.