Inert Dinuclear Polypyridylruthenium(II) Complexes as Antimicrobial Agents

With the widespread use of antibiotics, the continuous development of drug resistance by pathogenic bacteria has become a worldwide concern and there is a need for developing new classes of antimicrobial agents. Inert ruthenium complexes have been investigated for their anticancer activity, due to their ability to interact with DNA, but few studies of their antimicrobial properties have been undertaken. This thesis aimed at exploring the potential of a class of inert dinuclear polypyridylruthenium(II) complexes containing a flexible linking ligand, [{Ru(phen)2}(µ-bbn)]4+ {“Rubbn”; where phen = 1,10-phenanthroline; bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12, 14 or 16)}, as novel antimicrobial agents.The in vitro susceptibility of the Rubbn complexes against a range of pathogenic bacteria was screened through the determination of the minimum inhibitory concentrations (MIC), the minimum bactericidal concentrations (MBC) and time-kill curve assays. The Rubbn complexes showed excellent bactericidal activities against both drug-susceptible and drug-resistant bacterial strains. In particular, Rubb12, Rubb14 and Rubb16 showed the best activity with MIC values of 1 mg/L against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and 2-4 and 8-16 mg/L against Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), respectively. The relative MBC to MIC values indicated that Rubb12 and Rubb16 are bactericidal, and from the time-kill curve experiments, the ruthenium complexes can kill the bacteria within 2-6 hours. The cytotoxicities of Rubbn against mammalian cells including erythrocytes, leukemia, liver and kidney cell lines were also examined. The Rubbn complexes were shown to be non-toxic against these human eukaryotic cells at concentrations significantly greater than the corresponding MIC values.A series of studies aimed at understanding the mechanism of the antimicrobial action of the Rubbn complexes were carried out using fluorescence spectroscopy, flow cytometry and wide-field high-magnification fluorescence microscopy. It was established that the antimicrobial activity of the Rubbn complexes is correlated with the level of cellular accumulation, which in turn is controlled by the lipophilicity, charge and charge separation. The Rubbn complexes enter bacterial cells in an energy-independent manner, and significantly depolarise and permeabilise the cellular membrane. Cellular localisation studies showed that one of the lead compounds, Rubb16, preferentially binds bacterial RNA in vivo and accumulates at bacterial ribosomes. It is proposed that the specific targeting and condensation of ribosomes when concentrated as polysomes halts translation, thereby interrupting protein synthesis in actively growing bacterial cells.The DNA-selective dye DAPI was used in a wide-field high-magnification fluorescence microscopy study to show that incubation of Rubb16 at concentrations four-fold higher than the MIC value condensed the chromosomal DNA in E. coli cells. An in vitro NMR spectroscopy study demonstrated that Rubb7 selectively bound a non-duplex destabilised segment contained with the DNA sequence 5'-d(CGCGATAAGCCGC•5'-GCGGCATTACGCG) with high affinity. Fluorescence spectroscopy was used to confirm that the decrease in antimicrobial activity observed when the MIC assays were carried out in serum-supplemented broth was due to the strong binding of the Rubbn complexes with the human serum albumin (HSA) and transferrin. A series of ruthenium complexes showed stronger binding to HSA than apo-transferrin but comparable or less than with holo-transferrin, with the binding affinity to all three proteins decreasing in the order trinuclear > dinuclear > mononuclear. The dinuclear complex Rubb12 displaced warfarin from HSA, tentatively suggesting that the ruthenium complexes bind at or near the warfarin-binding site, Sudlow’s site 1.A preliminary in vivo toxicity and pharmacokinetic study of the Rubbn complexes was conducted using a murine model. Intramuscular administration gave the highest MTD for Rubb12, with no apparent toxicity being observed at a dose of 64 mg/kg. Rubb12 accumulated mainly in the liver, and the excretion rate of the ruthenium complex from the body was slow, suggesting that cumulative liver damage could be the major mechanism of toxicity. The potential of using a macrocyclic host called cucurbit[10]uril as a delivery system for the Rubbn complexes was also examined, and the results demonstrated that encapsulation of the ruthenium complexes did provide a small decrease in toxicity with the amount accumulating in the liver being significantly reduced.This study demonstrates the significant potential of the Rubbn complexes as a new class of antimicrobial agent. Furthermore, the information provided in this thesis on the biological interactions of cationic polynuclear metal complexes in bacterial and eukaryotic cells may also assist the general development of transition metal complexes as therapeutic agents