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Abstract
Despite tremendous progress in modern medicine in the last century and the successful obliteration of many diseases, there are still many diseases challenging human health, and consequently, there is a need for the development of new drugs. Ruthenium-based complexes have been widely examined for their anti-cancer and anti-microbial properties. Due to their unique properties, the interactions of ruthenium complexes with different biological targets, particularly with nucleic acids (DNA and RNA) have been extensively investigated. However, the protein binding and enzyme inhibition properties of ruthenium complexes are less well explored. This thesis aimed at exploring the enzyme inhibition properties of a class of inert oligonuclear polypyridylruthenium(II) complexes (Rubbn) containing the flexible linking ligand bbn, {where bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 12 and 16)} and particularly in their potential as novel anti-parasitic agents.
A series of mono-, di-, tri- and tetra-nuclear complexes have been synthesised and their ability to inhibit acetylcholinesterase enzymes (AChEs) in various parasite extracts was examined. The Rubbn complexes selectively inhibited the acetylcholinesterase (AChE) at sub-micromolar concentrations (IC₅₀< 1µM), but showed no inhibitory activity against two other enzymes, alkaline phosphatase (AP) and phosphodiesterase (PDE). The ruthenium complexes showed differential activity against the different molecular forms and orthologues of AChE that exist in the various parasites. The in vitro susceptibility of the Rubbn complexes against different life stages of Schistosoma mansoni (S. mansoni) and Trichuris muris (T. muris) was investigated. The complexes showed excellent activity against each of the intra-mammalian stages of both parasites and the activity of the complexes was correlated with AChE inhibition, particularly against S. mansoni. A series of studies was conducted to understand the mechanism of the anti-parasitic action of the Rubbn complexes. Treatment with Rubbn complexes caused paralysis in the parasites due to AChE inhibition. At sub-lethal concentrations, the Rubbn complexes significantly affected the surface and neuronal AChE activity whereas the AP and PDE enzyme activities remained unaffected. The ruthenium complexes also damaged the tubercules (glycogen stores) of the parasite and inhibited glucose uptake at sub-lethal concentrations, further supporting AChE as the mechanism of anti-parasitic activity.
The cytotoxicity of Rubbn complexes was examined against the eukaryotic cell line H69, and were shown to be less toxic compared to the previously used AChE inhibitor drug dichlorvos. The in vivo toxicity of the most effective complexes (Rubb₁₂-tri, Rubb₇-tnl and Rubb₁₂-mono) in the in vitro assays were evaluated in healthy mice. Rubb₁₂-tri and Rubb₇-tnl had maximum tolerated doses of 4 mg/kg and 10 mg/kg, respectively, by intravenous administration, whereas Rubb₁₂-mono was tolerated at a 10 mg/kg oral dose.
Finally, the in vivo efficacy of the Rubbn complexes was investigated in infected mice. The Rubb₁₂-tri complex significantly reduced the worm burden in S. mansoni infected mice and recovered worms displayed modest but significant reduction in surface AChE activity. The ruthenium complexes did not affect the egg burden in mice, however the eggs collected from Rubb₁₂-tri and Rubb₇-tnl treated mice had significantly reduced hatching abilities and abnormal morphology, compared to eggs collected from control mice. On the other hand, Rubb₁₂-mono treatment significantly reduced both the worm burden and egg burden in T. muris infected mice.
Serum protein binding properties of Rubbn complexes were investigated in order to confirm that the decrease in anti-schistosomal activity observed when the assays were carried out in serum-supplemented media was due to the strong interaction of the Rubbn complexes to serum proteins. The ruthenium complexes showed high levels of serum protein binding, and specifically high affinity for HSA, especially the tri- and tetra-nuclear complexes.
To further understand the enzyme inhibitory properties of the Rubbn complexes, the effect of the ruthenium complexes on the activities of the phosphatase enzymes MKP-1, MKP-3 and CDC25B were examined. The Rubbn complexes showed moderate selectivity against MKP-3 compared to MKP-1 and CDC25B. The inhibitory activity of Rubbn complexes against the phosphatase enzymes was shown to be MKP-3 > MKP-1 > CDC25B. The results of the study also suggested that the tetra-nuclear complexes have the potential to regulate the amount of phosphatase enzyme (MKP-1) synthesised in live cancer cells. However, the Rubbn complexes were less active (between 60- and 200-fold) against the phosphatase enzymes,compared to parasite AChE. The combined results of the enzyme inhibition studies highlight the selectivity of the Rubbn complexes for parasite AChE.
The results of this study demonstrates the significant potential of the oligonuclear polypyridylruthenium(II) complexes as anti-parasitic agents. More importantly, to the best of my knowledge, this is the first investigation on inhibitory properties of Rubbn complexes against parasite AChEs and the information provided in this thesis will advance our knowledge in the understanding of enzyme inhibition properties of ruthenium-based complexes and in developing them as therapeutic agents.