A thiol-reactive Ru(II) ion, not CO release, underlies the potent antimicrobial and cytotoxic properties of CO-releasing molecule-3

Abstract

Carbon monoxide (CO)-releasing molecules (CORMs), mostly metal carbonyl compounds, are extensively used as experimental tools to deliver CO, a biological ‘gasotransmitter’, in mammalian systems. CORMs are also explored as potential novel antimicrobial drugs, effectively and rapidly killing bacteria in vitro and in animal models, but are reportedly benign towards mammalian cells. Ru-carbonyl CORMs, exemplified by CORM-3 (Ru(CO)3Cl(glycinate)), exhibit the most potent antimicrobial effects against Escherichia coli. We demonstrate that CORM-3 releases little CO in buffers and cell culture media and that the active antimicrobial agent is Ru(II), which binds tightly to thiols. Thus, thiols and amino acids in complex growth media – such as histidine, methionine and oxidised glutathione, but most pertinently cysteine and reduced glutathione (GSH) – protect both bacterial and mammalian cells against CORM-3 by binding and sequestering Ru(II). No other amino acids exert significant protective effects. NMR reveals that CORM-3 binds cysteine and GSH in a 1:1 stoichiometry with dissociation constants, Kd, of about 5 μM, while histidine, GSSG and methionine are bound less tightly, with Kd values ranging between 800 and 9000 μM. There is a direct positive correlation between protection and amino acid affinity for CORM-3. Intracellular targets of CORM-3 in both bacterial and mammalian cells are therefore expected to include GSH, free Cys, His and Met residues and any molecules that contain these surface-exposed amino acids. These results necessitate a major reappraisal of the biological effects of CORM-3 and related CORMs.

Mycobacteria tolerate carbon monoxide by remodelling their respiratory chain

“…The difference between these findings may result from the use of CORM-3 for CO delivery in the former study. CORM-3 is known to exert cytotoxic effects independent of CO, making the role of cytochrome bd-I oxidase in CO resistance in E. coli uncertain (12). As such, our current work represents the first definitive evidence that bacterial cytochrome bd oxidases display inherent resistance to CO.…”

“…Despite the suggestion that CO has antibacterial potential against pathogenic mycobacteria and numerous other bacterial species (5, 13, 46), surprisingly little is known about physiological and biochemical effects of gaseous CO on the bacterial cell. The uncertainty regarding the effects of CO on bacteria is confounded by the fact that much of the work testing the effects of CO has been performed using CORMs, which have antibiotic activity in addition to the effects of CO release (12). In this work we have shown that M. smegmatis has a high level of resistance to CO that requires relatively few changes to its proteome.…”

“…Studies treating bacteria with CO-releasing molecules (CORMs) have reported more acute toxicity and a bactericidal mode of action, which was attributed to CO (10, 11). However, subsequent analysis of the effect of CORMs strongly suggests that this toxicity is largely due to the transition metal complex that constitutes many CORMs, rather than toxicity of CO (12, 13). Further investigation is required to determine the extent to which CO is toxic to bacteria, the molecular targets of toxicity, and how bacteria are able to grow at high partial pressures of CO.…”

Cytotoxicity of Mn-Based photoCORMs of Ethynyl-α-Diimine Ligands Against Different Cancer Cell Lines: The Key Role of CO-Depleted Metal Fragments

“…The role of thiols in rich media was not explained until recently in a study that demonstrated that active antimicrobial agent of CORM-3 is not CO but Ru 2+ , which binds tightly to thiols [58]. Thus, thiols, amino acids and other sulphur-rich species in complex growth media protect bacteria against CORM-3 by binding and sequestering the metal ion.…”

Design and Biological Evaluation of Manganese‐ and Ruthenium‐Based Hybrid CO‐RMs (HYCOs)

“…[4a] However,t he kinetics measured for the Ru-based hybridsw ere significantly faster than for their Mn-based analogues.T his suggests that under our conditions, Ru-based HYCOs are less stable than their Mn-based counterparts, whatever the nature of the ligand. [19] Althoughn oc lear explanation was apparent, compounds incorporating linker B exhibit faster kinetics of CO release.…”