‘Carbon-Monoxide-Releasing Molecule-2 (CORM-2)’ Is a Misnomer: Ruthenium Toxicity, Not CO Release, Accounts for Its Antimicrobial Effects

Abstract

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological ‘gasotransmitter’, in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (<0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 μM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 μM; His, Kd 130 μM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 μM; GSSG, Kd 25,000 μM). The toxicity of low, but potent, levels (15 μM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.

Head‐to‐Head Comparison of Selected Extra‐ and Intracellular CO‐Releasing Molecules on Their CO‐Releasing and Anti‐Inflammatory Properties

“…It was found that the ruthenium-based CORM-2 and CORM-3 mainly liberate CO 2 (besides only little CO), confirming the results of Poole and co-workers that the use of these first-generation CORMs as reference CORMs may no longer be appropriate. [12] Noteworthy, no CO 2 was released from the iron-based AT-and ET-CORMs after enzymatic hydrolysis. Based on the aforementioned observation that extraand intra-cellular CO delivery yield similar anti-inflammatory properties, our study suggests that the use of specific membrane associated enzymatic activity may pave the way for tissue-targeted CO delivery.…”

“…[11] Also, Poole and co-workers have recently shown that CORM-2 only releases negligible amounts of CO (< 0.1 mol CO per mol CORM-2) and concluded that the biological effects of CORM-2 and related CORMs should be re-examined in the light of these data. [12]…”

A bioinspired carbon monoxide delivery system prevents acute kidney injury and the progression to chronic kidney disease

“…5 ). Southam, M. et al also showed that CO derived from the CORM-2 was nearly exhausted after a 4 h period of incubation in DMSO [ 67 ]. We further confirmed that the treatment of HK-2 cells with iCORM-2 did not activate HIF-1α, AMPK, and Nrf-2, all of which were activated by the CORM-2 treatment (S. Fig.…”

CORM-3 induces DNA damage through Ru(II) binding to DNA

“…It is still not clear how Ru complexes enter cells, though one possibility is that they use iron transport mechanisms [ 14 ]. Once inside cells, ruthenium is able to bind to proteins (via cysteine thiols [ 16 , 17 ] or histidines [ 18 ]), to free thiols such as glutathione [ 16 , 17 ], and also to DNA. In this behaviour it shows interesting differences as compared with platinum, which binds mainly to DNA, and tends to form square planar complexes, in particular linking the N7 positions of adjacent guanines, while ruthenium forms octahedral complexes [ 14 ].…”

“…Numerous studies have shown that CORM-3 is toxic to bacterial cells but shows little toxicity to mammalian cells, which has stimulated research on modes of action of the Ru-based CORMs that have focussed on the toxicity of CO. However, recently we showed that CORM-3, as well as the related molecule CORM-2, in fact release very little CO in standard growth conditions [ 16 , 17 , 22 ]. Furthermore, we showed that the bacterial toxicity is due to the ruthenium(II) ion and probably not to CO at all.…”