Ruthenium, Not Carbon Monoxide, Inhibits the Procoagulant Activity of Atheris, Echis, and Pseudonaja Venoms

Abstract

The demonstration that carbon monoxide releasing molecules (CORMs) affect experimental systems by the release of carbon monoxide, and not via the interaction of the inactivated CORM, has been an accepted paradigm for decades. However, it has recently been documented that a radical intermediate formed during carbon monoxide release from ruthenium (Ru)-based CORM (CORM-2) interacts with histidine and can inactivate bee phospholipase A2 activity. Using a thrombelastographic based paradigm to assess procoagulant activity in human plasma, this study tested the hypothesis that a Ru-based radical and not carbon monoxide was responsible for CORM-2 mediated inhibition of Atheris, Echis, and Pseudonaja species snake venoms. Assessment of the inhibitory effects of ruthenium chloride (RuCl3) on snake venom activity was also determined. CORM-2 mediated inhibition of the three venoms was found to be independent of carbon monoxide release, as the presence of histidine-rich albumin abrogated CORM-2 inhibition. Exposure to RuCl3 had little effect on Atheris venom activity, but Echis and Pseudonaja venom had procoagulant activity significantly reduced. In conclusion, a Ru-based radical and ion inhibited procoagulant snake venoms, not carbon monoxide. These data continue to add to our mechanistic understanding of how Ru-based molecules can modulate hemotoxic venoms, and these results can serve as a rationale to focus on perhaps other, complementary compounds containing Ru as antivenom agents in vitro and, ultimately, in vivo.

The role of carbon monoxide, heme oxygenase 1, and the Nrf2 transcription factor in the modulation of chronic pain and their interactions with opioids and cannabinoids

“…69 Thus, a radical intermediate formed during carbon monoxide release from ruthenium (Ru)based CORM-2 is likely responsible for the CORM-2-mediated inhibition of diverse snake and lizard venoms. 70,71 Other findings also demonstrated that CORM-2 activates nonselective cation current in endothelial cells independently of carbon monoxide release, 72 and the potent antimicrobial and cytotoxic properties of CORM-3 are produced by Ru-based CORM-3. 73 Regarding the analgesic effects produced by CORM-2 and CORM-3 in animals with chronic pain, the treatments with inactive CO-RMs (iCORM-2 and iCORM-3) did not produce any antiallodynic and antihyperalgesic effects during neuropathic pain.…”

Modulation of Diverse Procoagulant Venom Activities by Combinations of Platinoid Compounds

“…In response to these new findings [ 7 , 8 ], it was subsequently reported that purified phospholipase A 2 isolated from bee venom was inhibited by CORM-2 via a CO-independent mechanism, with inhibition of the venom by CORM-3 (tricarbonylchloro(glycinato)ruthenium) likely also dependent on a Ru(II)-based mechanism [ 9 ]. Thereafter, anticoagulant metalloproteinase activity in venoms collected from mambas was found to be inhibited by CORM-2 by a similar CO-independent mechanism [ 10 ], and, finally, the procoagulant activity exerted by metalloproteinases and serine proteases was found to be inhibited by CORM-2 and ruthenium(III) chloride (RuCl 3 ) [ 11 ]. Thus, as suggested by the works of the past several months [ 9 , 10 , 11 ], it is the ruthenium species, not CO, that are binding to key anticoagulant/procoagulant venom enzymes in a heme-independent and perhaps irreversible fashion.…”

“…Thereafter, anticoagulant metalloproteinase activity in venoms collected from mambas was found to be inhibited by CORM-2 by a similar CO-independent mechanism [ 10 ], and, finally, the procoagulant activity exerted by metalloproteinases and serine proteases was found to be inhibited by CORM-2 and ruthenium(III) chloride (RuCl 3 ) [ 11 ]. Thus, as suggested by the works of the past several months [ 9 , 10 , 11 ], it is the ruthenium species, not CO, that are binding to key anticoagulant/procoagulant venom enzymes in a heme-independent and perhaps irreversible fashion.…”

“…The platinum (Pt)-based compounds cisplatin and carboplatin also bind histidine and methionine [ 20 ]. These binding characteristics of Ru compounds to specific amino acid residues may explain why CORM-2 and RuCl 3 separately have inhibited snake venom and isolated enzyme activities [ 1 , 2 , 3 , 4 , 5 , 6 , 9 , 10 , 11 ], as highly conserved histidines and disulfide bridges that are critical to function are found in snake venom metalloproteinases (MP) [ 21 , 22 ], snake venom serine proteases (SP) [ 23 , 24 ] and phospholipase A 2 (PLA 2 ) [ 25 , 26 ]. Taken as a whole, small molecular weight Ru-based or Pt-based compounds may inhibit anticoagulant/procoagulant snake venom activity by binding to a hereto unappreciated Achilles’ heel of highly conserved amino acid residues essential to functions shared across multiple enzyme types.…”

“…The structures of CORM-2, CORM-3, RuCl 3 and carboplatin are displayed in Figure 1 . It is of note that CORM-2 and RuCl 3 have been found to have similar or no inhibitory effects on various procoagulant venoms when tested separately [ 11 ]. This finding opened the possibility that the Ru-based and Pt-based compounds may bind to the same critical amino acid residue, with perhaps different affinity to different residues that are enzymatically important, or perhaps to more than two molecular sites on any given enzyme.…”