Abstract 17004: Five-coordinate H64Q Neuroglobin as a Ligand-trap Antidote for Carbon Monoxide Poisoning
Background: Carbon monoxide (CO) poisoning is a leading cause of poisoning deaths worldwide, with no available antidotal therapy. Neuroglobin (Ngb) is a six-coordinate hemoprotein, with the heme iron coordinated by two histidine residues. We mutated the distal histidine to glutamine (H64Q), combined with three surface thiol substitutions, forming a five-coordinate heme protein (Ngb-H64Q-CCC). This molecule exhibits an unusually high affinity for gaseous ligands, with a P50 value for oxygen of 0.05 mmHg. This finding informs our primary hypothesis that a mutant five-coordinated Ngb binds CO with very high affinity and can be developed as a novel specific antidotal therapy to bind, chelate and eliminate CO for the treatment of CO poisoning.
Methods and Results: We measured CO binding affinities from recombinant Ngb molecules and from hemoglobin (Hb) using laser flash photolysis. It was found Ngb-H64Q-CCC bound CO almost 500 times stronger than Hb. We evaluated CO transfer from carboxy-Hb (CO-Hb) to Ngb-H64Q-CCC under anaerobic and aerobic conditions. The half-lives of CO dissociation from free Hb and red blood cells in the presence of Ngb-H64Q-CCC were calculated via single exponential fits. Incubation of Ngb-H64Q-CCC with 100% CO-saturated cell–free or human red blood cell-encapsulated Hb reduced the half-life of carboxy-Hb (CO-Hb) to 0.11 and 0.41 minutes respectively, compared with 222 and 99 minutes in air. In a moderate CO poisoning mouse model, after 50 minutes of CO exposure blood CO-Hb levels plateaued at 64 ± 1%, which dropped at the first 5min after CO stopped by an average of 35.0 ± 2.1% in Ngb-H64Q-CCC treated group versus 13.3 ± 0.6% in PBS group and 27.4 ± 1.6% in the group that received 100% oxygen inhalation (P < 0.05), followed by rapid renal elimination of CO-bound Ngb-H64Q-CCC. Moreover, Ngb-H64Q-CCC infusion in a lethal CO poisoning mouse model significantly lowered mortality rate (20% in Ngb-H64Q-CCC vs. 100% in PBS group) and recovered heart rate and blood pressure.
Conclusion: The current studies identify a novel specific antidotal therapy for CO poisoning that rapidly scavenge CO from RBCs and tissues within minutes, and improve survival as well, providing for a potential paradigm changing approach to the most common human poisoning.
Author Disclosures: L. Wang: Research Grant; Significant; 1R01HL125886. I. Azarov: None. J.J. Rose: None. Q. Xu: None. X.N. Huang: None. A.M. Belanger: None. Y. Wang: None. L. Guo: None. C. Liu: None. K.B. Ucer: None. C.F. McTiernan: None. C.P. O’Donnell: None. S. Shiva: None. J. Tejero: Research Grant; Significant; 1R01HL125886. D.B. Kim-Shapiro: None. M.T. Gladwin: Research Grant; Significant; 1R01HL125886.
- © 2016 by American Heart Association, Inc.