Development of a carbon monoxide scavenging hemoprotein as a novel antidotal therapy to treat inhaled CO poisoning

PROJECT SUMMARY/ABSTRACT Accidental carbon monoxide (CO) poisoning is the leading cause of human poisoning in the United States, resulting in approximately 50,000 cases and at least 1,500 deaths annually. No point-of-care antidotal therapy exists for CO poisoning to date, and conventional treatments are limited to inhalation of 100% normobaric oxygen

or hyperbaric oxygen. While these therapies enhance CO clearance, delays in patient diagnosis and transport contribute to excess morbidity and mortality. Consequently, a fast-acting CO scavenger that can be deployed in the field, ambulance, or emergency room could significantly increase survival and long-term outcomes for

patients. Given that CO binds tightly to ferrous heme, our lab seeks to develop a hemoprotein-based CO scavenger that can bind and eliminate CO as a novel therapy for CO poisoning. Based on preliminary studies of recombinant hemoproteins, we have identified four key criteria for a safe and efficacious hemoprotein-based

CO scavenger: (1) high (nanomolar) CO affinity to maximize CO scavenging from physiological heme sites, (2) CO selectivity to minimize competitive inhibition by oxygen binding, (3) thermal and chemical stability to prevent heme release and adverse reactivity, and (4) redox stability of the Fe(II) heme to prevent autooxidation

to the inactive, Fe(III) heme state. Early investigations of the regulator of CO metabolism (RcoM) protein, a CO- sensing transcription factor from soil microbes, suggest that this protein exhibits high CO affinity and unprecedented selectivity for CO over oxygen. The primary objective of this proposal is to develop RcoM into a

safe and efficacious CO scavenger that will serve as an improved therapeutic treatment for CO poisoning. In Aim 1, we will utilize in vitro spectroscopic methods developed in our lab to identify 1) the minimum functional RcoM subunit, and 2) key amino acid residues that confer high CO affinity, selectivity, and heme stability. In

addition to characterizing basic biochemical properties, we will assess the ability of recombinantly expressed RcoM variants to scavenge CO from hemoglobin in CO-saturated red blood cells in vitro. In Aim 2, we will evaluate the safety and efficacy of two recombinant RcoM truncates in vivo. We will assess systemic and organ-

specific effects of intravenous RcoM delivery in healthy mice in vivo and quantify the ability of RcoM to reverse hemodynamic collapse and prevent death in a preclinical mouse model of CO poisoning previously developed in our laboratory. Completion of the proposed aims will advance our fundamental understanding of hemoprotein

ligand selectivity while also advancing the translational development of a novel antidotal therapy to treat inhaled CO poisoning. These outcomes, in addition to career development, mentored training, and didactic coursework, will ultimately provide me with the technical expertise, background knowledge, and leadership skills necessary

to accomplish my long-term academic career goal of directing a research team to study CO-dependent signaling mechanisms relevant to human health and disease.