By Aaron Johnson
Jesus Tejero
More than 1,500 Americans die from carbon monoxide (CO) poisoning each year, and more than 50,000 seek emergency treatment.
Researchers at the University of Pittsburgh, along with colleagues at the University of Maryland, created a novel protein therapy for CO poisoning that could eventually be carried by emergency responders to immediately help patients. Their findings were published in the Proceedings of the National Academy of Science (PNAS) on Aug. 5.
Jesus Tejero, associate professor of medicine, and his lab at Pitt’s Heart, Lung, Blood, and Vascular Medicine Institute developed RcoM-HBD-CCC, a protein-based therapy for CO poisoning, with Mark Gladwin’s group at the University of Maryland.
RcoM-HBD-CCC is intravenously administered to leach CO from hemoproteins in blood, sequestering it until the body excretes the CO-bound protein in urine. Their findings show that RcoM-HBD-CCC provides a safe, rapid clearance of CO that is unparalleled by other heme-based therapeutics.
The researchers at Pitt and Maryland study the interactions between hemoproteins and their ligands, including CO, oxygen (O2), and nitric oxide (NO), and employ or modify them for therapeutic purposes.
RcoM, short for “regulator of CO metabolism,” is a CO sensor protein isolated from the bacterium Paraburkholderia xenovorans. These bacteria express the RcoM protein to sense nanomolar amounts of CO through their heme-binding domain. Once CO binds to the heme, it activates the other region of the protein, called the DNA-binding domain, to initiate gene expression related to CO metabolism. Tejero and Gladwin leveraged this interaction as a potential therapeutic for CO poisoning by using only an engineered version of the heme-binding domain (HBD), which gave rise to RcoM-HBD-CCC.
When we breathe in and fill our lungs with air, hemoglobin in red blood cells is loaded with oxygen. Then, as blood circulates through arteries, oxygen diffuses into tissues and sustains cellular respiration.
CO, in contrast, is toxic. This colorless, odorless gas is a byproduct of incomplete combustion of carbon sources, like gasoline, natural gas, and coal. CO binds to hemoglobin approximately 100-times stronger than it binds to O2. If we are exposed to CO during a fire, CO steals O2’s “seat” on hemoglobin and other heme proteins as it circulates through our bloodstream. The resulting oxygen deprivation in tissues, called hypoxia, leads to symptoms of dizziness, headache, shortness of breath, confusion and/or a loss of consciousness.
The faster CO is removed from the bloodstream, the better the outcome will be. The go-to treatment for CO poisoning is breathing 100% oxygen through a mask, which is widely available. In some cases, patients may recover in a hyperbaric oxygen chamber, which encapsulates patients, elevates atmospheric pressure, and provides 100% oxygen. But there are only about 300 facilities providing it in the United States.
Current therapies certainly decrease CO levels, but it remains difficult to fully clear, or scavenge, CO from the bloodstream and cells. CO can “bounce” from hemoglobin to other hemoproteins in the cell and abrogate their function, like those in mitochondria. CO exposure may lead to long-term neurological or cardiac deficits, so novel therapies that sequester and remove CO from circulation and tissues may prevent these harmful effects. RcoM is unique in this sense because of its affinity and unparalleled specificity for CO compared to other hemoproteins.
“If your protein binds CO with very high affinity, but it binds oxygen too, you have so much oxygen in the blood that it will [also] catch oxygen and will compete,” Tejero said.
Using a method called stopped-flow electronic absorption spectroscopy, the researchers demonstrate that RcoM-HBD-CCC binds CO nearly 50 times more tightly than hemoglobin.
“RcoM-HBD-CCC’s affinity for CO is so high, it will bind as soon as it gets into the bloodstream, so the limit is how fast you can infuse the protein and how much,” Tejero said.
RcoM-HBD-CCC’s specificity for CO over O2 is also unique. Hemoproteins, like RcoM and hemoglobin, that bind O2 also scavenge NO from circulation. NO stimulates vasorelaxation and increases blood flow. Up to this point, intravenously administered hemoproteins scavenge NO and cause the recipient to become hypertensive, which has precluded the development of prior hemoglobin-based therapies.
In the researchers’ preclinical models, the intravenous administration of RcoM-HBD-CCC had no hypertensive effect, which is a significant finding and a novel contribution to the field. As control, the researchers infused hemoglobin, which scavenges NO.
“[When we infuse hemoglobin], blood pressure goes up because it scavenges NO, but we see that infusing RcoM-HBD-CCC doesn’t increase the blood pressure,” said Tejero.
“This was quite significant because when people were trying to invent hemoglobin-based oxygen carriers or artificial blood…they always caused hypertension when they were infused because of NO scavenging.... High blood pressure is linked to worse outcomes, so those applications never got approved [by the Food and Drug Administration in the United States].”
Although still in development, RcoM’s unique properties are paving the way for future studies in Tejero’s lab.
“We think this [nonhypertensive effect] is very promising to use RcoM also as a base to develop artificial oxygen carriers…that’s the future of this project, and we are already working to make this into an oxygen carrier.”
Tejero and his colleagues aim to scale up the recombinant production of RcoM-HBD-CCC and gear up for phase I clinical trials.
“We think RcoM-HBD-CCC [will be] a treatment for CO poisoning…that can be infused as soon as possible…. Ideally, it will be something that’s stocked in fire departments or ambulances for fast responses to CO poisoning,” Tejero said.
Tejero and Gladwin are also board members of Globin Solutions, a Pitt spin-off biotechnology company focused on developing antidotes to CO poisoning and other medical countermeasures based on technology licensed from Pitt and National Institutes of Health.