The therapeutic potential of signaling gases in COVID-19

Coronavirus disease 2019 (COVID-19) is an extremely contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID-19 pandemic has infected over 764 million people and caused the death of almost 7 million people globally.1 SARS-CoV-2 is a large, enveloped, positivesense, single-stranded RNA virus that enters host cells via the angiotensin-converting enzyme 2 receptors that are abundantly expressed on the respiratory epithelium. Once infected, host cells initiate an immune response resulting in the local recruitment of T-lymphocytes, monocytes, and neutrophils which in turn emits an assortment of cytokines. In most instances, the local immune response resolves the infection leading to mild to moderate flu-like symptoms such as fever, headache, cough, and fatigue. However, in severe COVID-19, overactivation of the immune system triggers a cytokine storm typified by the massive release of proinflammatory cytokines that mediates widespread lung inflammation and the development of pneumonia that can progress to acute respiratory distress syndrome. Moreover, the ensuing rise in circulating cytokines causes endothelial dysfunction, vasospasm, and thrombosis leading to vascular occlusion, multiorgan failure, and death. While the development of vaccines and antivirals have dramatically reduced SARS-CoV-2 infection, disease severity, and mortality, adaptive mutations in the viral genome generates viral variants that may limit the effectiveness of currently available antivirals and vaccines, underscoring the demand for additional therapeutic options in treating this deadly disease.

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) are small, lipid soluble gaseous molecules that were historically viewed as highly noxious environmental hazards. However, research conducted in the last few decades established that these gases are generated endogenously and serve as important signaling molecules in a multitude of physiological processes.2 NO is generated from arginine by a collection of enzymes known as NO synthase (NOS): neuronal NOS plays a role in synaptic plasticity and the central control blood pressure, inducible NOS is a critical player in host defense, and endothelial NOS preserves vascular health. CO is produced from heme by two distinct isoforms of heme oxygenase. Heme oxygenase-1 is a ubiquitously distributed and highly inducible isozyme that functions in an adaptive manner to maintain cellular redox balance and function, while heme oxygenase-2 is a constitutively expressed isoform that serves a key role in neurotransmission. H₂S is largely generated from cysteine by three different enzymes (cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfur transferase) that exhibit selective tissue distribution. These three signaling gases elicit comparable cellular and physiological functions often involving similar molecular targets. Intriguingly, emerging work has documented a deficiency in circulating levels of signaling gases in patients with COVID-19 compared to non-infected controls.3 Their low availability in COVID-19 may reflect reduced synthesis in response to inflammation and/or increased consumption by reactive oxygen species. Furthermore, diminished NO and H₂S levels have been identified as independent prognostic biomarkers of COVID-19 infection.3

Signaling gases possess a broad spectrum of antiviral activity and serving as part of the innate immune system they constitute the first line of defense against invading pathogens. NO directly inhibits the replication and entry of viruses into the host by modifying the viral replication machinery or host proteins. Recently, NO was shown to inhibit the replication of SARS-CoV-2 and the cytopathic effect of the virus.4 Enzymatic assays suggest that the antiviral effect is mediated via the inhibition of SARS-CoV-2 3CL protease activity via the nitration of cysteine in the active site of the enzyme. Phase 2 clinical studies have also corroborated the antiviral action of NO in patients with mild COVID-19.5 H₂S also acts as a direct antiviral agent against a series of enveloped RNA viruses by inhibiting membrane fusion and replication and may be effective against SARS-CoV-2 by suppressing the expression of transmembrane protease serine 2, a surface protein involved in viral entry.6 Furthermore, CO inhibits the replication of viruses by activating the cellular 3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway and repressing the activation of nuclear factor-κΒ.7 Thus, the use of signaling gases may be beneficial in fighting the initial stages of SARS-CoV-2 infection.

The vascular dysfunction and organ damage that occurs in severe COVID-19 is triggered by the onset of a cytokine storm that unleashes an uncontrolled wave of proinflammatory cytokine production and inflammation. Thus, strategies that moderate the force of this inflammatory storm represent a promising option for reducing the severity of disease. Significantly, signaling gases may dampen the hyperinflammation seen in patients with severe COVID-19. All three gases elicit potent anti-inflammatory effects and promote the repair of injured tissues.8 They suppress the synthesis of a wide range of pro-inflammatory cytokines by blocking the activation of nuclear factor-κΒ and stimulate the expression of the anti-inflammatory cytokine, interleukin-10. In addition, they downregulate the expression of adhesion molecules on both endothelium and leukocytes, and prevent the recruitment, infiltration, and activation of leukocytes within blood vessels. They also support the resolution of inflammation by stimulating the formation of a host of pro-resolution mediators. These gases also induce the repolarization of pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype that drives tissue repair. By blocking apoptosis, NO, CO, and H₂S also limits the extent of tissue damage caused by an inflammatory and oxidative environment.

A principal cellular target of the cytokine storm is the vascular endothelium.9 Pro-inflammatory mediators increase vascular permeability, induces capillary leakage, and unleashes another barrage of inflammatory cytokine secretion by endothelial cells (ECs). They also stimulate leukocyte recruitment and infiltration and can lead to EC death, which further increases vascular permeability. The endothelium also undergoes a prothrombotic transformation involving the loss of the glycocalyx and the synthesis of various effectors that stimulate platelet adhesion, complement activation, and fibrin formation. In addition, the inflammatory environment increases oxidative stress and reduces NO bioavailability resulting in impaired endothelium-dependent vasodilation. Endothelial injury and dysfunction are widespread encompassing many organs and may be the underlying mechanism for both pulmonary and extrapulmonary symptoms of COVID-19. In the lung, COVID-19-induced endothelial damage and dysfunction compromises barrier function leading to capillary leak and pulmonary edema. EC death also causes loss of vascular integrity resulting in alveolar hemorrhage, while EC malfunction stimulates thrombosis and fibrin formation causing vascular occlusion and ultimately producing respiratory failure.

Notably, all three gasotransmitters elicit beneficial effects in the circulation and lung.10 They all act as vasodilators and increase NO bioavailability by elevating endothelial NOS expression and/or decreasing oxidative stress. In addition, they restore endothelium-dependent vasodilation in various pathological states and prevent damage to ECs exposed to various inimical stimuli. Moreover, potent antithrombotic effects have been noted with NO, CO, and H₂S in several preclinical models of thrombosis. In the lung, all three gases improve ventilation by serving as bronchodilators. They also preserve epithelial barrier function by limiting apoptosis and reduce lung injury during mechanical ventilation. This latter point is noteworthy as many hospitalized patients with COVID-19 require invasive mechanical ventilation which comes with a higher risk of lung damage, thrombosis, secondary infection, and mortality. Strikingly, these three gases evoke salutary effects in animal models of infection, inflammation, acute lung injury, and cardiovascular disease, underscoring the potential therapeutic importance of restoring signaling gas levels in COVID-19 patients.

Multiple approaches may be used to deliver signaling gases in COVID-19.10 Inhaled NO is approved for use in persistent pulmonary hypertension in newborns and has been granted emergency access by the U.S. Food and Drug Administration for use in COVID-19. Initial studies reported variable effects of inhaled NO on pulmonary function and outcomes in COVID-19 patients likely due to differences in dosage regimens, patient characteristics, and infection stage and severity. Several clinical trials are presently underway, and they may further clarify the dosing requirements and efficacy of inhaled NO in distinct COVID-19 patient populations. Although inhalation of CO has been effectively utilized in numerous animal studies, its translation to the clinic has lagged due to safety concerns and the use of suboptimal dosing regimens. Similar concerns related to toxicity and the feasibility of achieving adequate dosing schedules has hindered the clinical use of inhaled H₂S. In addition, gas inhalation therapy restricts the actions of gases to the lung and consequently does not address other organs that are affected by COVID-19. In this respect, the use of donor molecules provides an avenue for the systemic delivery of signaling gases. Numerous NO-, H₂S- and CO-releasing molecules have been synthesized that possess distinct biophysical properties, half-life, and release kinetics. In addition, gas-releasing molecules have been developed that liberate a controlled amount of gas in response to specific environmental stimuli. Furthermore, the use of organic-based click-and-release prodrugs that employ a chemical reaction to generate a specific gas provides another modality for systemic gas delivery. The incorporation of gases into polymeric nanoparticles that allows for stable, sustained release of the gas in a targeted manner is also under investigation. Moreover, dietary approaches may be utilized to raise circulating levels of NO and H₂S. Recent work has highlighted the utility of nitrate supplementation with beet juice to raise NO levels and improve endothelial function and blood pressure in patients with hypertension.11 Alternatively, ingestion of vegetables rich in organosulfur compounds such as garlic, onions, and broccoli may restore plasma H₂S levels in patients with COVID-19. The supplementation of diets with precursor amino acids such as arginine and cysteine may also be used to augment circulating concentrations of NO and H₂S, respectively.

Aside from increasing the concentration of signaling gases in COVID-19, the biological potency of these gases could be enhanced. Since many of the effects of these gases are mediated by a rise in intracellular 3',5'-cyclic guanosine monophosphate, strategies that target this cyclic nucleotide may be helpful. In particular, the use of phosphodiesterase type 5 inhibitors such as sildenafil, which antagonizes the hydrolyses of 3',5'-cyclic guanosine monophosphate, will potentiate the effects of signaling gases. Interestingly, sildenafil also stimulates the production of signaling gases making it a particularly attractive drug.12 In addition, highly potent stimulators of soluble guanylate cyclase that amplify the biological actions of NO and CO have been developed and may be used in treating the respiratory and vascular complications associated with COVID-19. While targeting a single signaling gas may be effective, a more holistic approach that restores the concentration of all three signaling gases may be advantageous. However, the interaction between the signaling gases is extremely complex influencing both the synthesis and biological actions of the signaling gas necessitating close monitoring of patients receiving any of these gases. Finally, other gaseous molecules including ammonia, hydrogen gas, sulfur dioxide, and methane may also be impacted by COVID-19, and this could contribute to disease progression.

In conclusion, signaling gases represent a promising therapy to mitigate viral infection, inflammation, endothelial dysfunction, thrombosis, and organ failure in COVID-19. These gases can be given via multiple delivery vehicles, including inhalation, synthetic gas donors, gas-encapsulated nanoparticles, gas-boosting agents, and dietary supplementation. This gas-based remedy may provide a robust approach in preventing the devastating clinical consequences of this ongoing global pandemic.

This work was supported by the National Institutes of Health, National, Heart, Lung, and Blood Institute, No. R01HL149727.