Therapeutic Functions of tDodSNO: Nitric Oxide Donor Activity and CaMKII Protein Interaction

Cardiovascular disease is a leading cause of death worldwide. The heart and vascular system are complex and highly regulated with various signalling molecules, such as nitric oxide (NO), playing a crucial role in vasodilation. Dysregulation of NO can lead to various pathology conditions and cell death through oxidative stress, disturb energy metabolism, DNA damage, activation of polymerase or dysregulation of systolic Ca⁺².Therfore, NO is produced by nitric oxide synthase enzyme (NOS), which converts the amino acid L-arginine into NO and citrulline in the presence of oxygen (O₂) and cofactor Nicotinamide Adenine Dinucleotide Phosphate (NADPH).

NO can bind to a haem group in soluble guanylate cyclase (sGC), which activates cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG). Additionally, at high concentration NO is involved in protein nitrosylation and post translational modification, as well as oxidative stress and peroxynitrite formation. CAMKII (Calcium/Calmodulin-dependent Protein Kinase II) is a protein involved in Ca²⁺ handling and cardiac contractility. It is

believed that NO regulates CaMKII through S- nitrosylation, which involves adding NO to the protein’s cysteine residue.

There is growing interest in developing a new medication to treat cardiovascular disease involving NO donor and NO/CaMKII signalling pathway using NO donors’ drugs. However, current NO donors are associated with systemic side effects, necessitating the development of new medications with fewer drawbacks. Our approach is to use the photolabile NO donor tDodSNO (tert-dodecane S-nitrosothiol), photoactive compound with higher metabolic stability.

We initially characterized the effect of photoactivation on NO release from tDodSNO using the NO-induced oxidation of oxy-myoglobin (MbO₂) to met-myoglobin (metMb). Experimental results identified tDodSNO (100μM) as a photolabile agent, with NO release increasing upon photoactivation. A safe concentration of tDodSNO was then established by exposing A549 cells to tDodSNO with different concentrations, with and without photoactivation, and driving (maximum inhibitory concentration) IC₅₀ values. No significant difference in IC₅₀ observed upon tDodSNO photoactivation also, the drug found to be safe at concentration below 10 µM when exposed to 20W/m² white light for 30-60 minutes. The effect of NO release from tDodSNO (100μM) diluted in either DMSO or Ca²⁺on CaMKII activation was then determined using the fluorescent construct Camui, a model for CaMKII activation. Here no significant difference in Camui activation were observed using different time and light intensities. These project findings expand our understanding of tDodSNO and its potential for use in the treatment of cardiovascular diseases.