Denufosol Tetrasodium

The recent pandemic caused by SARS-CoV-2 has led the world to a standstill, causing a medical and economic crisis worldwide. This crisis has triggered an urgent need to discover a possible treatment strategy against this novel virus using already-approved drugs. The main protease (Mpro) of this virus plays a critical role in cleaving the translated polypeptides that makes it a potential drug target against COVID-19. Taking advantage of the recently discovered three-dimensional structure of Mpro, we screened approved drugs from the Drug Bank to find a possible inhibitor against Mpro using computational methods and further validating them with biochemical studies. The docking and molecular dynamics study revealed that DB04983 (denufosol) showed the best glide docking score, -11.884 kcal/mol, and MM-PBSA binding free energy, -10.96 kcal/mol. Cobicistat, cangrelor (previous computational studies in our lab), and denufosol (current study) were tested for the in vitro inhibitory effects on Mpro. The IC₅₀ values of these drugs were ∼6.7 μM, 0.9 mM, and 1.3 mM, respectively, while the values of dissociation constants calculated using surface plasmon resonance were ∼2.1 μM, 0.7 mM, and 1.4 mM, respectively. We found that cobicistat is the most efficient inhibitor of Mpro both in silico and in vitro. In conclusion, cobicistat, which is already an FDA-approved drug being used against HIV, may serve as a good inhibitor against the main protease of SARS-CoV-2 that, in turn, can help in combating COVID-19, and these results can also form the basis for the rational structure-based drug design against COVID-19.

T he life-shortening genetic disease cystic fibrosis (CF) is caused by mutations in the CF gene on both alleles, resulting in failure of a defective CF transmembrane conductance regulator (CFTR) glycoprotein to normally regulate chloride and bicarbonate flux at the airway surface. According to a leading theory of CF pathogenesis, the “volume depletion” hypothesis, this abnormal anion transport leads to reduced airway surface liquid, airway dehydration, and impairedmucociliary clearance, resulting in vulnerability to airway obstruction, microbial infection, and inflammation. In 1991, investigators at the University of North Carolina discovered that triphosphate nucleotides, such as adenosine5’-triphosphate and uridine-5’-triphosphate, stimulated chloride secretion in both normal and CF respiratory epithelia, offering a potential bypass mechanism for defective CFTR. Triphosphate nucleotides transduce a signal by binding to purinergic receptors on respiratory epithelial cells specifically responding to purine and pyrimidine nucleotides, termed P2Y2 receptors. This leads to release of intracellular calcium and activates calcium-dependent chloride channels (CaCCs) distinct from CFTR. In vitro tissue culture studies have confirmed that adenosine-5’-triphosphate and uridine5’-triphosphate can restore liquid transport in CF cultures within a few minutes, enhance tracheal mucus velocity in sheep models, and acutely increase sputum volume and mucociliary clearance in smokers. Additional actions of P2Y2 agonists also have been reported from cell culture experiments, including stimulation of ciliary beat frequency, increased mucin secretion from goblet cells and surfactant from type II alveolar cells, and inhibition of epithelial sodium absorption, the net sum of these plausibly resulting in improved airway hydration and increased mucociliary transport that should be beneficial in treating CF. Native triphosphate nucleotides are rapidly metabolized by ectonucleotidases in an autocrine/paracrine manner, however. This has led to the development of dinucleotides, INS365 and INS37217, that are more resistant to enzymatic degradation as assessed in 2 preclinical models: ex vivo exposure of the nucelotides to CF sputum samples and in vitro addition to human nasal ciliated epithelial cell cultures. In these models, these dinucleotides had sufficient stability to merit consideration for drug development, with INS37217 (later named denufosol) outperforming INS365. Denufosol’s