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Targeting iNOS and associated proteins in neutrophils: An in-silico and ex-vivo approach for anti-sepsis therapy
Sarvjeet Das1, Anshu Kumar1, Gaurang Agarwal1
1Inflammation Research Laboratory, Department of Life Science, Parul Institute of Applied Science, Faculty of Applied Sciences, Parul University, Vadodara, Gujarat 391760, India.
Background:
Previous reports from the lab have demonstrated the role of inducible nitric oxide synthase (iNOS) and associated proteins in sepsis-induced neutrophilic hyperinflammation. This study aimed to identify FDA-approved drugs that inhibit iNOS and associated proteins to attenuate neutrophil-induced excessive inflammation.
Methods:
An in-silico approach involving STRING and Cytoscape was used to map protein-protein interactions (PPI). The FDA-approved drugs that target these proteins were retrieved from DrugBank. Candidate drugs were identified from the DrugBank database using a target sequence of iNOS and associated proteins. The human iNOS and associated proteins were investigated for interacting partners using the STRING database (highest confidence >0.90). The UCSF Chimera and Discovery Studio were used to perform molecular docking and Molecular Dynamics (MD) simulation to evaluate drug binding affinities. Thirty sepsis patients and twenty healthy volunteers were enrolled. The effect of high-scoring candidate drugs was assessed under an ex-vivo model by isolating neutrophils from sepsis patients and healthy volunteers. Levels of reactive oxygen species (ROS), nitric oxide (NO), neutrophil extracellular traps (NETs) and phospho-p38MAPK activity following Phorbol 12-myristate 13-acetate (PMA), Escherichia coli, Staphylococcus aureus, and LPS stimulation in the presence or absence of candidate drugs were also evaluated.
Results:
The result of the PPI network was developed by using the Cytoscape plugin. The molecular docking and MD simulation analysis showed that dexamethasone and curcumin have high binding affinities to iNOS and associated proteins. Ex-vivo studies showed that neutrophils from sepsis patients had elevated ROS, NO, NET, and phospho-p38MAPK levels, which were significantly attenuated by dexamethasone + curcumin treatment (p < 0.01 for ROS, NO, and NET; p < 0.001 for phospho-p38MAPK).
Conclusion:
Results obtained from in silico and ex-vivo studies showed that dexamethasone and curcumin have the potential to inhibit neutrophilic functions attributed to iNOS and associated proteins. These findings support their potential for repurposing as targeted immunomodulatory therapies in sepsis management.
