Identification and mechanistic exploration of structural and conformational dynamics of NF-kB inhibitors: rationale insights from in silico and in vitro studies

Abstract

Increased expression of target genes that code for proinflammatory chemical mediators results from a series of intracellular cascades triggered by activation of dysregulated NF-κB signaling pathway. Dysfunctional NF-kB signaling amplifies and perpetuates autoimmune responses in inflammatory diseases, including psoriasis. This study aimed to identify therapeutically relevant NF-kB inhibitors and elucidate the mechanistic aspects behind NF-kB inhibition. After virtual screening and molecular docking, five hit NF-kB inhibitors opted, and their therapeutic efficacy was examined using cell-based assays in TNF-α stimulated human keratinocyte cells. To investigate the conformational changes of target protein and inhibitor-protein interaction mechanisms, molecular dynamics (MD) simulations, binding free energy calculations together with principal component (PC) analysis, dynamics cross-correlation matrix analysis (DCCM), free energy landscape (FEL) analysis and quantum mechanical calculations were carried out. Among identified NF-kB inhibitors, myricetin and hesperidin significantly scavenged intracellular ROS and inhibited NF-kB activation. Analysis of the MD simulation trajectories of ligand–protein complexes revealed that myricetin and hesperidin formed energetically stabilized complexes with the target protein and were able to lock NF-kB in a closed conformation. Myricetin and hesperidin binding to the target protein significantly impacted conformational changes and internal dynamics of amino acid residues in protein domains. Tyr57, Glu60, Lys144 and Asp239 residues majorly contributed to locking the NF-kB in a closed conformation. The combinatorial approach employing in silico tools integrated with cell-based approaches substantiated the binding mechanism and NF-kB active site inhibition by the lead molecule myricetin, which can be explored as a viable antipsoriatic drug candidate associated with dysregulated NF-kB.

Communicated by Ramaswamy H. Sarma

Keywords:

• Dynamic cross-correlation matrix
• free-energy landscape
• molecular dynamics simulation
• NF-kB
• principal component analysis
• quantum mechanics

Acknowledgments

The authors thank the Department of Biosciences and Bioengineering, IIT Roorkee, India, and the Department of Pharmacy, Central University of Rajasthan, India, for facilitating their research.

Authors’ Contributions

Amit Kumar Srivastava: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing - original draft, review & editing; Shubham Srivastava: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing - original draft; Viney Kumar: Data curation, Formal analysis, Investigation, Validation, Visualization, Review & editing; Souvik Ghosh: Data curation, Investigation, Visualization; Review; Siddharth Yadav: Investigation, Visualization; Review; Ruchi Malik: Formal analysis, Investigation, Resources, Software, Visualization, Review & editing; Partha Roy: Formal analysis, Investigation, Resources, Visualization, Review & editing; Ramasare Prasad: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, review & editing. All authors reviewed and approved the final version of the article and agreed on data accuracy, integrity, and intellectual content.

Disclosure statement

The authors declare that they have no conflict of interest.

Geolocation Information

This study was conducted in the Department of Biosciences and Bioengineering, IIT Roorkee, India, and the Department of Pharmacy, Central University of Rajasthan, India.

Additional information

Funding

Amit Kumar Srivastava gratefully acknowledges the receipt of Junior and Senior research fellowships from the Ministry of Education (formerly the Ministry of Human Resource Development), Government of India.