Mutational analysis of SARS-CoV-2 ORF6-KPNA2 binding interface and identification of potent small molecule inhibitors to recuse the host immune system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surfaced on 31 December, 2019, and was identified as the causative agent of the global COVID-19 pandemic, leading to a pneumonia-like disease. One of its accessory proteins, ORF6, has been found to play a critical role in immune evasion by interacting with KPNA2 to antagonize IFN signaling and production pathways, resulting in the inhibition of IRF3 and STAT1 nuclear translocation. Since various mutations have been observed in ORF6, therefore, a comparative binding, biophysical, and structural analysis was used to reveal how these mutations affect the virus's ability to evade the human immune system. Among the identified mutations, the V9F, V24A, W27L, and I33T, were found to have a highly destabilizing effect on the protein structure of ORF6. Additionally, the molecular docking analysis of wildtype and mutant ORF6 and KPNA2 revealed the docking score of - 53.72 kcal/mol for wildtype while, -267.90 kcal/mol, -258.41kcal/mol, -254.51 kcal/mol and -268.79 kcal/mol for V9F, V24A, W27L, and I33T respectively. As compared to the wildtype the V9F showed a stronger binding affinity with KPNA2 which is further verified by the binding free energy (-42.28 kcal/mol) calculation. Furthermore, to halt the binding interface of the ORF6-KPNA2 complex, we used a computational molecular search of potential natural products. A multi-step virtual screening of the African natural database identified the top 5 compounds with best docking scores of -6.40 kcal/mol, -6.10 kcal/mol, -6.09 kcal/mol, -6.06 kcal/mol, and -6.03 kcal/mol for tophit1-5 respectively. Subsequent all-atoms simulations of these top hits revealed consistent dynamics, indicating their stability and their potential to interact effectively with the interface residues. In conclusion, our study represents the first attempt to establish a foundation for understanding the heightened infectivity of new SARS-CoV-2 variants and provides a strong impetus for the development of novel drugs against them.