Mechanistic Insights into the SARS-CoV-2 Nucleocapsid Protein: Structure, Dynamics, and Inhibitors Screening through In Silico and In Vitro Strategies

The nucleocapsid (N) protein of SARS-CoV-2 is an essential structural protein for viral replication, assembly, and immune modulation. The N protein is more structurally conserved than the spike protein, making it a promising target for antiviral drug development. To identify novel inhibitors, a high-throughput virtual screening of 65,000 antiviral compounds was conducted against both domains of the N protein using AutoDock Vina. Top candidates were prioritized based on binding energy and key molecular interactions, followed by ADMET evaluation via SwissADME. Ten compounds that met these criteria were subjected to 200 ns molecular dynamics simulations using GROMACS 2022.2, revealing four compounds (C4, C7, C8, and C9) with the highest binding affinities and stability. Fluorescence spectroscopy confirmed that the N protein’s tertiary structure remains unchanged upon compound binding. Isothermal titration calorimetry (ITC) measurements indicated binding by hydrogen-bond formation, with compounds 7 and 9 displaying the highest Gibbs free energies ( -8.67*104 and -4.36*103 cal/mol). Surface plasmon resonance (SPR) analysis corroborated the fluorescence spectroscopy and ITC findings, showing that compounds 7, 9, 8, and 4 exhibit the highest binding constants (1.67*10-4, 4.50*10-4, 2.92*10-4, and 7.77*10-4 M, respectively), while compound 9 demonstrates a faster dissociation rate, indicating lower stability. Liquid–liquid phase separation (LLPS) assays revealed that the N protein forms spherical biocondensate in the presence of poly U, and compounds 4, 7, 8, and 9 deform condensate formation. These results, confirmed by fluorescence microscopy and Förster resonance energy transfer, suggest functional inhibition of the N protein, positioning C4, C7 and C8 as promising antiviral candidates. Collectively, these findings support further evaluation of these inhibitors in cell-based and in vivo studies. Advancing these compounds holds significant promise for developing therapeutics targeting the highly conserved N protein, thereby complementing existing vaccine approaches and addressing the urgent challenge posed by neutralization-escape mutants.