Structural basis for suramin binding to the C-terminal domain of the SARS-CoV-2 nucleocapsid proteinOA

The global threat posed by COVID-19 persists,largely due to the high mutability of SARS-CoV-2 and the limited availability of effective antiviral therapeutics.The nucleocapsid(N)protein of SARS-CoV-2 is an attractive drug target because of its high degree of sequence conservation and essential role in viral replication.In this study,we show that suramin,a polysulfonated antiviral compound,binds to the C-terminal domain(N-CTD)of the N protein and interferes with its interaction with RNA.Biolayer interferometry(BLI)shows that suramin has a higher binding affinity for N-CTD(K d,3.30μM)than for RNA(Kd,10.12μM).Electrophoretic mobility shift assays(EMSAs)further confirms that suramin effectively displaces RNA from N-CTD.NMR titration experiments and site-directed mutagenesis identify theα1-η1 helix(residues 248–262)as the primary suramin binding region,with residues K256,R259 and R262 playing critical roles in ligand recognition.In addition,NMR relaxation and model-free analyses reveal that theα1-η1 helix is highly flexible on the picosecond to nanosecond timescale,a dynamic feature that likely facilitates ligand binding.Furthermore,ITC and EMSA experiments demonstrate that suramin can bind to the full-length N protein at multiple sites and dissociate RNA from the N protein.Taken together,these findings provide structural and biophysical insights into the mechanism of action of suramin and establish a rational basis for the development of targeted antiviral therapies against SARS-CoV-2.