Structural insights into the RNA binding inhibitors of the C-terminal domain of the SARS-CoV-2 nucleocapsid

The SARS-CoV-2 nucleocapsid (N) protein is an essential structural element of the virion, playing a crucial role in enclosing the viral genome into a ribonucleoprotein (RNP) assembly, as well as viral replication and transmission. The C-terminal domain of the N-protein (N-CTD) is essential for encapsidation, contributing to the stabilization of the RNP complex. In a previous study, three inhibitors (ceftriaxone, cefuroxime, and ampicillin) were screened for their potential to disrupt the RNA packaging process by targeting the N-protein. However, the binding efficacy, mechanism of RNA binding inhibition, and molecular insights of binding with N-CTD remain unclear. In this study, we evaluated the binding efficacy of these inhibitors using isothermal titration calorimetry (ITC), revealing the affinity of ceftriaxone (18 {+/-} 1.3 M), cefuroxime (55 {+/-} 4.2 M), and ampicillin (28 {+/-} 1.2 M) with the N-CTD. Further inhibition assay and fluorescence polarisation assay demonstrated RNA binding inhibition, with IC50 ranging from 10.4 to 12.4 M and KD values between 24 and 32 M for the inhibitors. Additionally, we also determined the inhibitor-bound complex crystal structures of N-CTD-Ceftriaxone (2.0 [A]) and N-CTD-Ampicillin (2.2 [A]), along with the structure of apo N-CTD (1.4 [A]). These crystal structures revealed previously unobserved interaction sites involving residues K261, K266, R293, Q294, and W301 at the oligomerization interface and the predicted RNA-binding region of N-CTD. These findings provide valuable molecular insights into the inhibition of N-CTD, highlighting its potential as an underexplored but promising target for the development of novel antiviral agents against coronaviruses. HighlightsO_LIThe inhibitors ceftriaxone, cefuroxime, and ampicillin-demonstrated high-affinity binding to the C-terminal domain (N-CTD) of the SARS-CoV-2 nucleocapsid (N) protein, effectively disrupting the formation of the N-CTD-RNA complex. C_LIO_LIComplex crystal structures of N-CTD with ceftriaxone and ampicillin revealed previously unobserved distinct binding sites. C_LIO_LIStructures reveal how the selected inhibitors disrupt the oligomerization of N-CTD and hinder the RNA packaging process of the virus. C_LI