Targeting the N-Terminus Domain of the Coronavirus Nucleocapsid Protein Induces Abnormal Oligomerization via Allosteric Modulation

Abstract

Epidemics caused by coronaviruses (CoVs), namely the severe acute respiratory syndrome (SARS) (2003), Middle East respiratory syndrome (MERS) (2012), and coronavirus disease 2019 (COVID-19) (2019), have triggered a global public health emergency. Drug development against CoVs is inherently arduous. The nucleocapsid (N) protein forms an oligomer and facilitates binding with the viral RNA genome, which is critical in the life cycle of the virus. In the current study, we found a potential allosteric site (Site 1) using PARS, an online allosteric site predictor, in the CoV N-N-terminal RNA-binding domain (NTD) to modulate the N protein conformation. We identified 5-hydroxyindole as the lead via molecular docking to target Site 1. We designed and synthesized four 5-hydroxyindole derivatives, named P4-1 to P4-4, based on the pose of 5-hydroxyindole in the docking model complex. Small-angle X-ray scattering (SAXS) data indicate that two 5-hydroxyindole compounds with higher hydrophobic R-groups mediate the binding between N-NTD and N-C-terminal dimerization domain (CTD) and elicit high-order oligomerization of the whole N protein. Furthermore, the crystal structures suggested that these two compounds act on this novel cavity and create a flat surface with higher hydrophobicity, which may mediate the interaction between N-NTD and N-CTD. Taken together, we discovered an allosteric binding pocket targeting small molecules that induces abnormal aggregation of the CoV N protein. These novel concepts will facilitate protein-protein interaction (PPI)-based drug design against various CoVs.

Computational exploration of the dual role of the phytochemical fortunellin: Antiviral activities against SARS-CoV-2 and immunomodulatory abilities against the host

“…Based on the available experimental and computational studies, we have chosen the druggable pocket located on the surface of the nucleocapsid N-terminal RNA binding domain (PDB ID: 6M3M ). The pocket has been shown to bind with the small molecule via Ala91, Thr92, Arg93, Arg94, Ile95, Asp104, Leu105, Ser106, Arg108, Tyr110, Tyr112, and Arg150 [ 40 , [117] , [118] , [119] , [120] ]. The drug Naproxen binds and interacts with the key residues Thr50, Ala51, Tyr112, and Arg150 [ 119 ].…”

The SARS-CoV-2 nucleoprotein associates with anionic lipid membranes

“…The N protein from SARS-CoV-2 and other betacoronaviruses can form oligomers (Chang, Chen et al 2013, Carlson, Asfaha et al 2020, Hsu, Chen et al 2022), which play a critical role in assembly and binding RNA in SARS-CoV-2 genome packaging and virus spread. To determine if N oligomers can also associate with anionic lipid membranes, we crosslinked N using a BS 3 cross linker (Figure 4A).…”

The SARS-CoV-2 envelope (E) protein forms a calcium- and voltage-activated calcium channel

“…Inhibitors of the nucleocapsid protein (N) and E have also been proposed (10,11). Of the four structural proteins, E is the least well-characterized in terms of structure and function.…”

“…Meanwhile, the SARS-CoV-2 main protease (M pro ) has been the focus in the design of small molecule antivirals (9). Inhibitors of the nucleocapsid protein (N) and E have also been proposed (10, 11). Of the four structural proteins, E is the least well-characterized in terms of structure and function.…”