Introduction
Small-molecule stabilization of protein–protein interactions (PPIs) is an extremely promising strategy in drug discovery. It can be used to treat cancers and viral infections.1−3 Stabilizing PPIs with small molecules may be allosteric or direct (also called orthosteric). This process alters the oligomerization equilibrium of the protein and enables small molecules to modulate its physiological function.4−7 The anticancer drug paclitaxel, for example, allosterically enhances microtubule structure assembly by binding to β-tubulin.8,9 On the other hand, rapamycin, another anticancer agent, binds directly to the interface between FKBP12 and mTOR and stabilizes the structure of the complex.10 The most well-characterized PPIs suitable as targets for drug development form natively under physiological conditions. However, non-native interactions, which may form under extreme circumstances such as inside a crystal lattice, are also potential drug targets. For example, nucleozin exerts its antiviral activity by stabilizing the non-native PPI interface between the two neighboring nucleoprotein trimers within the influenza virus, which results in abnormal protein oligomerization and loss of viral viability.11
Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the betacoronavirus (β-CoVs) family. It causes severe respiratory distress with a high mortality rate in humans.12−14 Recently, a closely related novel coronavirus, coronavirus disease 2019 (COVID-19), caused an outbreak of pneumonia in Wuhan, which further underscored the risk of CoVs to the global public health.15,16 However, there is no effective treatment for CoVs. Thus, there is an urgent need to develop new antiviral agents against CoVs.14,17 MERS-CoV packages its genome in a nucleocapsid (N) protein and forms a ribonucleoprotein (RNP) complex. The RNP is essential for viral transcription and assembly. Several studies suggested that the modulation of CoV N protein oligomerization by small molecules is a feasible antiviral drug development strategy.18,19 The CoV N protein is organized into the N-terminal domain (NTD) and the C-terminal domain (CTD), with both domains participating in RNA binding.20,21 All CoV N-NTD structures are folded in a monomeric conformation. In contrast, the CoV N-CTDs are always dimeric and are responsible for N protein oligomerization via protein–protein interactions.22,23
Here, we report the crystal structure of MERS-CoV N-NTD in a non-native dimeric configuration. We used the non-native dimer interface as the target in virtual screening for an orthosteric stabilizer. To this end, we considered the binding scores and hydrophobic complementarity of the acquired poses, and further selected the potential leads P1–P3 from Acros and ZINC drug databases. Of these, only 5-benzyloxygramine (P3) had both antiviral and stabilizing activities on the N protein. Small-angle X-ray scattering (SAXS) and cell-based assays showed that P3 induces abnormal full-length N protein oligomerization in vitro and at the cell level. We also described the structure of MERS-CoV N-NTD complexed with 5-benzyloxygramine and revealed its stabilizing mechanism. Our findings provide insight into the development of a new therapeutic approach based on stabilizing a non-native protein interaction interface. It may lead to the discovery and development of new treatments for various infectious diseases.