In Silico Discovery of Small Molecule Modulators Targeting the Achilles' Heel of SARS-CoV-2 Spike Protein

Abstract

The spike protein of SARS-CoV-2 has been a promising target for developing vaccines and therapeutics due to its crucial role in the viral entry process. Previously reported cryogenic electron microscopy (cryo-EM) structures have revealed that free fatty acids (FFA) bind with SARS-CoV-2 spike protein, stabilizing its closed conformation and reducing its interaction with the host cell target in vitro. Inspired by these, we utilized a structure-based virtual screening approach against the conserved FFA-binding pocket to identify small molecule modulators of SARS-CoV-2 spike protein, which helped us identify six hits with micromolar binding affinities. Further evaluation of their commercially available and synthesized analogs enabled us to discover a series of compounds with better binding affinities and solubilities. Notably, our identified compounds exhibited similar binding affinities against the spike proteins of the prototypic SARS-CoV-2 and a currently circulating Omicron BA.4 variant. Furthermore, the cryo-EM structure of the compound SPC-14 bound spike revealed that SPC-14 could shift the conformational equilibrium of the spike protein toward the closed conformation, which is human ACE2 (hACE2) inaccessible. Our identified small molecule modulators targeting the conserved FFA-binding pocket could serve as the starting point for the future development of broad-spectrum COVID-19 intervention treatments.

Figure 1

Overall structures of the prefusion SARS-CoV-2 spike protein in different conformational states. (A) Schematic diagram of SARS-CoV-2 spike protein monomer representing domain organization. NTD: N-terminal domain; RBD: receptor-binding domain; RBM: receptor-binding motif; SD1: subdomain 1; SD2: subdomain 2; FP: fusion peptide; HR1: heptad repeat 1; CH: central helix; CD: connector domain; HR2: heptad repeat 2; TM: transmembrane domain; CT: cytoplasmic tail. (B) Cryo-EM structures of spike protein with the closed state (left, PDB ID: 6XR8) and the open state (right, PDB ID: 7A94). One protomer of spike protein is represented as ribbons, and the other two protomers are displayed as density maps. All the molecular images are rendered in UCSF Chimera.

Figure 2

Comparison of the conservation of (A) residues in the FFA-binding pocket (PDB ID: 7E7B) and (B) residues in the RBD-hACE2 interface (PDB ID: 6M0J). Mutation frequencies of residues, derived from the GISAID database (https://www.gisaid.org/) on November 16, 2022, were mapped on the 3D structure of SARS-CoV-2 RBD. Variable residues (i.e., with high mutation frequencies) are displayed as green sticks, while conserved residues (i.e., with low mutation frequencies) are depicted as maroon sticks. Labels of residues associated with variants of concern (VOCs) are bolded.

Figure 3

Structure-based discovery of small molecule modulators against the FFA-binding pocket. (A) SILCS FragMaps overlaid on the surface of spike protein with the OA, with a GFE cutoff of −1.4 kcal/mol and −1.9 kcal/mol at the top and bottom, respectively. FragMaps for the aliphatic carbon, aromatic carbon, hydrogen bond acceptor, and acetate oxygen are displayed as green, purple, red, and orange mesh, respectively. The OA is shown in a cyan stick to indicate the position of FraMaps in the FFA-binding pocket. The surface of the two protomers is displayed as transparent salmon and cornflower blue, respectively. Note that unrelated maps outside the pocket were removed for clear visualization. (B) Flowchart of structure-based hierarchical virtual screening.

Figure 4

Hits identified by the hierarchical virtual screening approach, with dozens of micromolar K_D values. (A) 2D structure and corresponding binding affinity of each hit. (B) Docking poses of hits in the FFA-binding pocket in the cryo-EM structure of OA bound SARS-CoV-2 spike protein (PDB ID: 7E7B). Small molecules are depicted as cyan sticks, and residues in the binding pocket are represented as sticks colored in salmon for one RBD and cornflower blue for the adjacent RBD. Backbone atoms of spike protein are hidden for clear visualization. Hydrogen bonding interactions are displayed as green dashed lines.

Figure 5

Cryo-EM structure determination. (A) The dose–response curves of LA, OA, ATRA, and SPC-14 binding to trimeric spike protein in the SPR assay. (B) Cryo-EM density maps of spike+SPC-14 (left panel) and apo spike system (right panel). (C) The complex structure of the SPC-14 bound spike is shown in a front view (left panel) and a top view (middle panel). The spike trimer is illustrated as ribbons colored in salmon, cornflower blue, and forest green for three monomers, respectively. Bound SPC-14 is displayed as cyan spheres. The right panel represents the binding mode of SPC-14 (cyan sticks). Additional density in the binding pocket is shown as mesh. Residues are in the stick representation, and their backbone atoms are hidden for clear visualization. (D) Comparison of the RBD trimer of the closed apo spike (magenta, PDB ID: 6VXX) and SPC-14 (cyan sticks) bound spike (cornflower blue, and forest green for three RBDs, respectively). (E) Comparison of the binding pockets of the SPC-14 bound spike (cyan), LA bound spike (cornflower blue, PDB ID: 6ZB5), OA bound spike (light gray, PDB ID: 7E7B), and ATRA bound spike (light green, PDB ID: 7Y42).