Abstract
The COVID-19 pandemic, driven by SARS-CoV-2, continues to present global health challenges due to viral mutations and immune escape mechanisms. While current therapies primarily target viral entry and replication proteins, therapeutic agents that directly inhibit the Spike receptor-binding domain (S-RBD) are lacking. This study aimed to utilize virtual screening and biological methods to identify potential therapeutic compounds with anti-SARS-CoV-2 activity. We conducted virtual screening and molecular docking of 3,014 compounds obtained from a customized drug library to identify candidates with high affinity for S-RBD. The biological activity of these candidates was assessed using cellular thermal shift assays, surface plasmon resonance, immunoprecipitation, immunofluorescent analysis, and pseudoviral invasion tests against three pseudoviral variants: 2019-nCoV, Delta, and Omicron. We identified that DOTAP chloride ($K_D$ = 49.94 µM), cefotiam hexetil hydrochloride ($K_D$ = 142.26 µM), melittin ($K_D$ = 34.98 µM) and teicoplanin ($K_D$ = 73.58 µM) exerted antiviral activity. These compounds demonstrated potent binding to S-RBD through hydrogen bonding and π-interactions. They effectively inhibited S-RBD/hACE2 binding and pseudoviral entry with different efficacy: DOTAP chloride (25 µM) inhibited pseudoviral entry of both the 2019-nCoV and Omicron strains, while cefotiam hexetil hydrochloride, melittin and teicoplanin showed broader spectrum activity across the variants. Our findings suggest that these four compounds could serve as potential therapeutic candidates for COVID-19 treatment.
Introduction
As SARS-CoV-2 continues to evolve, mutations in its Spike protein—specifically the Receptor-Binding Domain (S-RBD)—allow the virus to "escape" the immune response triggered by previous infections or vaccines. While many current drugs target the virus's ability to replicate once inside a cell, there is a critical shortage of "entry inhibitors" that can prevent the virus from docking with the human ACE2 receptor in the first place.
The challenge is finding molecules that are "variant-proof." Because the Spike protein is highly mutable, researchers sought compounds that target conserved residues or utilize multimodal mechanisms (like electrostatic interference) to maintain efficacy even as the virus changes its "shape."
Methodology
The study employed a "dry-lab to wet-lab" pipeline to narrow down thousands of candidates:
- Virtual Screening: Researchers used MOE software to perform molecular docking of 3,014 compounds from a customized drug library. They targeted 10 specific "hotspot" residues on the S-RBD known to be critical for ACE2 binding.
- Biophysical Validation: The top 36 "hits" from the computer model were tested using CETSA (Cellular Thermal Shift Assay) to see if they physically stabilized the Spike protein when heated, a hallmark of direct binding.
- Kinetic Analysis: Surface Plasmon Resonance (SPR) was used to calculate the equilibrium dissociation constants ($K_D$), measuring exactly how "sticky" these compounds are to the Spike protein.
- Functional Assays: The team used Co-Immunoprecipitation (Co-IP) to prove the compounds could physically disrupt the S-protein/hACE2 complex and pseudovirus invasion tests to evaluate if the drugs stopped viral infection in live cells.
Structural data for the S-RBD were obtained from the Protein Data Bank (PDB IDs: 6M0J, 7V8B, and 7T9L). Efficacy was evaluated against three distinct lineages: the original 2019-nCoV, Delta (B.1.617.2), and Omicron (BA.5).
Results and Discussion
The virtual screening process identified several high-affinity candidates. Tables 1 and 2 detail the docking scores for the top-performing compounds.
From the initial pool of 3,014 compounds, four potent inhibitors were identified:
- Melittin: Showed the highest affinity ($K_D = 34.98\ \mu\text{M}$) and achieved an impressive 85.1% inhibition of the Spike-ACE2 complex formation at 1.25 µM, though it exhibited significant cytotoxicity at higher concentrations.
- DOTAP chloride: ($K_D = 49.94\ \mu\text{M}$) Effectively inhibited entry for both ancestral and Omicron strains at 25 µM. As a cationic lipid typically used for mRNA delivery, its positive charge allows it to interfere electrostatically with the Spike-ACE2 interface.
- Teicoplanin ($K_D = 73.58\ \mu\text{M}$) and Cefotiam hexetil ($K_D = 142.26\ \mu\text{M}$): Both showed broad-spectrum activity across all tested variants.
These compounds utilize multimodal inhibition, combining direct hydrogen bonding, $\pi$-H interactions, and host receptor modulation. This "Swiss Army Knife" approach likely contributes to their sustained effectiveness against mutated variants.
Conclusion and Future Directions
While these results are promising, the study was conducted in vitro. Future in vivo (animal) studies are necessary to determine the efficacy and safety of these compounds in a living system. Toxicity concerns, particularly for Melittin and certain antibiotics at higher doses, must be addressed through chemical modifications or optimized delivery methods, such as intranasal administration. Overcoming bottlenecks related to delivery and stability will be crucial for translating these findings into clinical treatments for COVID-19.