Discovery of thiadiazole-based small-molecule inhibitors of SARS-CoV-2 spike-ACE2 interaction through integrated computational prediction and experimental validation

Abstract

The SARS-CoV-2 spike protein enables viral entry by binding to the human ACE2 receptor, making it a critical target for antiviral intervention. In this study, five small molecules derived from 1,3,4-thiadiazole scaffolds were identified as potential inhibitors of the spike-ACE2 interaction using structure-based virtual screening and experimental validation. All five compounds were predicted to bind within the receptor-binding domain of the spike protein and disrupted ACE2 attachment. Simulations confirmed stable binding for all compounds without structural destabilization of the spike protein. Binding reduced the flexibility of key receptor-binding loops, maintained overall compactness of the spike structure, and restricted large-scale conformational shifts compared to the unbound protein. Energy analysis showed that hydrophobic contacts and electrostatic interactions contributed to stable complex formation. Of the five compounds, NS1 and NS2 demonstrated the strongest binding affinities (− 49.17 and − 47.74 kcal mol−¹), stable binding behavior over extended simulations, and consistent structural stabilization of the spike protein. In vitro, NS1 and NS2 inhibited the spike-ACE2 interaction with IC₅₀ values of 2162.77 nM and 2946.28 nM, respectively. Both compounds exhibited low cytotoxicity in human BJ fibroblast cells and predicted oral bioavailability based on pharmacokinetic modeling. We identified NS1 and NS2 as promising lead compounds capable of directly targeting the spike-ACE2 interface. Our results support small-molecule inhibition of spike-ACE2 binding as a viable antiviral approach and highlight the identified thiadiazole scaffold as a starting point for future optimization and preclinical development.