Single molecule force spectroscopy for evaluating inhibitors of SARS-CoV-2 variants of concern.

The binding between the SARS-CoV-2 Spike protein and its cellular receptor ACE2 is essential for viral entry and infection and, therefore, represents a critical target for antiviral intervention. Single-molecule force spectroscopy (SMFS), conducted using atomic force microscopy (AFM), allows direct investigation of Spike–ACE2 interactions at the single-molecule level, providing insights into binding mechanisms not easily captured by conventional methods. This review discusses recent applications of SMFS to assess molecular inhibitors of Spike–ACE2 interactions, focusing specifically on soluble ACE2 and glycan-binding lectins (Clec4g, hCLEC4G, and engineered H84T-Banlec). These inhibitors demonstrated potent, concentration-dependent activity against multiple SARS-CoV-2 variants, including Delta and Omicron, achieving IC₅₀ values in the low nanomolar range (soluble ACE2: 0.25–0.38 nM; Clec4g/hCLEC4G: 36–58 nM; H84T-Banlec: 2.6–5.2 nM). Notably, single-molecule IC₅₀ measurements closely correlated with EC₅₀ values from cell-based assays, validating the physiological relevance and predictive capability of the SMFS method. Collectively, these findings underscore the utility of AFM-SMFS as a powerful approach for inhibitor evaluation and antiviral discovery, particularly for strategies targeting conserved structural features of the Spike protein to achieve broad-spectrum efficacy against current and emerging viral variants.