Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

The study addresses how SARS-CoV-2 engages the ACE2 receptor at the earliest stages of infection and quantifies the dynamics of this interaction at the single-molecule level. Context: SARS-CoV-2 causes COVID-19 and, like SARS-CoV, uses the spike (S) glycoprotein to bind host cells via ACE2. Entry requires coordinated receptor binding and proteolytic activation of S. While structural studies identified the ACE2–RBD interface, direct single-molecule evidence of the binding dynamics and energetics was lacking. Purpose: to measure the kinetics and thermodynamics of S1 subunit and RBD binding to ACE2 on purified model surfaces and living cells, and to assess whether ACE2-derived peptides can inhibit this interaction. Importance: defining the strength, rate constants, and inhibition of the ACE2–S interactions informs understanding of viral attachment stability and supports therapeutic design of attachment inhibitors.

Prior work established ACE2 as the receptor for SARS-CoV and SARS-CoV-2, including cryo-EM structures of ACE2 in complex with the SARS-CoV-2 RBD and evidence for ACE2 dimerization engaging S-proteins. The S1 subunit mediates receptor binding while S2 mediates fusion, with host proteases activating S for membrane fusion. Single-molecule AFM approaches have previously characterized virus–receptor interactions for other systems, and reported high-affinity associations for SARS-CoV–ACE2. Alternative host attachment factors such as sialylated glycans and RGD-binding integrins have been suggested for coronaviruses, motivating tests of their contribution to early binding.

- Force-distance (FD) curve-based atomic force microscopy (AFM) was used to probe interactions between SARS-CoV-2 S1 subunit or isolated RBD (ligands tethered to AFM tips) and ACE2 receptors immobilized on model surfaces, as well as on living A549 cells with or without ACE2 overexpression (A549-ACE2). - Dynamic force spectroscopy (DFS) was performed by varying retraction speeds/loading rates and contact times to obtain rupture force versus loading rate dependencies. The Bell–Evans model (two-state energy landscape) was fitted to estimate kinetic off-rates (koff) and distance to transition state (xβ). Binding probability (BP) was measured as a function of contact time to extract association parameters and derive Kd (via koff/kon under pseudo-first-order assumptions considering the effective encounter volume). - Single- versus multiple-bond events were assessed via rupture force histograms across loading rate windows, fitting with multiple Gaussians to identify most probable single-bond forces. - Validation on living cells: Correlative AFM–confocal microscopy was used to locate A549 and A549-ACE2 cells; adhesion maps (BP) and rupture forces were measured in fast force-volume and peak force tapping modes across different loading regimes. - Competition/blocking assays on cells tested potential alternative receptors by adding 9-O-acetyl-sialoglycans and cyclic RGD (cRGD) peptides to compete with sialic acid– and integrin-mediated interactions. - Inhibition studies employed ACE2-derived peptides ([22–44], [22–57], [22–44–8–351–357], [351–357]) incubated at 1–100 µM with S1/RBD-functionalized tips to quantify reductions in BP on ACE2-coated surfaces and then applied to living cells. - Additional kinetic confirmation was performed by biolayer interferometry (BLI/BLItz) to independently measure affinities. - Computational support included molecular dynamics simulations (GROMACS, Amber99-ILDN, TIP3P water) using ACE2–RBD structures (e.g., PDB 6M0J/6M0Y) to model peptide–S1 interactions and hydrogen bonding over hundreds of nanoseconds. - Reagent preparation included functionalization of AFM cantilevers with S1 or RBD proteins, fabrication of ACE2-coated model surfaces, and synthesis of a 9‑O‑acetyl sialic acid derivative; detailed imaging and force-curve acquisition parameters (spring constants, scan sizes, frequencies) are specified.

- The SARS-CoV-2 S1 subunit and its RBD bind ACE2 via the same interface and exhibit similar single-bond kinetic and thermodynamic parameters. - Dynamic force spectroscopy showed rupture force increasing linearly with the logarithm of the loading rate, consistent with the Bell–Evans model. - Distance to the energy barrier (xβ): 0.81 ± 0.05 nm (S1) and 0.79 ± 0.04 nm (RBD), indicating similar energy landscapes for both ligands. - Kinetic off-rates (koff): 0.008 ± 0.005 s−1 (S1) and 0.009 ± 0.006 s−1 (RBD), in agreement with values from surface plasmon resonance for ACE2–S/RBD interactions. - Association analysis from BP versus contact time yielded pseudo-first-order association constants leading to dissociation constants Kd ≈ 120 nM for both S1–ACE2 and RBD–ACE2, evidencing high-affinity binding. - On living cells, S1 exhibited higher binding probability and adhesion on A549-ACE2 (ACE2-overexpressing) cells than on control A549 cells; DFS data on cells aligned well with purified receptor measurements, supporting physiological relevance. - Even ACE2-low control cells displayed a baseline BP (~10%), suggesting additional attachment factors. Blocking experiments with 9‑O‑acetyl-sialoglycans and cRGD reduced binding, implicating sialylated glycans and integrins as potential auxiliary attachment partners. - ACE2-derived peptides inhibited S1/RBD–ACE2 binding in a concentration-dependent manner. Peptides mimicking the ACE2 N-terminal helix ([22–44] and [22–57]) were most effective, achieving >70% reduction in binding probability at higher concentrations and exhibiting IC50 values in the micromolar range on purified systems. - In cell assays, the [22–57] peptide (100 µM) significantly decreased BP on A549-ACE2 cells (approximately 70% reduction), lowering binding to near control-cell levels.

These measurements provide direct single-molecule evidence that the SARS-CoV-2 S protein engages ACE2 predominantly through its RBD with high affinity and slow dissociation, explaining the stability of the initial virus–cell attachment. The close match of kinetic parameters for S1 and isolated RBD indicates that the RBD–ACE2 interface dominates the interaction, while the rest of S1 does not substantially alter single-bond energetics under the tested conditions. Validation on living A549-ACE2 cells confirms that purified-surface findings translate to a cellular context and that multivalency and receptor distribution likely further stabilize attachments on virions. The detection of residual binding on ACE2-low cells and its reduction by sialic acid analogs and cRGD supports the role of auxiliary receptors (sialylated glycans and integrins) in early attachment, potentially enhancing initial contact prior to ACE2 engagement. Importantly, ACE2-derived peptides targeting the RBD–ACE2 interface effectively disrupt binding at the single-molecule level and on cells, nominating them as promising leads for attachment inhibitors that avoid perturbing endogenous ACE2 physiology compared with soluble ACE2 therapies.

The study quantitatively maps the energy landscape and kinetics of SARS-CoV-2 S1/RBD binding to ACE2 at the single-molecule level on purified receptors and living cells, establishing a high-affinity interaction (Kd ~120 nM) dominated by the RBD–ACE2 interface. It demonstrates that ACE2-derived peptides, especially those mimicking the N-terminal helix ([22–44], [22–57]), can potently inhibit this binding (>70% reduction, µM IC50) and reduce cellular binding to baseline, highlighting their potential as therapeutic attachment inhibitors. Future work should optimize peptide stability and affinity, evaluate broad-spectrum activity across variants, assess synergy with blockers of auxiliary receptors, and validate antiviral efficacy and safety in infection models and in vivo.

- Cellular assays used A549 cells, which naturally exhibit low ACE2 expression and limited permissiveness to SARS-CoV-2; overexpression models may not fully recapitulate native airway physiology. - AFM measures single-bond mechanics under controlled geometries and may not capture the full multivalent architecture and dynamics on intact virions and complex mucosal environments. - Inhibition was assessed primarily by reductions in binding probability/force; direct viral neutralization or infection assays were not reported here. - Kinetic parameters were derived under specific buffer, temperature, and surface-attachment conditions that can influence absolute rates and affinities. - Peptide efficacy and stability in biological fluids, potential off-target effects, and pharmacokinetics remain to be established.