Oxalic acid blocked the binding of spike protein from SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants to human angiotensin-converting enzymes 2

Abstract

An epidemic of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. Moreover, the emergence of SARS-CoV-2 variants of concern, such as Delta and Omicron, has seriously challenged the application of current therapeutics including vaccination and drugs. Relying on interaction of spike protein with receptor angiotensin-converting enzymes 2 (ACE2), SARS-CoV-2 successfully invades to the host cells, which indicates a strategy that identification of small-molecular compounds to block the entry is of great significance for COVID-19 prevention. Our study evaluated the potential efficacy of natural compound oxalic acid (OA) as an inhibitory agent against SARS-CoV-2 invasion, particular on the interaction of the receptor binding domain (RBD) of Delta and Omicron variants to ACE2. By employing a competitive binding assay in vitro, OA significantly blocked the binding of RBDs from Delta B.1.617.2 and Omicron B.1.1.529 to ACE2, but has no effect on the wide-type SARS-CoV-2 strain. Furthermore, OA inhibited the entries of Delta and Omicron pseudovirus into ACE2 high expressing-HEK293T cells. By surface plasmon resonance (SPR) assay, the direct bindings of OA to RBD and ACE2 were analyzed and OA had both affinities with RBDs of B.1.617.2 and B.1.1.529 and with ACE2. Molecular docking predicted the binding sites on the RBD-ACE2 complex and it showed similar binding abilities to both complex of variant Delta or Omicron RBD and ACE2. In conclusion, we provided a promising novel small-molecule compound OA as an antiviral candidate by blocking the cellular entries of SARS-CoV-2 variants.

Fig 1. Chemical structure of oxalic acid.

Fig 2. Effects of OA on the interaction between ACE2 and SARS-CoV-2 Spike RBD from Delta (B.1.617.2) and Omicron (B.1.1529).

Representative inhibitory curves of ACE2 binding to SARS-CoV-2 RBDs of Delta RBD (A), Omicron RBD (B) in the presence of OA determined by ELISA.

Fig 3. Effect of OA on ACE2^h cell viability.

ACE2^h cells were pretreated with different doses of OA and incubated for 24 h. Viability of ACE2^h cells was detected at OD_450nm. The proliferation ration was calculated. The experiments were repeat three times. Data are presented as mean ± SD.

Fig 4. The effect of OA on the entry of SARS-COV-2 Delta (B.1.617.2) pseudovirus into ACE2^h cells.

(A) The entrance of SARS-CoV-2 Delta pseudovirus into ACE2^h cells was evaluated by the luciferase activity after treated with ACE2-Fc and different concentrations of OA. (B) Inhibition rate was calculated and showed in curves. (C) Representative images were photographed at × 4 magnification using the fluorescence microscope. Data were presented as mean ± S.D. **p < 0.01, ***p < 0.001, compared with control.

Fig 5. The effect of OA on the entry of SARS-COV-2 Omicron (B.1.1.529) pseudovirus into ACE2^h cells.

(A) The entrance of SARS-CoV-2 Delta pseudovirus into ACE2^h cells was evaluated by the luciferase activity after treated with ACE2-Fc and different concentrations of OA. (B) Representative images were photographed at × 4 magnification using the fluorescence microscope. Data are presented as mean ± S.D. *p <0.05, **p < 0.01, ***p < 0.001, compared with control.

Fig 6. The binding characters of OA on ACE2 and RBDs of Delta and Omicron.

The SPR titration curves of OA interacted with ACE2 protein (A), Delta RBD protein (B) and Omicron RBD protein (C) were shown. The affinity constants of OA to Delta, Omicron and ACE2 protein were calculated and presented (D).

Fig 7. SARS-CoV-2 Delta or Omicron spike protein in complex with human ACE2.

Delta (A) and Omicron (B) RBD binding to ACE2 interface were marked.

Fig 8. Molecular docking of OA with RBD-ACE2 binding interfaces.

The predicted binding sites of OA to the complex of ACE2 with Delta (A) or Omicron (B) RBD were shown.