Microfluidic Diffusion Sizing Applied to the Study of Natural Products and Extracts That Modulate the SARS-CoV-2 Spike RBD/ACE2 Interaction

Abstract

The interaction between SARS-CoV-2 spike RBD and ACE2 proteins is a crucial step for host cell infection by the virus. Without it, the entire virion entrance mechanism is compromised. The aim of this study was to evaluate the capacity of various natural product classes, including flavonoids, anthraquinones, saponins, ivermectin, chloroquine, and erythromycin, to modulate this interaction. To accomplish this, we applied a recently developed a microfluidic diffusional sizing (MDS) technique that allows us to probe protein-protein interactions via measurements of the hydrodynamic radius (R_h) and dissociation constant (K_D); the evolution of R_h is monitored in the presence of increasing concentrations of the partner protein (ACE2); and the K_D is determined through a binding curve experimental design. In a second time, with the protein partners present in equimolar amounts, the R_h of the protein complex was measured in the presence of different natural products. Five of the nine natural products/extracts tested were found to modulate the formation of the protein complex. A methanol extract of Chenopodium quinoa Willd bitter seed husks (50 µg/mL; bisdesmoside saponins) and the flavonoid naringenin (1 µM) were particularly effective. This rapid selection of effective modulators will allow us to better understand agents that may prevent SARS-CoV-2 infection.

Keywords: dissociation constant; hydrodynamic radius; protein-protein interaction.

Figure 4

Chemical structures of the tested natural products. The figure depicts the major compounds present in tested commercial extracts [91,94,95,96,97]; their actual levels were inferred from the analysis certificates issued by the selling companies. For the extracts of Rhei radix and Ginkgo biloba L., chemical structures of major compounds are presented to exemplify their complex compositions [96,98]. For Chenopodium quinoa bitter seed husks, compounds were identified and quantified in μg HCe/g (μg hederacoside C equivalents per g of dry weight) by LC-MS-MS according to [91] (mean ± standard deviation; n = 3; limit of detection, 0.83 µg/g; limit of quantification, 2.53 µg/g). PA: phytolaccagenic acid; Hed: hederagenin; SA: serjenic acid; AG489 and AG487 refer to aglycones with a specific m/z; Glc: glucose; Ara: arabinose; Gal: galactose, Xyl: xylose; Hex: hexose; Pent: pentose.

Figure 4

Chemical structures of the tested natural products. The figure depicts the major compounds present in tested commercial extracts [91,94,95,96,97]; their actual levels were inferred from the analysis certificates issued by the selling companies. For the extracts of Rhei radix and Ginkgo biloba L., chemical structures of major compounds are presented to exemplify their complex compositions [96,98]. For Chenopodium quinoa bitter seed husks, compounds were identified and quantified in μg HCe/g (μg hederacoside C equivalents per g of dry weight) by LC-MS-MS according to [91] (mean ± standard deviation; n = 3; limit of detection, 0.83 µg/g; limit of quantification, 2.53 µg/g). PA: phytolaccagenic acid; Hed: hederagenin; SA: serjenic acid; AG489 and AG487 refer to aglycones with a specific m/z; Glc: glucose; Ara: arabinose; Gal: galactose, Xyl: xylose; Hex: hexose; Pent: pentose.

Figure 1

Microfluidic diffusional sizing determination of K_D for the spike RBD (20 nM)/ACE2 complex (DMSO, 1% v/v; room t°). R_h as a function of ACE2 concentration. ACE2, 180 pM to 750 nM; spike RBD, 20 nM; mean ± standard deviation (n = 4).

Figure 2

R_h modulation of the spike RBD (20 nM)/ACE2 (20 nM) complex by various natural products and extracts. R_h variation (%) of the spike RBD/ACE2 complex as a function of the tested natural products and extracts. Microfluidic diffusional size. Mean ± standard deviation. n = 3.

Figure 3

Schematic view of possible 3D conformational changes that the spike RBD/ACE2 complex could adopt in the presence of natural compounds, with their influence on R_h. The initial conformation of the spike RBD/ACE2 complex is represented in the center (PDB 6M0J); SARS-CoV-2 spike RBD is depicted in orange and ACE2 in green. (A) An increase in R_h could be due to distension of the complex; (B) An increase in R_h could be due to clustering of natural compounds on proteins; (C) A decrease in R_h could be due to collapse or folding of the complex; (D) A decrease in R_h could be due to (partial) separation of the 2 proteins.

Figure 5

Diagram showing the interior of a microfluidic chip and the travel of the sample after injection on the loading pedestal (adapted from [100,101]).