Glucocorticoids Bind to SARS-CoV-2 S1 at Multiple Sites Causing Cooperative Inhibition of SARS-CoV-2 S1 Interaction With ACE2

Dexamethasone may reduce mortality in COVID-19 patients. Whether dexamethasone or endogenous glucocorticoids, such as cortisol, biochemically interact with SARS-CoV-2 spike 1 protein (S1), or its cellular receptor ACE2, is unknown. Using molecular dynamics (MD) simulations and binding energy calculations, we identified 162 druggable pockets in various conformational states of S1 and all possible binding pockets for cortisol and dexamethasone. Through biochemical binding studies, we confirmed that cortisol and dexamethasone bind to S1. Limited proteolysis and mass spectrometry analyses validated several MD identified binding pockets for cortisol and dexamethasone on S1. Interaction assays indicated that cortisol and dexamethasone separately and cooperatively disrupt S1 interaction with ACE2, through direct binding to S1, without affecting ACE2 catalytic activity. Cortisol disrupted the binding of the mutant S1 Beta variant (E484K, K417N, N501Y) to ACE2. Delta and Omicron variants are mutated in or near identified cortisol-binding pockets in S1, which may affect cortisol binding to them. In the presence of cortisol, we find increased inhibition of S1 binding to ACE2 by an anti-SARS-CoV-2 S1 human chimeric monoclonal antibody against the receptor binding domain. Whether glucocorticoid/S1 direct interaction is an innate defence mechanism that may have contributed to mild or asymptomatic SARS-CoV-2 infection deserves further investigation.


Supplementary figure 2:
Structures of cortisol and dexamethasone. A. 2D structure of cortisol. B. 2D structure of dexamethasone. C. 3D structure of cortisol in ball and stick model. D. 3D structure of dexamethasone in ball and stick model.

Supplementary figure 3:
Analysis of dynamics between each monomer of SARS-CoV2 spike glycoprotein trimer. The final pdb from the 10 µs simulation was used for this analysis. The three monomeric structural components (chains-A, B and C) of the trimer were overlayed as pairs to visualize the dynamics in one with respect to the other. The protein chain is shown in cartoon and the glycan chains are shown in stick representations. (A) Overlay of chain A (green) and chain B (cyan); (B) Overlay of chain A (green) and chain C (magenta); (C) Overlay of the three chains. These overlays reveal that the glycan chains and the RBD domains are dynamic, while the NTD domains are relatively stable.

Supplementary figure 4:
The effect of cortisol and dexamethasone on the thermal stability of SARS-CoV-2 S1. (A) Line plot showing solubility of SARS-CoV-2 S1 at increasing temperatures from 37°C to 90°C in the presence or absence of cortisol (10 nM or 100 nM). Soluble fraction was quantitated by densitometric analysis of SARS-CoV-2 S1 protein bands on an SDS-PAGE gel stained with Zn-Imidazole and plotted as a percentage of band intensity at 37°C (100% solubility). (B) Line plot showing solubility of SARS-CoV-2 S1 at 85C relative to 37C in the presence or absence of cortisol in cortisol:S1 molar ratios of 0:1, 10:1, 100:1.
Supplementary movie: SARS CoV2 Spike protein trimer with predicted high affinity pockets using F pocket. The pocket (in white) is the unique to each S1 monomer and the pockets (in blue) are formed between domains of different S1 monomers. Table 1: Full list of peptides identified by mass spectrometry (MS) following limited proteolysis of SARS-CoV-2 S1 incubated with vehicle or cortisol. Peptides highlighted blue are unique peptides only present in either vehicle or cortisol sample due to cortisol binding at these sites either facilitating or preventing proteolysis of these sites. These experimentally identified binding sites of cortisol coincide with those identified by our in-silico molecular simulation validating our hypothesis that cortisol (and possibly other glucocorticoids) can bind to multiple pockets on S1 which is likely to cause S1 denaturation and decreased affinity for its receptor, ACE2. Y: peptide detected by MS in the sample; N: peptide not detected by MS in the sample. Supplementary proteomics raw data are provided as a supplementary excel spread sheet.

Peptides
Vehicle Cortisol