Screening of Mpro Protease (SARS-CoV-2) Covalent Inhibitors from an Anthocyanin-Rich Blueberry Extract Using an HRMS-Based Analytical Platform

Abstract

Background: The viral main protease (M^pro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of M^pro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract.

Methods: The experiments focus on finding MS covalent inhibitors of M^pro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme's catalytic site.

Results: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known M^pro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the M^pro pocket observed was also evaluated by computational studies.

Conclusions: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind M^pro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.

Keywords: Mpro; SARS-CoV-2; blueberry; delphinidin-3-glucoside; mass spectrometry; metabolomics.

Figure 1

Structure formulae of the three most abundant components of BPE, in two of which the catechol/pyrogallol portion is highlighted.

Figure 2

Extracted ionic currents for delphinidin-3-glucoside/galactoside having m/z 465.1033 (upper panel), and for the corresponding delphinidin-3-glucoside/galactoside-Cys adduct having m/z 584.10740 (lower panel), obtained from the re-elaboration of the chromatogram acquired at the 2 h time point.

Figure 3

Time-dependent peak areas of BPE’s compounds (left panels) and Cys-BPE’s compound adducts (right panels) at different time points.

Figure 4

Peak area values as a function of time for the aglycones present in BPE (left panels, A,C) and the glycosidic forms (right panels, B,D); (panels C,D) correspond to normalized trends. Normalization considers the relative abundance of each ion; more specifically, the AUC was divided for the relative abundance.

Figure 5

Total ionic current (TIC) of recombinant M^pro incubated with BPE and sequentially digested with trypsin and chymotrypsin. Above is shown the M^pro sequence with the peptide portions identified by nLC-HR-MS/MS analysis highlighted in green. Symbols above the sequence indicate modifications detected as follows: C, carbamidomethylation; O, oxidation; * delphinidin-3-glucoside/galactoside Micheal adduct on His163 or His164.

Figure 6

Fragmentation spectrum of the [M + 3H]³⁺ precursor ion at m/z 1128.15894 identified by computational analysis (* modified fragment ion); the corresponding theoretical fragmentation pattern, obtained by means of the software Proteome Discoverer, is reported in Table S1 (Supplementary Materials).

Figure 7

Fragmentation spectrum of the [M + 2H]²⁺ precursor ion at m/z 1356.56018 identified by computational analysis (* modified fragment ion); the corresponding theoretical fragmentation pattern obtained by means of the software Proteome Discoverer is reported in Table S2 (Supplementary Materials).

Figure 8

(A) Antiviral effect (expressed as % of inhibition) of baicalin and delphinidin-3-glucoside (μM) against SARS-CoV-2 measured by qRT-PCR assay. The inhibition of viral replication in Vero E6 cells by baicalin and delphinidin was expressed as the reduction of the viral load in the culture media. (B) Virucidal activity was confirmed by plaque reduction assays expressed as PFU/mL. Data were obtained from three independent experiments, and are expressed as mean ± SD.

Figure 9

(A) Docking pose of delphinidin-3-glucoside (yellow sticks) within M^pro active site. (B) Representative structure of the MD simulation performed on delphinidin-3-glucoside–M^pro complex (green) superimposed to the starting coordinates obtained from docking (cyan). The residues of the binding pocket are displayed as sticks, while blue dashed lines represent H-bond interactions.