Abstract: LETTER
Reframing quercetin as a promiscuous inhibitor against
SARS-CoV-2 main protease
Haohao Yana,1, Rui Zhanga,1, Xiaoping Liua, Yanchang Wangb,2
, and Yunyu Chena,2
Fig. 1. Inhibitory effect of quercetin on SARS-CoV-2 Mpro in vitro. (A) The chemical structure of quercetin. (B) Inhibition of Mpro by quercetin using FRET assay.
(C) The fluorescence quenching effect of quercetin on MCA-AVLQ fragment in the FRET assay. MCA: 7-methoxycoumarin-4-acetic acid. (D) The comparison
between Mpro inhibition and fluorescence quenching effect of quercetin in the FRET assay. (E) Inhibition of Mpro by quercetin using FP assay. The FRET and FP
assays were carried out as previously described (4, 5, 7). The IC50 value of quercetin was shown. Nirmatrelvir (PF-332, 1 μM) and DMSO served as the positive and
negative controls, respectively. (F and G) Inhibition of Mpro by quercetin using ddRFP assay. The time course trajectories of ddRFP biosensor in the presence of
quercetin at the indicated concentrations were recorded every minute for 30 min by a microplate reader (BioTek). A fluorescent ddRFP biosensor produces a
high RFU value in the presence of Mpro inhibitors, whereas the cleavage by Mpro generates two separate RFP fragments with a low RFU signal. In the gel-based
assay, the ddRFP biosensor (55 kDa) can be cleaved by Mpro (34 kDa) to generate RFP-A1 (top band, 29 kDa) and RFP-B1 fragments (bottom band, 26 kDa). The
testing concentration of quercetin was 50, 100, or 200 μM. Nirmatrelvir (PF-332, 10 μM) and DMSO were used as the positive and negative controls, respectively.
The ddRFP assay was performed based on our previous publications (6, 7).
Traditional Chinese medicine has made contributions to the
treatment of coronavirus disease 2019 (COVID-19) because
of its favorable efficacy, such as Huashi Baidu decoction
(Q-14) (1, 2). Recently, quercetin, a main component of Q-14,
has been identified as a potent inhibitor against severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) using an integrative pharmacological strategy,
and its inhibitory effect on Mpro is examined by the fluorescence resonance energy transfer (FRET) assay with the half-
maximal inhibitory concentration (IC50) value of 22.47 μM (3).
Considering the potential of quercetin in COVID-19 treatment, a rigorous validation for its Mpro inhibition is
necessary.
We have developed a systematic high-throughput screening (HTS) platform for the discovery and assessment of Mpro
inhibitors, including FRET, fluorescence polarization (FP), and
dimerization-dependent red fluorescent protein (ddRFP)
assays (4–7). With these assays, we previously demonstrated
that baicalein is a nonspecific Mpro inhibitor (7). Herein, we
rigorously evaluated the inhibition of Mpro by quercetin
in vitro using these HTS assays (Fig. 1A). To ensure the
PNAS
2023
Vol. 120
No. 37
e2309289120
reliability of these assays, nirmatrelvir (PF-07321332, PF-332)
served as a positive control in the presence of dithiothreitol
(DTT) (7). Using FRET assay, our results showed that quercetin
exhibits apparent inhibition against Mpro (IC50 = 42.81 μM)
(Fig. 1B). However, the presence of quercetin at the testing
concentrations was able to quench the fluorescence signal
of MCA-AVLQ fragment, which is generated by the cleaved
FRET substrate (Fig. 1C). Importantly, this quenching effect
Author affiliations: aInstitute for Drug Screening and Evaluation, Wannan Medical..
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