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. 2022 Oct:206:105389.
doi: 10.1016/j.antiviral.2022.105389. Epub 2022 Aug 17.

Punicalagin as an allosteric NSP13 helicase inhibitor potently suppresses SARS-CoV-2 replication in vitro

Lian Lu  1 , Yun Peng  2 , Huiqiao Yao  1 , Yanqun Wang  3 , Jinyu Li  4 , Yang Yang  5 , Zhonghui Lin  6
Affiliations

Punicalagin as an allosteric NSP13 helicase inhibitor potently suppresses SARS-CoV-2 replication in vitro

Lian Lu et al. Antiviral Res. 2022 Oct.

Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) helicase NSP13 plays a conserved role in the replication of coronaviruses and has been identified as an ideal target for the development of antiviral drugs against SARS-CoV-2. Here, we identify a novel NSP13 helicase inhibitor punicalagin (PUG) through high-throughput screening. Surface plasmon resonance (SPR)-based analysis and molecular docking calculation reveal that PUG directly binds NSP13 on the interface of domains 1A and 2A, with a KD value of 21.6 nM. Further biochemical and structural analyses suggest that PUG inhibits NSP13 on ATP hydrolysis and prevents it binding to DNA substrates. Finally, the antiviral studies show that PUG effectively suppresses the SARS-CoV-2 replication in A549-ACE2 and Vero cells, with EC50 values of 347 nM and 196 nM, respectively. Our work demonstrates the potential application of PUG in the treatment of coronavirus disease 2019 (COVID-19) and identifies an allosteric inhibition mechanism for future drug design targeting the viral helicases.

Keywords: Helicase; Inhibitor; NSP13; Punicalagin; SARS-CoV-2.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Establishment of HTS assay. (A) Schematic representation of FRET-based DNA unwinding assay. NSP13 protein is depicted as 3D structural model. The complete sequences for 5ˊ flap and Trap-DNA are listed in Table S1. (B) Real-time monitoring of DNA unwinding activity of various concentrations of NSP13. Values are means ± SD, n = 3.
Fig. 2
Fig. 2
The inhibitory effects of PUG on NSP13. (A) The chemical structure of PUG. (B) Dose-inhibition curve for PUG in the FRET-based DNA unwinding assay. Values are means ± SD, n = 3. (C) Native polyacrylamide gel analysis of NSP13 helicase activity in the presence of increasing concentrations of PUG. Asterisk (*) indicates FAM-labeled DNA strands.
Fig. 3
Fig. 3
Direct interaction between PUG and NSP13. (A) SPR measurement of the binding affinity between PUG and NSP13. (B) The binding site of PUG on NSP13 as revealed by molecular docking calculation. NSP13 is shown in surface representation, with each domain labeled and shown in different colors. PUG is shown in stick. The ssRNA/DNA and NTP binding regions are indicated by dashed line and circle, respectively. (C) Detailed interaction between PUG and NSP13. NSP13 is shown in cartoon representation with the same color scheme in (B). The key residues involving in PUG binding are labeled and shown in sticks. Hydrogen bonds are indicated by black dashed lines. (D, E) Effects of E319A (D) and E375A (E) mutations on the inhibitory potency of PUG compared to the wild type (WT). Values are means ± SD, n = 3.
Fig. 4
Fig. 4
Comparisons of PUG with punicalin (PUL) and ellagic acid (EA) on the DNA unwinding activity of NSP13. (A) Structures of PUL and EA. (B) FRET-based measurement of NSP13 helicase activity in the presence of indicated compounds or DMSO (Veh). Values are means ± SD, n = 3. (C) Native polyacrylamide gel analysis of DNA unwinding by NSP13 in the presence of indicated compounds. Asterisk (*) indicates FAM-labeled DNA strands and pound (#) indicates the fluorescence emitted by the compound EA.
Fig. 5
Fig. 5
The inhibition mechanism of PUG against NSP13. (A) Determination of ATP hydrolysis by NSP13 in the presence of indicated compounds using the luciferase-coupled ATP assay. Values are means ± SD, n = 3. (B) The enzyme kinetics analysis of NSP13 in the presence of various concentrations of PUG. Values are means ± SD, n = 3. (C) Effects of various compounds on the DNA binding activity of NPS13 detected by EMSA assay. (D) Quantification of DNA binding in (C). (E) Comparison of the APO (blue, PDB ID: 7NIO) and AMP-PNP-bound (pink, PDB ID: 7NNO) structures of NSP13. Major conformational changes are indicated by red arrows. Dashed line indicates the ssRNA/DNA binding region. (F) Schematic representation summarizing the mechanism of action for NSP13 inhibition by PUG.
Fig. 6
Fig. 6
Antiviral activity of PUG in Vero cells. (A, B) Cytotoxicity of PUG to A549-ACE2 (A) and Vero (B) cells measured by CCK-8 assay. Values are means ± SD, n = 3. (C, D) Dose-response curve of viral inhibition by PUG. A549-ACE2 (C) or Vero (D) cells were infected with SARS-COV-2 in the presence of various concentrations of PUG for 48 h, then the viral yields were determined by qRT-PCR assay. Values are means ± SD, n = 3. (E, F) Indirect immunofluorescence analysis of SARS-COV-2 virus in the cells upon the treatment of PUG at the indicated concentrations. A549-ACE2 (E) or Vero (F) Cells were immunostained 48 h post-infection for the SARS-CoV-2 nucleocapsid protein (red) and cell nuclei (blue).
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