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GSK3326595 is a promising drug to prevent SARS-CoV-2 Omicron and other variants infection by inhibiting ACE2-R671 di-methylation - PubMed

GSK3326595 is a promising drug to prevent SARS-CoV-2 Omicron and other variants infection by inhibiting ACE2-R671 di-methylation

Zhongwei Li et al. J Med Virol. 2023 Jan.

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused COVID-19 epidemic is worsening. Binding of the Spike1 protein of SARS-CoV-2 with the angiotensin-converting enzyme 2 (ACE2) receptor mediates entry of the virus into host cells. Many reports show that protein arginine methylation by protein arginine methyltransferases (PRMTs) is important for the functions of these proteins, but it remains unclear whether ACE2 is methylated by PRMTs. Here, we show that PRMT5 catalyses ACE2 symmetric dimethylation at residue R671 (meR671-ACE2). We indicate that PRMT5-mediated meR671-ACE2 promotes SARS-CoV-2 receptor-binding domain (RBD) binding with ACE2 probably by enhancing ACE2 N-glycosylation modification. We also reveal that the PRMT5-specific inhibitor GSK3326595 is able to dramatically reduce ACE2 binding with RBD. Moreover, we discovered that meR671-ACE2 plays an important role in ACE2 binding with Spike1 of the SARS-CoV-2 Omicron, Delta, and Beta variants; and we found that GSK3326595 strongly attenuates ACE2 interaction with Spike1 of the SARS-CoV-2 Omicron, Delta, and Beta variants. Finally, SARS-CoV-2 pseudovirus infection assays uncovered that PRMT5-mediated meR671-ACE2 is essential for SARS-CoV-2 infection in human cells, and pseudovirus infection experiments confirmed that GSK3326595 can strongly suppress SARS-CoV-2 infection of host cells. Our findings suggest that as a clinical phase II drug for several kinds of cancers, GSK3326595 is a promising candidate to decrease SARS-CoV-2 infection by inhibiting ACE2 methylation and ACE2-Spike1 interaction.

Keywords: ACE2; PRMT5; RBD; SARS-CoV-2; arginine methylation.

© 2022 Wiley Periodicals LLC.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

PRMT5‐mediated ACE2‐SDMA modification promotes ACE2‐RBD interaction. (A) Co‐IP‐Flag or Co‐IP‐GFP detected the amount of ACE2‐RBD association by western blot assays in Flag‐ACE2 and GFP‐RBD coexpression HEK‐293T cells after treated with PRMT5 specific inhibitor GSK3326595 (100 nM) or PRMT1 specific inhibitor AMI‐1 (8.8 μM) for 48 h. (B) SDMA and MMA of ACE2 and interaction of PRMT5 with ACE2 were assessed after IP‐Flag‐ACE2 assays in Flag‐ACE2 overexpressing HEK‐293T cells. (C, D) The amount of ACE2‐SDMA and ACE2‐MMA modification and ACE2‐PRMT5 interaction were detected after IP‐Flag‐ACE2 or IP‐GFP‐RBD assays in Flag‐ACE2 overexpressing HEK‐293T cells (C) or A549 cells (D) following treated with DMSO or GSK3326595 (100 nM). (E, F) The amount of ACE2‐SDMA and ACE2‐MMA modification, ACE2 binding with PRMT5 and ACE2 binding with RBD were assessed by IP‐Flag‐ACE2 or IP‐GFP‐RBD in coexpression of Flag‐ACE2, Myc‐PRMT5, and GFP‐RBD HEK‐293T cells (E) or A549 cells (F). ACE2, angiotensin‐converting enzyme 2; Co‐IP, coimmunoprecipitation; MMA, monomethyl‐arginine; PRMT, protein arginine methyltransferases; RBD, receptor‐binding domain; SDMA, symmetric dimethyl‐arginines.

Figure 2
Figure 2

PRMT5‐mediated ACE2‐R671 SDMA modification is required for ACE2‐RBD interaction. (A, B) MS analysis of Flag‐ACE2 methylation. The fragmentation of the ACE2 peptides AYEWNDNEMYLFmeR (A) and NQMILFGEEDVmeR (B) were identified two methylated sites at R644 and R671. (C) Detection of SDMA‐ACE2 levels in HEK‐293T cells overexpression of WT ACE2, R644K/R671K mutant ACE2, respectively. (D) In vitro methylation assays. Purified GST‐tag fusion proteins of GST‐ACE2‐WT (601−744 aa), were incubated with Myc‐PRMT5 in the presence of SAM (1 μM/μl). SDMA methylation of GST‐ACE2 was detected via western blot by anti‐SDMA antibody and total amounts of proteins were visualized by Coomassie Blue staining (dot: the position of GST‐ACE2‐WT [601−744 aa]; asterisks: the position of Myc‐PRMT5). (E) In vitro methylation assays using Myc‐PRMT5 and SAM (1 μM/μl) to test the methylation of GST‐ACE2‐WT (601−744 aa), GST‐ACE2‐R671K (601−744 aa) proteins (dot: the position of GST‐ACE2‐WT (601−744 aa); asterisks: the position of Myc‐PRMT5). (F) Detection of ACE2‐RBD association levels by IP‐Flag and IP GFP assays in coexpression of GFP‐RBD and Flag‐ACE2‐WT HEK‐293T cells or coexpression of GFP‐RBD and Flag‐ACE2‐R671K HEK‐293T cells, respectively. (G) The amount of ACE2‐SDMA, ACE2‐PRMT5 interaction, and ACE2‐RBD association were assessed by IP‐Flag‐ACE2 or IP‐GFP‐RBD in coexpression of GFP‐RBD and Flag‐ACE2‐WT/R671K A549 cells. (H) GST pull‐down assays to detect ACE2‐RBD association in vitro. First, the GST‐ACE2 and PRMT5 methylated‐GST‐ACE2 were pulled down with the HEK‐293T‐GFP‐RBD lysates, respectively. Then, the GST‐ACE2 SDMA midofication and GFP‐RBD binding with GST‐ACE2 or methylated‐GST‐ACE2 were detected by western blot, respectively. ACE2, angiotensin‐converting enzyme 2; MS, mass spectrometry; PRMT, protein arginine methyltransferases; RBD, receptor‐binding domain; SDMA, symmetric dimethyl‐arginines.

Figure 3
Figure 3

PRMT5‐mediated ACE2‐R671 SDMA modification facilitates ACE2‐N322 glycosylation and subsequently ACE2‐RBD interaction. (A) Detection the amount of ACE2‐RBD and ACE2‐PRMT5 interactions in HEK‐293T cells coexpression of GFP‐RBD and Flag‐ACE2‐WT/R671K/N322Q/(R687K + N322Q), respectively. (B) The interaction of ACE2‐RBD and ACE2‐PRMT5 were assessed by western blot after IP‐Flag‐ACE2 in HEK‐293T cells overexpression of GFP‐RBD and Flag‐ACE2 following treated by DMSO or GSK3326595 (100 nM). (C−E) Assessment of cell proliferation ability by CCK‐8 assays in BEAS‐2B cells, HK2 cells, and A549 cells after treated with GSK3326595 in different concentrations (100, 50, 25, 10 nM). (F, G) Detection of ACE2‐RBD interaction by western blot after IP‐GFP in coexpressing GFP‐RBD and Flag‐ACE2 HEK‐293T cells (F) or A549 cells (G) after treated with GSK3326595 in different concentrations (100, 50, 25, 10 nM). ACE2, angiotensin‐converting enzyme 2; PRMT, protein arginine methyltransferases; RBD, receptor‐binding domain; SDMA, symmetric dimethyl‐arginines.

Figure 4
Figure 4

GSK3326595 prevents the Spike1 proteins of SARS‐CoV‐2 variants from binding with ACE2 through inhibiting PRMT5‐mediated ACE2‐R671 SDMA modification. (A−C) ACE2‐His‐S1 and ACE2‐PRMT5 interaction were detected by IP‐Flag‐ACE2 assays after His‐S1‐Omicron protein (A) or His‐S1‐Delta protein (B) or His‐Beta protein (C) incubating with Flag‐ACE2‐WT HEK‐293T cells lysate or Flag‐ACE2‐R671K HEK‐293T cell lysates, respectively. (D−F) ACE2‐His‐S1 and ACE2‐PRMT5 interaction were detected by IP‐Flag‐ACE2 assays after His‐S1‐Omicron protein (D) or His‐S1‐Delta protein (E) or His‐Beta protein (F) incubating with Flag‐ACE2 HEK‐293T cell lysates which have been treated with DMSO or GSK3326595 (100 nM), respectively. (G−I) Detection of the levels of ACE2 binding with His‐Omicron (G) or His‐Delta (H) or His‐Beta (I) proteins by western blot after IP‐Flag in Flag‐ACE2 HEK‐293T cells which have been treated with GSK3326595 in different concentrations (100, 50, 25 nM). ACE2, angiotensin‐converting enzyme 2; PRMT, protein arginine methyltransferases; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SDMA, symmetric dimethyl‐arginines; S1, Spike1.

Figure 5
Figure 5

GSK3326595 suppresses SARS‐CoV‐2 spike pseudovirus infection. (A, B) Detection of SARS‐CoV‐2 spike pseudovirus infection in Flag‐ACE2‐overexpressing HEK‐293T cells (A) or A549 cells (B) after treated with GSK3326595 in different concentrations (100, 50, 25, 10 nM). (C−F) Detection of SARS‐CoV‐2 spike pseudovirus infection in Flag‐ACE2‐overexpressing HEK‐293T cells (C, D) or A549 cells (E, F) after knocking down PRMT5 expression. (G−J) Assessment of SARS‐CoV‐2 spike pseudovirus infection in Flag‐ACE2‐WT/R671K‐overexpressing HEK‐293T cells (G, H) or Flag‐ACE2‐WT/R671K‐overexpressing A549 cells (I, J). (K, L) The SARS‐CoV‐2 spike pseudovirus infection ability was detected after overexpression of PRMT5 with different dose Myc‐PRMT5 plasmids (1, 2, 3, 4, 5 μg) in HEK‐293T‐Flag‐ACE2 cells, respectively. ACE2, angiotensin‐converting enzyme 2; PRMT, protein arginine methyltransferases; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 6
Figure 6

A proposal model for PRMT5‐mediated meR671‐ACE2 enhances SARS‐CoV‐2 infection. PRMT5‐mediated ACE2 di‐methylation at the R671 site promotes SARS‐CoV‐2 Spike1 binding with ACE2 and subsequent infection of host cells. PRMT5‐specific inhibitor GSK3326595 can attenuate SARS‐CoV‐2 infection by inhibiting ACE2‐R671 di‐methylation. ACE2, angiotensin‐converting enzyme 2; PRMT, protein arginine methyltransferases; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

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