Xiaolei Liu, Anurag Verma, Gustavo Garcia Jr, Holly Ramage, Anastasia Lucas, Rebecca L. Myers, Jacob J. Michaelson, William Coryell, Arvind Kumar, Alexander W. Charney, Marcelo G. Kazanietz, Daniel J. Rader, Marylyn D. Ritchie, Wade H. Berrettini, David C. Schultz, Sara Cherry, Robert Damoiseaux, Vaithilingaraja Arumugaswami, and Peter S. Klein

PNAS October 19, 2021 118 (42) e2113401118

Significance

COVID-19 is taking a major toll on personal health, healthcare systems, and the global economy. With three betacoronavirus epidemics in less than 20 y, there is an urgent need for therapies to combat new and existing coronavirus outbreaks. Our analysis of clinical data from over 300,000 patients in three major health systems demonstrates a 50% reduced risk of COVID-19 in patients taking lithium, a direct inhibitor of glycogen synthase kinase-3 (GSK-3). We further show that GSK-3 is essential for phosphorylation of the SARS-CoV-2 nucleocapsid protein and that GSK-3 inhibition blocks SARS-CoV-2 infection in human lung epithelial cells. These findings suggest an antiviral strategy for COVID-19 and new coronaviruses that may arise in the future.

Abstract

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35–0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type–dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

See: https://www.pnas.org/content/118/42/e2113401118

Fig. 1. The SARS-CoV-2 N protein is phosphorylated by GSK-3 in two conserved consensus sites. (A) The RS domains of SARS-CoV-2 (amino acids 176 to 206) and SARS-CoV N proteins are 90% identical and contain tandem sets of SXXXS motifs, labeled “a” and “b.” Consensus site serines and threonines are in bold; red indicates sites shown previously by mass spectroscopy to be phosphorylated (1, 8). (B) Alignment of RS domains in N proteins from pathogenic CoVs showing conservation of repeated SXXXS motifs (“S” in motif represents serine or threonine) and a highly conserved arginine 3 residues before the putative priming sites (SRXXS). Blue indicates β-CoV; orange: α-CoV; green: γ-CoV. (C) SARS-CoV-2 N was expressed in 293T cells. Cells were treated 24 h after transfection with LiCl for an additional 18 h and then harvested and subjected to SDS/PAGE and immunoblotting for N protein (CoV2-N), phosphorylated glycogen synthase (pGS), or β-actin as a loading control. Alkaline phosphatase (AP) treatment of cell lysates increases electrophoretic mobility. LiCl inhibits N phosphorylation with IC₅₀ ∼10 mM. Ctl, nontreated control; NT, nontransfected; “phos” indicates phosphorylated N protein; “unphos” indicates dephosphorylated N. (D) SARS-CoV-2 N expressing 293T cells were treated at 24 h posttransfection with BIM-I or CHIR99021 (CHIR) at the indicated concentrations. Cell lysates were harvested after 18 h and immunoblotted as in C.