Targeting the Pentose Phosphate Pathway for SARS-CoV-2 Therapy

Abstract

SARS-CoV-2 is causing the coronavirus disease 2019 (COVID-19) pandemic, for which effective pharmacological therapies are needed. SARS-CoV-2 induces a shift of the host cell metabolism towards glycolysis, and the glycolysis inhibitor 2-deoxy-d-glucose (2DG), which interferes with SARS-CoV-2 infection, is under development for the treatment of COVID-19 patients. The glycolytic pathway generates intermediates that supply the non-oxidative branch of the pentose phosphate pathway (PPP). In this study, the analysis of proteomics data indicated increased transketolase (TKT) levels in SARS-CoV-2-infected cells, suggesting that a role is played by the non-oxidative PPP. In agreement, the TKT inhibitor benfooxythiamine (BOT) inhibited SARS-CoV-2 replication and increased the anti-SARS-CoV-2 activity of 2DG. In conclusion, SARS-CoV-2 infection is associated with changes in the regulation of the PPP. The TKT inhibitor BOT inhibited SARS-CoV-2 replication and increased the activity of the glycolysis inhibitor 2DG. Notably, metabolic drugs like BOT and 2DG may also interfere with COVID-19-associated immunopathology by modifying the metabolism of immune cells in addition to inhibiting SARS-CoV-2 replication. Hence, they may improve COVID-19 therapy outcomes by exerting antiviral and immunomodulatory effects.

Keywords: 2-deoxy-d-glucose; COVID-19; SARS-CoV-2; antiviral therapy; benfooxythiamine; oxythiamine; pentose phosphate pathway.

Figure 1

Glycolysis and pentose phosphate pathway (PPP) are deregulated during SARS-CoV-2 infection. (A) Schematic of proteomic analysis of SARS-CoV-2 infected Caco-2 cells. (B) Heatmap of changes in protein abundance of components of glycolysis and pentose phosphate pathway in SARS-CoV-2 infected Caco-2 cells at 24 h post infection. A Z score transformation was performed such that red and blue represent high and low protein abundance, respectively. The plot was performed using the heatmaps2 function of the gplots package of the R suite. (C) Pathway depiction of main regulators of glycolysis and PPP using BioRender.

Figure 2

SARS-CoV-2 infection causes metabolic shift towards glycolysis. Caco-2 cells were infected with untreated or UV-inactivated SARS-CoV-2/FFM7 at an MOI of 0.01 for 24 h before measurement. (A–C) Pathway-specific ATP production (A) derived from mitochondrial respiration (B) and glycolysis (C) measurement. Mean + SD from three biological replicates are displayed. Statistical significance was determined through one way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns: not significant. (D–F) Measurement of mitochondrial respiration and glycolytic function under stress conditions (Mito stress Test). Mean + SD from three biological replicates are displayed. ECAR, extracellular acidification rate; OCR, oxygen consumption rate.

Figure 3

Targeting the pentose phosphate pathway (PPP) in SARS-CoV-2-infected cells. (A,B) Effects of the TKT inhibitor benfooxythiamine (BOT) on virus replication and cell viability in SARS-CoV-2 strain FFM1 and FFM7 (MOI 0.01)-infected Caco2 cells 24 h post infection. Caco-2 cells were pre-treated with different concentrations of BOT for 24 h. Percentage of viral inhibition was evaluated by spike (S) protein staining, and cell viability was measured by MTT assay. The IC50 and CC50 values were determined using the curve regression function of GraphPad Prism 8. Both plots represent mean ± SD of the three independent experiments. (C) Representative images illustrating SARS-CoV-2 S protein levels in SARS-CoV-2/FFM1 and SARS-CoV-2/FFM7 (MOI 0.01) infected Caco-2 cells treated with BOT 24h post infection. (D) BOT treatment strategy of air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells infected with SARS-CoV-2/FFM7 (MOI 1). (E) Quantification of viral genomic RNA copy numbers in apical washes five days post infection. Bars represent mean ± SD of the three biological replicates. (F) Cytotoxicity assay measuring LDH levels in apical washes of BOT-treated HBE ALI cultures in comparison to untreated control. Bars display mean ± SD of the three biological replicates. *** p ≤ 0.001.

Figure 4

Inhibition of viral infection in benfooxythiamine (BOT)-treated cells in combination with 2-deoxy-d-glucose (2DG). Caco-2 cells were pre-treated with different concentration of BOT for 24 h. Then, the 2DG at concentration 5mM or 10 mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. (A) Representative images illustrating immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. (B) Quantification of illustrating immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Values represent the mean ± SD of the three independent experiments. p-values were determined with a two-sided unpaired t-test. *** p ≤ 0.001 (C) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. SARS-CoV-2/FFM7 RNA copy numbers used the RNA-polymerase (RdRp) gene by qRT-PCR of RdRp gene. Values represent mean ± SD of the three independent experiments. p-values were determined with a two-sided unpaired t-test. ns: not significant; * p ≤ 0.05; ** p ≤ 0.01. Effects of BOT in combination with 2DG on cell viability are provided in Supplementary Figure S1. (D) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com (accessed on 18 August 2021).