Heme oxygenase-1 inducer hemin does not inhibit SARS-CoV-2 virus infection

Abstract

Antiviral agents with different mechanisms of action could induce synergistic effects against SARS-CoV-2 infection. Some reports suggest the therapeutic potential of the heme oxygenase-1 (HO-1) enzyme against virus infection. Given that hemin is a natural inducer of the HO-1 gene, the aim of this study was to develop an in vitro assay to analyze the antiviral potency of hemin against SARS-CoV-2 infection. A SARS-CoV-2 infectivity assay was conducted in Vero-E6 and Calu-3 epithelial cell lines. The antiviral effect of hemin, and chloroquine as a control, against SARS-CoV-2 virus infection was quantified by RT-qPCR using specific oligonucleotides for the N gene. Chloroquine induced a marked reduction of viral genome copies in kidney epithelial Vero-E6 cells but not in lung cancer Calu-3 cells. Hemin administration to the culture medium induced a high induction in the expression of the HO-1 gene that was stronger in Vero-E6 macaque-derived cells than in the human Calu-3 cell line. However, hemin treatment did not modify SARS-CoV-2 replication, as measured by viral genome quantification 48 h post-infection for Vero-E6 and 72 h post-infection for the Calu-3 lineages. In conclusion, although exposure to hemin induced strong HO-1 up-regulation, this effect was unable to inhibit or delay the progression of SARS-CoV-2 infection in two epithelial cell lines susceptible to infection.

Keywords: Antiviral effect; Heme oxygenase-1 induction; Hemin; SARS-CoV-2 infection.

Graphical abstract

Fig. 1

Effects of hemin administration on rate limiting enzymes of heme synthesis and catabolism. A) Regulation of heme synthesis and potential antiviral effect of hemin administration. The first and rate limiting step in heme synthesis is the conversion of glycine and succinyl-coenzyme A to form δ-aminolevulinic acid (ALA), catalyzed by ALA synthase-1 (ALAS1). Heme oxygenase-1 (HO-1) is the inducible isoform of HO responsible for the oxidative cleavage of heme groups to yield carbon monoxide (CO) and biliverdin, which is subsequently reduced to bilirubin by the cytosolic biliverdin reductase enzyme. CO is a strong vasodilatory, anti-inflammatory and immunomodulatory agent and both biliverdin reductase B and bilirubin are efficient scavengers of Reactive Oxygen Species (ROS) and reduce the formation of peroxidation products. Fold-change expression of HO-1 in B) Vero-E6 and C) Calu-3 cell lines was measured after exposure to 64.4 µM of hemin. Immunoblot analyses of HO-1 protein levels in D) Vero-E6 and E) Calu-3 cell lines treated with two hemin concentrations (21.5 μM and 64.4 μM) for 48 h. F) Fold change of ALAS1 expression after hemin exposure. Results were plotted as mean ± SD from three experiments. *** p < 0.001 vs untreated control condition.

Fig. 2

Hemin did not inhibit SARS-CoV-2 infection in Vero-E6 and Calu-3 cell lines. A) Quantification of SARS-CoV-2 genomes determined by RT-qPCR of supernatants collected at 48 h for Vero-E6 cells or at 72 h for Calu-3 cells in the absence (untreated control) or presence of hemin (64.4 µM) added 24 h before infection (hemin pre-treatment) or at the same time as infection (hemin). B) A similar study was performed using chloroquine (10 µM). In all conditions, the drug was maintained during the incubation period and the medium changed every 24 h. Pre-treat: pre-treatment. CQ: chloroquine. Results were plotted as mean ± SD (one representative experiment is shown of at least two with similar results). *** p < 0.001 vs untreated control condition.