AMPK inhibitor, compound C, inhibits coronavirus replication in vitro

Abstract

The coronavirus disease (COVID-19) pandemic has resulted in more than six million deaths by October 2022. Vaccines and antivirals for severe acute respiratory syndrome coronavirus 2 are now available; however, more effective antiviral drugs are required for effective treatment. Here, we report that a potent AMP-activated protein kinase (AMPK) inhibitor, compound C/dorsomorphin, inhibits the replication of the human coronavirus OC43 strain (HCoV-OC43). We examined HCoV-OC43 replication in control and AMPK-knockout (KO) cells and found that the virus replication decreased in AMPK-KO cells. Next, we examined the effect of the AMPK inhibitor, compound C on coronavirus replication. Compound C treatment efficiently inhibited the replication and decreased the coronavirus-induced cytotoxicity, further inhibiting autophagy. In addition, treatment with compound C in combination with chloroquine synergistically inhibited coronavirus replication. These results suggest that compound C can be considered as a potential drug candidate for COVID-19.

Fig 1. Coronavirus replication is decreased in AMPK-knockout (KO) cells.

(A) The expression level of coronavirus proteins in AMPK-KO cells. HEK293T control cells and AMPK-KO cells were infected with the indicated dilutions of HCoV-OC43 coronavirus. Cells were collected after 72 h of infection and equal amount of cell lysates were subjected to western blotting with the indicated antibodies. (B) The expression level of coronavirus proteins in the media of AMPK-KO cells. Media of virus-infected cells were collected and probed with anti-HCoV-OC43 antibody. Coomassie staining of media was used as a loading control. (C and D) The level of OC43 proteins were quantitated and denoted as a graph in cell lysates (C) showing mean ± standard error and in media (D) showing mean ± standard deviation. Control vs KO cells: *, P < 0.05, n = 5. (E) HEK293T control and AMPK-KO cells were infected with coronavirus (10⁻³ dilution), and RNA was collected from the media. The RNA level of RdRp gene, M gene and N gene was evaluated using quantitative RT-PCR. Control vs KO cells: *, P < 0.05, **, P < 0.01, ***, P < 0.005, n = 4.

Fig 2. Compound C treatment inhibits coronavirus replication.

(A) RD cells were infected with coronavirus (10⁻³ dilution) and incubated with the indicated concentrations of compound C. Cells and media were collected after 72 h of infection and probed with anti-HCoV-OC43 antibody. Ponceau S staining was used as a loading control for media western blot. (B) The expression of coronavirus protein was q uantitated and denoted as a graph. Control vs compound C treated: *, P < 0.05, ***, P < 0.005, n = 4. (C and D) RD cells were infected with coronavirus, and incubated with the indicated concentration of compound C. After 72 h of infection, RNA was collected from the cells (C) and media (D) and the RNA level of RdRp gene, M gene and N gene was evaluated by quantitative RT-PCR. Control vs compound C treated: *, P < 0.05, **, P < 0.01, ***, P < 0.005, n = 4.

Fig 3. Compound C treatment inhibits the coronavirus infection.

(A) HEK293T cells and RD cells were incubated with coronavirus (10⁻³ dilution), and with the indicated concentrations of compound C. Cells were fixed 48 h after infection and stained with anti-HCoV-OC43 antibody. Bars: 10 μM. (B and C) Coronavirus infected cells were counted and the percentage of infection was calculated in HEK293T cells (B) and RD cells (C). Control vs compound C treated: ***, P < 0.005, n = 13. (D) Compound C treatment induced HCoV-OC43 cytotoxicity. RD cells were infected with mock or HCoV-OC43 and treated with control or compound C followed by agarose overlay. To visualize the cytotoxicity, the infected cells were fixed and stained with crystal violet. (E) RD cells were infected with mock or HCoV-OC43 and incubated with control or compound C for 5 days. Cell viability was measured by MTT assay. ***, P < 0.005, n = 4.

Fig 4. Combination of compound C (CC) and chloroquine (CQ) treatment synergistically inhibits the coronavirus infection.

(A) HEK293T cells were incubated with coronavirus (10⁻³ dilution), and incubated with the indicated concentrations of CC and CQ. Cells were collected 48 h after infection and subject to western blot with anti-HCoV-OC43 antibody. (B) The level of OC43 proteins were quantitated and denoted as a graph. **, P < 0.01, ***, P < 0.005, n = 3. (C and D) The expression levels of p62 and LC3 were quantitated and denoted as a graph. **, P < 0.01, n = 3. (E) HEK293T cells were incubated with coronavirus (10⁻³ dilution), and incubated with the indicated combinations of CC and CQ. Cells were fixed 48 h after infection and stained with anti-HCoV-OC43 antibody. Bars, 10 μM. (F) Coronavirus infected cells were calculated in HEK293T cells and denote in graph. ***, P < 0.005, n = 6. (G) Schematic diagram showing the action of compound C and chloroquine on the autophagy.