Antidepressant and Antipsychotic Drugs Reduce Viral Infection by SARS-CoV-2 and Fluoxetine Shows Antiviral Activity Against the Novel Variants in vitro

Abstract

Repurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants. Several antidepressants and antipsychotic drugs with different primary mechanisms of action were tested in ACE2/TMPRSS2-expressing human embryonic kidney cells against the infection by SARS-CoV-2 spike protein-dependent pseudoviruses. Some of these compounds were also tested in human lung epithelial cell line, Calu-1, against the first wave (B.1) lineage of SARS-CoV-2 and the variants of concern, B.1.1.7, B.1.351, and B.1.617.2. Several clinically used antidepressants, including fluoxetine, citalopram, reboxetine, imipramine, as well as antipsychotic compounds chlorpromazine, flupenthixol, and pimozide inhibited the infection by pseudotyped viruses with minimal effects on cell viability. The antiviral action of several of these drugs was verified in Calu-1 cells against the B.1 lineage of SARS-CoV-2. By contrast, the anticonvulsant carbamazepine, and novel antidepressants ketamine, known as anesthetic at high doses, and its derivatives as well as MAO and phosphodiesterase inhibitors phenelzine and rolipram, respectively, showed no activity in the pseudovirus model. Furthermore, fluoxetine remained effective against pseudoviruses with common receptor binding domain mutations, N501Y, K417N, and E484K, as well as B.1.1.7 (alpha), B.1.351 (beta), and B.1.617.2 (delta) variants of SARS-CoV-2. Our study confirms previous data and extends information on the repurposing of these drugs to counteract SARS-CoV-2 infection including different variants of concern, however, extensive clinical studies must be performed to confirm our in vitro findings.

Keywords: SARS-CoV-2; alpha variant; antidepressants (AD); beta variant; delta variant; fluoxetine.

FIGURE 1

Protocols used in the study. Protocol I represents the experiments in HEK293T-ACE-TMPRSS2 cell line, while Protocol II corresponds to the experiments conducted in Calu-1 cell line. created with BioRender.com.

FIGURE 2

The effect of antidepressant drugs on HEK293T-ACE2-TMPRSS2 cells challenged with SARS-CoV-2 pseudotyped viruses. Treatment with (A) fluoxetine (luciferase assay: n = 18–26; cell viability: n = 6; IC50 = 5.992 µM), (B) citalopram (luciferase assay: n = 16–22; cell viability: n = 6; IC50 = 27.51 µM), (C) paroxetine (luciferase assay: n = 12; cell viability: n = 5; IC50 = 12.55 µM), (D) fluvoxamine (luciferase assay: n = 14–24; cell viability: n = 6; IC50 = 10.54 µM), (E) venlafaxine (luciferase assay: n = 11–12; cell viability: n = 5; IC50 = 36.35 µM), (F) reboxetine (luciferase assay: n = 12; cell viability: n = 6; IC50 = 17.69 µM), (G) clomipramine (luciferase assay: n = 12–24; cell viability: n = 6; IC50 = 0.75 µM), (H) imipramine (luciferase assay: n = 6–18; cell viability: n = 6; IC50 = 3 µM), and (I) desipramine (luciferase assay: n = 12; cell viability: n = 5; IC50 = 8.097 µM) significantly reduced luciferase reporter activity. At higher concentrations, all the compounds, except (E) venlafaxine and (F) reboxetine reduced ATP levels in the luminescent cell viability assay after 24 h incubation. *p < 0.05 from control group (0). Data represented as mean ± SEM.

FIGURE 3

The effect of antidepressant drugs on SARS-CoV-2 (B.1) infection in Calu-1 cells. (A,B) Fluoxetine (n = 6), (D,E) citalopram (n = 4–6), (G,H) reboxetine (n = 5–6), and (J,K) clomipramine (n = 6) reduced the genome copy number of SARS-CoV-2 measured by qRT-PCR and also the amount of infectious viral particles detected by TCID50 assay after 48 h of treatment. (M) Imipramine (n = 6) reduced the genome copy number, while (N) failed to reduce TCID50 (n = 6). (P,Q) Venlafaxine (n = 5–6) was ineffective for changing the genome copy number of SARS-CoV-2 and TCID50. (C,F,I,L,R) All the compounds, (O) except imipramine, reduced the viability of Calu-1 cells infected with the native SARS-CoV-2 (n = 6). Genome copies were plotted in linear scale, while TCID50 values in log scale. Fluoxetine: FLX, Citalopram: CIT, Reboxetine: RBX, Clomipramine: CMI, Imipramine: IMI, Venlafaxine: VEN. *p < 0.05 from control group (0) at corresponding time point. Data represented as mean ± SEM.

FIGURE 4

Antiviral activity of antipsychotics in HEK293T-ACE2-TMPRSS2 cells challenged with pseudotyped viruses and in Calu-1 cells infected with SARS-CoV-2 (B.1). Decline of luciferase reporter activity was observed at multiple doses of (A) chlorpromazine (luciferase assay: n = 8–20; cell viability: n = 8; IC50 = 0.972 µM), (B) flupenthixol (luciferase assay: n = 12; cell viability: n = 5; IC50 = 1.072 µM), and (C) pimozide (luciferase assay: n = 12; cell viability: n = 5; IC50 = 4.539 µM) following 24 h incubation suggesting reduced infection. Treatment of cells with (A) chlorpromazine, (B) flupenthixol, and (C) pimozide combined with viral infection decreased cell viability indicated by the ATP level. (D) Treatment with chlorpromazine for 48 h decreased the genome copy number of SARS-CoV-2 (n = 5–6) in qRT-PCR, but (E) failed to reduce the amount of infectious virus in TCID50 assay (n = 6). (F) The highest dose of chlorpromazine treatment increased the toxicity in infected Calu-1 cells indicated by the ATP level. Genome copies were plotted in linear scale, while TCID50 values in log scale. Chlorpromazine: CPZ. *p < 0.05 from control group (0). #p < 0.05 from control group (0) at corresponding time point. Data represented as mean ± SEM.

FIGURE 5

Antiviral activity of fluoxetine against the mutant pseudo-viruses and the native variants of SARS-CoV-2. Treatment with fluoxetine (10 μM, 24 h) significantly reduced the luciferase reporter activity in HEK293T-ACE2-TMPRSS2 cells infected with (A) WT (grey, n = 16), N501Y (blue, n = 18), K417N (red, n = 18), (B) E484K (green, n = 8–9) and (C) triple mutant (N501Y/K417N/E484K, orange, n = 13–16), viral particles. Fluoxetine (10 µM) reduced genome copy number and TCID50 in Calu-1 cells infected with (D,E) B.1.1.7 (alpha), (G,H) B.1.351 (beta), and (J,K) B.1.617.2 (delta) variants, measured by qRT-PCR and TCID50 assay, respectively (n = 6). (F,I,L) Fluoxetine also slightly reduced the Calu-1 viability in cells infected with the variants (n = 6) measured by the ATP levels. Fluoxetine: FLX and wild type: WT. *p < 0.05 from control group (veh). #p < 0.05 from control group (0). Data represented as mean ± SEM.