The antipsychotic drug lurasidone inhibits coronaviruses by affecting multiple targets

Abstract

Coronaviruses (CoVs) share key genomic elements critical for viral replication, suggesting the feasibility of developing therapeutics with efficacy across different viruses. In a previous work, we demonstrated the antiviral activity of the antipsychotic drug lurasidone against both SARS-CoV-2 and HCoV-OC43. In this study, our investigations on the mechanism of action of lurasidone suggested that the drug exhibits antiviral activity by targeting the papain-like protease (PL-Pro) of both viruses, and the Spike protein of SARS-CoV-2, thereby hampering both the entry and the viral replication. In vitro assays demonstrate that lurasidone significantly reduces viral load in infected cells, showing that the drug is a promising candidate for further development as a dual-action antiviral, offering a potential new strategy in the fight against COVID-19 and other coronavirus-related diseases.

Keywords: ACE2 interaction; Spike protein; coronaviruses; dual-target compound; papain-like protease.

Figure 1

Effect of lurasidone on SARS-CoV-2 replication and inactivation. ToA experiments: 5 µM lurasidone was added to Huh-7-ACE2 cells for 1 hour before infection (1 h PRE), for 1 hour during infection (CO), or after 1, 4, 18 hours after virus inoculum (POST). Cells were treated with an equivalent volume of DMSO as a control (DMSO). Subsequently, supernatants were collected at 24 hours post-infection, and virus samples were titrated. Viral titers are expressed as mean PFU/mL and shown as average values with standard deviation and p-values, measured with a paired two-tailed t-test. Significant p-values, calculated from at least 2 independent experiments, are indicated by asterisks (**p < 0.01; *p < 0.05).

Figure 2

Effect of Lurasidone on pseudoviral transduction. (A) Percentage of GFP-positive HEK293-ACE2 cells pre-incubated for 4 h with 1 or 5 µM lurasidone and infected with pseudovirus particles exposing SARS-CoV-2 Wuhan, B.1.1.7 UK or B.1.351 South Africa (SA) Spike protein. Control cells were pre-incubated with an equivalent volume of DMSO (Vehicle). Data are the mean ± SD of GFP-positive cells compared to the control. *** p < 0.001 vs the corresponding vehicle according to one-way ANOVA and Bonferroni’s post hoc test. (B) Representative fluorescence microscopy images of cells treated or not with lurasidone and infected with pseudovirus displaying the different SARS-CoV-2 Spike isoforms on the envelope. Scale bar = 100 µm.

Figure 3

Effect of lurasidone on ACE2 expression and ACE2 binding. (A) Quantitative analysis of representative Western blot performed on HEK293-ACE2 cell lysates incubated for 3, 6 or 24 hours with 5 µM lurasidone. Control cells were treated with an equivalent volume of DMSO (Vehicle). ACE2 quantification is expressed as the percentage of the mean volume of the ACE2 band immunoreactivity/activity of the vehicle at the same time point. Data are the mean ± SD of three separate experiments. (B) Direct binding of lurasidone to rhACE2. SPR sensorgram revealed no binding signal after the injection of 10 µM lurasidone on immobilized rhACE2 (blue line) compared with the curve obtained after 10 nM SARS-CoV-2 Spike RBD Wuhan injection, used as a positive control (black line). The results shown are corrected with the data obtained on the reference channel.

Figure 4

Modeling of lurasidone in the interface between Spike-ACE2. SARS-CoV-2 RBD complex (PDB 6M0J) showing (A) lurasidone poses docked with DiffDock-L (rank 1 in black) highlighting the putative binding site at the interface between Spike (in green) and ACE2 (in white). A secondary low-ranked site is on the side of Spike protein (gray); (B) overview of the putative binding site as seen from the RBD: ACE2 interface. The 10 top ranked DiffDock poses are shown, with rank 1 in black. The residues characterizing the WT, B.1.1.7 and B.1.351 SARS-CoV-2 strains are shown in red (K417 and E484) or in bright green (N501).

Figure 5

Investigation of the mechanism of action of lurasidone against HCoV-OC43. (A) Time-of-addition experiments. The drug at 50 µM was added to cells for 1 hours before infection (pre-treatment), for 1 hour during infection (co-treatment), or after 1-4-18 hours from virus inoculum (post-treatment); subsequently, supernatants were collected 24 hours post-infection, and virus samples were titrated. Treated and control (UT) samples were compared with one-way ANOVA. On the y-axis, viral titers are expressed as FFU/mL and shown as mean ± SEM for three independent experiments. UT, untreated. n.s., not significant. *** p < 0.001. (B) Entry assay. Cells were treated with serial dilutions of lurasidone (100–0.4 μM) during virus entry, and viral infectivity was assessed 16 hours post-infection by immunostaining. The percent infection (%) was calculated by comparing treated and untreated wells. Error bars represent SEMs for three independent experiments.

Figure 6

Putative binding site of lurasidone on PL-Pro proteins from SARS-CoV-2 (6WZU) and HCoV-OC43 (AF2) both colored by domain: ubiquitin-like in yellow, thumb in blue, finger in red and palm in green. (A) Front view (left) of SARS-CoV-2 showing lurasidone poses obtained with DiffDock (rank 1 in black) and the catalytic amino acids in light blue; side view (right) of the same structure; all the docking poses are located between the loop of the palm and the one of the thumb domains, nearby the catalytic site. (B) Front view (left) of HCoV-OC43 showing lurasidone poses (rank 1 in black) and the catalytic amino acids in light blue; side view (right) of the same model; again, all the docking poses are located between palm and the thumb loops, nearby the catalytic site.