WO2021249982A1

WO2021249982A1 - Antiviral compound

Info

Publication number: WO2021249982A1
Application number: PCT/EP2021/065247
Authority: WO
Inventors: Alberto BOLDRINI; Luca TERRUZZI; Tania MASSIGNAN; Lidia PIERI; Andrea ASTOLFI
Original assignee: Sibylla Biotech S.R.L.
Priority date: 2020-06-08
Filing date: 2021-06-08
Publication date: 2021-12-16
Also published as: IT202000013564A1

Abstract

The subject of the present invention is Artefenomel or its pharmaceutically acceptable salts for use in the treatment of pathologies related to overexpression and / or over activity of ACE2 and / or receptor activity of ACE2 (Angiotensin-converting enzyme 2). In a preferred form, it is claimed Artefenomel or its pharmaceutically acceptable salts for use in the treatment of SARS-related coronavirus infections. A further object of the present invention is a pharmaceutical composition which comprises Artefenomel or its pharmaceutically acceptable salts for use in the treatment of pathologies related to overexpression and / or over activity of ACE2 and / or the receptor activity of ACE2.

Description

“Antiviral compound”

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The present invention relates to 4- (2- {4 - [(1s, 3s, 4's) -dispiro [adamantane- 2,2'- [1,3,5] trioxolane-4',1"- cyclohexane]-4" - yl] phenoxy} ethyl) morpholine or a pharmaceutically acceptable salt thereof, for use in the treatment of diseases related to overexpression and / or over activity of ACE2 and / or the receptor activity of ACE2 (Angiotensin-converting enzyme 2). The invention also relates to an association and a pharmaceutical composition comprising said 4- (2- {4 - [(1s, 3s, 4's) -dispiro [adamantane-2,2 '- [1,3,5] trioxolane-4 ', 1"- cyclohexane] -4"- yl] phenoxy} ethyl) morpholine or a pharmaceutically acceptable salt thereof for use in the treatment of diseases related to overexpression and / or over activity of ACE2 and / or the receptor activity of ACE2 (Angiotensin-converting enzyme 2).

Background

SARS-related coronavirus, or SARS-CoV, is a coronavirus that infects humans, a member of the genus Betacoronavirus, subgenus Sarbecovirus. It is a single-stranded RNA virus that enters the host cell by binding, via a surface protein, called spike, to the ACE2 receptor (Ge XY et al. 2013 Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature, 503: 535-538).

There are hundreds of strains of SARS-CoV, only some of these are responsible for diseases in humans. Coronaviruses generally involve mild forms affecting the upper airways, however at least 3 strains of the virus have caused outbreaks of severe respiratory disease in humans: SARS-CoV, which caused a SARS outbreak between 2002 and 2003, MERS-CoV, identified in 2012 as the cause of Middle East Respiratory Syndrome and SARS-CoV-2, which caused the 2019-2020 COVID-19 pandemic (Alexander E et al. 2020 The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nature Microbiology, 5: 536-544).

4- (2- {4 - [(1s, 3s, 4's) -dispiro [adamantane-2,2¹- [1,3,5] trioxolane-4', 1" - cyclohexane] -4" - yl ] phenoxy} ethyl) morpholine, called Artefenomel, is a synthetic trioxolane which has been shown to be effective in the treatment of P. falciparum and P. Vivax malaria, with a better safety and pharmacokinetic profile than other antimalarial peroxides (Ashley EA, Phyo AP 2018 Drugs in development for malaria. Drugs. 78: 861-79; Moherle JJ et al. 2013 First-in- man safety and pharmacokinetics of synthetic ozonide OZ439 demonstrates an improved exposure profile relative to other peroxide antimalarials. Br J Clin Pharmacol. 75: 524-37).

Given the rapid and global spread of the virus, and the danger of the same, there is a strong need to readily have molecules effective against it.

Description

The authors of the present invention have surprisingly observed that Artefenomel reduces ACE2 protein levels.

Therefore, it forms an object of the present invention Artefenomel or its pharmaceutically acceptable salts for use in the treatment of pathologies related to overexpression and / or over activity of ACE2 and / or receptor activity of ACE2 (Angiotensin-converting enzyme 2).

The authors also demonstrated that Artefenomel has a virucidal effect on SARS-CoV-2. Therefore, in a preferred form, the object of the present invention is Artefenomel or its pharmaceutically acceptable salts for use in the treatment of Coronavirus infections.

Brief drawing description

Figure 1: ACE2 expression in the indicated different cell lines, highlighted with anti-ACE2 antibody.

Figure 2: Vero cells exposed to Artefenomel (SIB-P012-M032) at the indicated concentrations. (A) effect on the expression of ACE2; (B) cell viability.

Figure 3: plasmids used in the experimental section: (A) MLV packaging vector, pc Gag-Pol; (B) env-encoding vector, pc SARS-CoV-2 spike AC and (C) MLV transfer vector, pc NCG.

Figure 4: Effect of Artefenomel on the transduction efficiency of a pseudo- typed retroviral vector in Vero (A, B, C) and HEK293#35 cells (D, E, F). (A, D) ACE2 expression; (B, E) Infection with pseudo-typed; (C, F) MTT.

Figure 5: ACE2 expression levels after treatment with Artefenomel at 10 mM (triangle) or 30 mM (circle) in a time course experiment in Vero cells.

Figure 6: ACE2 mRNA (A, C) and protein (B, D) expression levels. (A, B) time course experiment; (C, D) dose-response experiment, after 48h Artefenomel treatment in Vero cells.

Figure 7: ACE1 expression levels after treatment with Artefenomel (M032) in Vero cells.

Figure 8: Chloroquine effect on (A) ACE2 expression; (B) cell viability.

Figure 9: Viability in uninfected Vero E6 cells (percentage values). Results show the extent of cell viability as determined by the neutral red uptake assay (A540) after 4 days. Data is normalized to the values observed in cells in the absence of test-items (“vehicle”, medium only). Results show the average of triplicate data points with the standard deviation (s.d.). Average and standard deviation values for cells treated with vehicle only are derived from six replicates. In the highest concentration, one data point was removed as an outlier.

Figure 10: Inhibition by Test-items of the CPE mediated by SARS-CoV-2 (percentage values). Values show the inhibition of the SARS-CoV-2 induced CPE, as a surrogate marker for virus replication. The values are normalized to the A540 values observed in uninfected cells after subtraction of the average absorbance observed in infected cells in the presence of vehicle. Values in uninfected cells (“mock”) are included for comparison (100% inhibition). Data plotted for test-items shows the average and standard deviation of triplicates. Bottom graphs display the dose-response observed with GS-441524 from both plates. Figure 11: IC50 values for Inhibition of SARS-CoV-2 CPE by Test-items- separate plates and overlay. Values indicate the percentage inhibition of the CPE induced by live SARS-CoV-2 (MEX-BC2/2020), as compared to samples incubated with no test-item (vehicle alone). Results show the average of triplicate data points run on two separate plates. When possible, data was modelled to a sigmoidal function using GraphPad Prism software fitting a normalized dose-response curve with a variable slope.

Figure 12: Microscopic evaluation of monolayers of Vero E6 cells after 96h infection with SARS-CoV-2 (MEX-BC2/2020). Images from infected cells (B- E), or mock-infected cells (A) are shown after infection for 4 days with SARS- CoV-2 in the absence or the presence of test-items and control inhibitors. (A) Mock-infected cells; (B) infection in the presence of vehicle alone; (C) infection with 10mM GS-441524; (D) infection with 100pM Artefenomel and (E) infection with 33mM Artefenomel.

Figure 13: IC50 values for Inhibition of SARS-CoV-2 CPE by GS-441524- separate plates. Values indicate the percentage inhibition of the CPE induced by live SARS-CoV-2 (MEX-BC2/2020), as compared to samples incubated with no test-item (vehicle alone). Results from two separate plates show single data points for GS-441524. Data was modelled to a sigmoidal function using GraphPad Prism software fitting a dose-response curve with a variable slope (four parameters). The authors of the present invention used the PPI- FIT methodology, capable of identifying molecules that reduce the expression of a target protein at the post-translational level. The PPI-FIT method, described in W02020021493, combines the simulation of protein folding mechanisms with virtual screening approaches, so as to predict the ability of compounds to block protein expression, acting on folding intermediates, stabilizing them. In this way the protein is recognized as incorrectly folded and therefore degraded.

The PPI-FIT methodology was applied to ACE2, obtaining the all-atom simulation of the entire sequence of events underlying the ACE2 folding path. The data obtained revealed the existence of a folding intermediate showing potentially druggable pockets, not present in the native conformation. A virtual screening campaign was then conducted aimed at these pockets, with the aim of identifying, among the compounds already in clinical use, molecules capable of decreasing the expression of ACE2.

Virtual screening led to the selection of 35 compounds predicted to bind the identified folding intermediate. Among these, Artefenomel, of Formula (I).

Formula (I)

In an in vitro model, the expression of ACE2 was evaluated following treatment with Artefenomel, in comparison with untreated control. The data showed a decrease in ACE2 expression levels following exposure to Artefenomel. Artefenomel decreases the expression of ACE 2 in a dose- response manner, where the dose-response is observed and it is significant starting from the 1 mM dose and up to the maximum tested dose, 300 mM. Therefore, form the subject of the present invention Artefenomel or its pharmaceutically acceptable salts for use in the treatment of SARS-related coronavirus infections. In one embodiment, said virus is SARS-CoV. In a further embodiment, said virus is SARS-CoV-2. In a further embodiment, said virus is MERS-CoV.

A further object of the present invention is a composition comprising Artefenomel or its pharmaceutically acceptable salts for the prevention or treatment of infectious diseases from a virus belonging to the SARS-related coronavirus family.

Said composition is an oral pharmaceutical preparation, a solution for injection, a nasal aerosol or an inhalant. For example, it is a tablet, a coated tablet, an effervescent tablet, a capsule, powder, granules, sugar-coated tablets, lozenges, pills, drops, suppositories, emulsion, inhalation mixture, aerosol, mouth spray, nose spray .

Said composition is administered by oral, intranasal, topical, rectal, bronchial, or parenteral administration, or by any clinically accepted method.

A further object of the present invention is a combined therapy for the treatment of a patient with SARS-related coronavirus infection, comprising the application of a pharmacologically acceptable dose of Artefenomel or its pharmaceutically acceptable salts with one or more drugs selected in the group comprising antivirals such as, for example, e lopinavir / ritonavir, ribavirin, oseltamivir, umifenovir, remdesivir, favipiravir, immunomodulators such as baricitinib, imatinib, dasatinib, cyclosporine, interferon b, interferon a, chloroquine, hydrochloroquine, nitostatazoxanide, camostat mesilate, corticosteroid, monoclonal antibodies against inflammatory cytokines such as, for example, tocilizumab, sarilumab, bevacizumab, fingolimod, eculizumab.

The following examples are purely for the purpose of illustrating the invention and are in no way to be understood as limiting the scope of the same, whose scope of protection is defined by the claims.

Examples

Materials and methods Cell cultures

Cells HEK293, HepG2, Huh-7, Vero (ATCC CCL-81) were grown in Dulbecco's minimal essential medium (Euroclone # ECB7501L) containing 10% inactivated fetal bovine serum (A56-FBS, Gibco # 10270), Penicillin / Streptomycin (Corning # 20-002-CI), non-essential amino acids (Euroclone # ECB3054D) and L-glutamine (Gibco ## 25030-024). The cells were passed into 100 mm² Petri dishes and divided every 3-4 days. The cells used in this study were not switched more than 20 times from the original stock. Compound and treatments

35 different compounds, selected in silico, have been tested in vitro. Each molecule, received as powder, was resuspended at 50, 30 or 15 mM in DMSO (Euroclone # APA36720250) or in Milli-Q water or Methanol. Stock solutions were prepared at 0.3, 3 and 30 mM (1000X). To treat cells, 1 pL of each stock solution was added to a well from a 24-well plate containing 1 ml_ of antibiotic-free cell culture medium, thus obtaining a final concentrations of 0.3, 3 and 30 pM. Controls were obtained by adding equivalent volumes of DMSO or Milli-Q water. The tested compound are listed in table 1 :

Table 1 :

Cell viability test (MTT)

Cells were seeded in 48-well plates at approximately 60% confluence. The compounds at different concentrations, or an equivalent volume of DMSO or MilliQ water as a control, was added after 24 hours. The medium was replaced on the second day and then removed after a total of 48 hours of treatment. The cells were then incubated with 5 mg / ml_ of 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) (Sigma # M5655-1G) in PBS for 15 minutes at 37 ° C. After carefully removing the MTT, the cells were resuspended in 100 pL of DMSO and the absorbance at 560 nm was read in a plate spectrophotometer, deriving the cell viability values.

Western blotting and antibodies

Cells were plated in 24-well plates at approximately 60% confluence. The compounds at different concentrations, or an equivalent volume of DMSO or MilliQ water as a control, was added after 24 hours. The medium was replaced on the second day and then removed after a total of 48 hours of treatment. Samples were lysed in lysis buffer (10mM Tris, pH 7.4, 0.5% NP- 40, 0.5% TX-100, 150mM NaCI, EDTA-free protease inhibitor cocktail). Protein quantification was performed by BCA kit (Thermo Fisher # 23225), according to the manufacturer's recommendations. A 40 pg aliquot of total protein was diluted 2: 1 in 4X Laemmli sample buffer containing 100 mM dithiothreitol, boiled 8 minutes at 95 ° C and loaded onto SDS-PAGE, using 12% acrylamide gel. After the run, the proteins were transferred onto the PVDF membrane. Membranes were blocked for 20 minutes in 5% (w / v) fat- free milk powder in Tris-buffered saline containing 0.01% Tween-20 (TBS-T). Membranes were exposed to anti-ACE2 antibody (1: 200) (AC18Z, Santa Cruz), 3% (w / v) of BSA in TBS-T overnight at 4 ° C, washed 3 times with TBS- T, then stained with a 1 : 3,000 dilution of horseradish conjugated anti mouse goat for 1 hour at room temperature. After 2 washes with TBS-T and one with Milli-Q water, the signals were detected using the ECL Select Western Blotting Detection Reagent (RPN2235, GE Healthcare) and visualized with the ChemiDocXRS Touch Imaging System (Bio-rad). Statistical analysis

Statistical analyses, performed with Prism software version 8.0 (GraphPad), included all data points obtained, with the exception of the experiments in which the negative and / or positive controls did not give the expected result. No test for outliers was employed. Results were expressed as mean ± standard errors. All data was analyzed with the one-way ANOVA test, including an assessment of data normality, and corrected by Dunnet's post- hoc test. The probability values (p) <0.05 were considered significant (*). The 50% inhibitory concentration (IC50) or the 50% lethal dose (LD50) was obtained by fitting the dose response curves to a sigmoidal function using a 4PL non-linear regression model.

Example 1: Evaluation of ACE2 expression in different cell lines.

The endogenous expression of ACE2 was evaluated in several cell lines, including HEK293, HepG2, Huh-7 and Vero. The results obtained by western blot show that ACE2 expression is higher in Vero cells (Figure 1). These cells were then used in subsequent experiments.

Example 2: Effect on ACE2 Expression Levels for Selected Compounds.

The in silico predicted 35 compounds have been tested on Vero cells. ACE2 expression levels after treatment have been evaluated by Western Blot. 9 molecules out of 35 were able to lower at least 30% of the expression of ACE2 (data not shown). The active compounds are: SIB-P002-M006, SIB- P002-M007, SIB-P002-M010, SIB-P002-M014, SIB-P002-M017, SIB-P002- M025, SIB-P002-M029, SIB-P002-M031 , SIB-P002-M032.

All the molecules were also tested for their intrinsic toxicity, as assayed by MTT. No relevant cell death was observed in the active compounds.

The more promising candidate compound, Artefenomel, was tested in a eight to ten-point (0.01-300 mM) dose-response fashion by western blotting and MTT assay.

Vero cells were exposed for 48 hours to Artefenomel (SIB-P002-M032) at different concentrations (0.01-300 pM) and the expression of the protein was evaluated by western blot. The cells exposed to Artefenomel were also analysed by MTT assay to assess the possible cytotoxicity of the molecule. The results show that Artefenomel potently suppresses ACE2 expression in a dose-dependent manner, already at the 1 pM dose (Figure 2A). Importantly, the drug did not show significant cytotoxicity even at the highest concentration tested, 300 pM (Figure 2B). Based on the observed dose-response effect of Artefenomel on the expression of ACE2, starting from 1 mM dose, as well as on the absence of cytotoxicity even at the highest concentration tested, Artefenomel has been surprisingly shown to act as a potent ACE2 inhibitor.

Example 3: Evaluation of inhibitory effects on a pseudo-typed retrovirus expressing the SARS-CoV-2 spike protein

Human Embryonic Kidney 293 (HEK293#35) cells overexpressing ACE2, and Vero cells were cultured as above indicated.

Stock solutions of selected compounds were prepared at 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 mM (1000X). To test ACE2 expression, cells were treated adding a 1 pl_ aliquot of each stock solution to a well from a 24-well plate containing 1 ml_ of cell medium (corresponding to final concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 or 300 mM). To perform the Spike- dependent fluorescent assay, 1 mI_ aliquot of each stock solution was added to a well from a 24-well plate containing 1 ml_ of cell medium (corresponding to final concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 or 300 mM) and then 100 mI_ of media plus compound was transferred into a 96-well plate. Vehicle controls were obtained by adding equivalent volumes of DMSO.

Retroviral particles expressing the SARS-CoV-2 spike protein were produced as follow. HEK293-T cells were seeded into 10 cm plates with selection medium (DMEM with G418 0,5 mg/mL). Once cells reached ~80% confluence, medium was replaced with DMEM containing 2,5% FBS. Cells were then transfected with three different plasmids (MLV transfer vector, pc NCG; MLV packaging vector, pc Gag-Pol, and env-encoding vector, pc SARS-CoV-2 spike AC; see figure 3). An encoding vector lacking the SARS- CoV-2 spike protein was used to obtain the control retroviral particles.

Three to six days after transfection, cells were pelleted by centrifuging at 2,000g for 5 min. Supernatants were filtered using a 0.45 pm filter, and ultracentrifuged at 20,000g for 2h. Pellets were resuspended in 1X PBS and stored at -80 °C.

On day 1, untransfected Vero cells or HEK293 cells stably expressing the human ACE2 were seeded on 96-well plates in DMEM medium (10% FBS, Pen/Strep, L-glutamine, non-essential amino acids). After 24 and 48 h, the medium was replaced with fresh medium containing each compound to be tested at the desired concentration. On day 4, a 3 pi aliquot of retroviral vectors expressing the SARS-CoV-2 spike protein, or control vectors, was added to each well. For Vero cells, this step was repeated a second time on day 5 in order to increase the number of transduced cells. Three days after transduction, cells showing the GFP fluorescence were detected with an EnSight™ Multimode Microplate Reader and cell viability was assayed by MTT on the same wells.

The effect of Artefenomel on the transduction efficiency of a pseudo-typed retroviral vector was tested in both Vero and FIEK293#35 cells.

Vero (figure 4A, B, C) and FIEK293#35 (figure 4D, E, F) cells exposed to SIB- P012-M032 compound at the indicated concentrations, were transduced with a pseudo-typed retroviral vector expressing the SARS-CoV-2 spike protein and a GFP reporter gene. Identical retroviral vectors missing the spike protein were used as controls. The number of transduced cells were quantified by detecting the GFP fluorescence using a plate reader and analyzed with the ImageJ software (NIH). The number of green dots was normalized on the number of cells within each well, estimated by using the MTT assay, and expressed as the percentage of the vehicle control (figure 4B, 4E). ACE2 protein levels and cell viability were tested after 48h of treatment with the compound by western blot (figure 4A, 4D) and by MTT (figure 4C, 4F) respectively. For each condition, means ± SD were calculated from at least 3 independent replicates. Statistical analyses were performed using the one-way ANOVA Dunnett^'s post-hoc test. Significant changes are indicated by an asterisk (*p < 0.05).

Artefenomel is able to lower the expression of ACE2 starting from the concentration of 0.3 mM in Vero cells and at 100 mM in FIEK293#35 cells. This compound inhibits the entrance of the pseudo-typed retroviral vector at 100 mM in both cell type but it showed toxicity only in FIEK293#35 cells starting from the concentration of 30 mM. The data confirm Artefenomel capability to lower the expression of ACE2 and this phenomenon translates in a reduced ability of cellular entry for a pseudo-typed retroviral vector expressing the SARS-CoV-2 spike protein.

Example 4: Evaluation of the effect of Artefenomel in a time course experiment

Vero cells were cultured as above indicated.

Cells were treated for 4h, 16h, 24h, 48h - (without changing media with fresh compound after 24h), 48h + (changing media after 24h), 72h and 96h without changing media. Indicated treatments were started 24h after plating. The number of cells plated for the different treatments are listed below:

4h treatment: 4X10⁵ 16-24h treatment: 1.8X10⁵ 48h treatment: 1.0X10⁵ 72h treatment: 6.0X10⁴ 96h treatment: 2.5X10⁴

Stock solutions were prepared at 10 and 30 mM (1000X). To test ACE2 expression cells were treated adding 1 pL of each stock solution to a well of a 24-well plate containing 1 ml_ of cell culture medium (corresponding to final concentrations of 10 and 30 pM). Controls were obtained by adding equivalent volumes of DMSO.

Results are shown in figure 5.

Expression level of ACE2 was tested by western blot in Vero cells after exposure to 10 or 30 pM of compound or vehicle (DMSO) at the indicated time points. ACE2 expression starts to decrease after 16h of treatment with Artefenomel, reaches the lowest level (35%) after 72hr. The expression increases again after 96h of treatment. For each condition, means ± SD were calculated from at least 3 independent replicates. ACE2 levels remain low for 96h suggesting that the molecule keeps the activity up to 4 days Example 5: Evaluation of the effect of Artefenomel on ACE2 RNA Vero cells were seeded in 24-well plates at ~60% confluency and treated after 24h. Cells were plated at the following confluence:

2-4-8h treatment: 4X10⁵ cells/well 16-24h treatment: 1.8X10⁵ cells/well 48h treatment: 1.0X10⁵ cells/well

Stock solutions of the compound were prepared at 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 or 50 mM (1000X). To treat cells, 1 pL aliquot of each stock solution was added to a well of a 24-well plate containing 1 ml_ of cell culture medium (corresponding to final concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 or 100 pM). Vehicle controls were obtained by adding equivalent volumes of DMSO. For time course experiments, cells were collected at 2-4-8-16 and 24h. In experiments performed at 48h only, media was replaced with fresh compounds after 24h of treatment and cells collected the next day.

Total RNA was extracted using the Total RNA Purification Plus kit (Norgen, #48400) according to manufacturer’s instructions. Equal amounts of RNA (500 ng) were reverse transcribed into cDNA with the SensiFast cDNA synthesis kit (Bioline, #BIO-65054). RT-qPCR was performed using the iQ SYBR Green Supermix (Bio-Rad, #1708882) in a CFX 96 RT-PCR Thermal cycler (Bio-Rad) following the program and the primers reported in table 2. All data was normalized using GAPDFI, FIPRT and b-actin as housekeeping genes. Relative mRNA levels were calculated using the 2-AACT method. Experiments were performed in one biological replicate, and for each sample two technical replicates were set up in the RT-qPCR. Three independent experiments were performed for each condition.

Table 2

RT-qPCR program

Results are shown in Figure 6.

ACE2 mRNA (figure 6A) and protein expression (figure 6B) were evaluated at 2-4-8-16 and 24 h after treatment. A dose-response analyses has been performed at 48 h after treatment (figure 6C, 6D). Results showed that the compound SIB-P012-M032 affects ACE2 expression at the mRNA and protein level similarly. A dose-dependent effect was observed for both the mRNA and protein, which were significantly decreased by the compound even at low concentrations.

Example 6: Evaluation of the effect of Artefenomel on ACE1 expression

Vero cells were cultured as indicated above. ACE1 expression was tested on the samples obtained at the 48h+ time point of the time course experiments detailed in Example 6.

Western blotting was performed as described above. For ACE1, blots were probed with anti-ACE1 (1:1000) (ab254222, abeam) in 5% (w/v) low fat milk in TBS-T overnight at 4 °C, washed 3 times with TBS-T 10 min each, then probed with a 1 :5,000 dilution of horseradish conjugated goat anti-rabbit for 1 h at RT.

The expression level of ACE1, as shown in figure 7, doesn’t change after treatment with Artefenomel, suggesting a specific effect of the compounds on ACE2.

Example 7 (comparative): Evaluation of the effect of chloroquine and derivatives on ACE2 expression

Chloroquine, a clinically approved drug effective against malaria, have recently attracted widespread interest as potential therapy for COVID-19. The drug has been reported to interfere with terminal glycosylation of ACE2, which may negatively regulate the virus-receptor binding and inhibit the infection.

In our screening, we tested Chloroquine diphosphate (SIB-P012-M001) as well as several related compounds, including Flydroxychloroquine Sulfate (SIB-P012-M024), Tafenoquine succinate (SIB-P012-M005), Mefloquine (SIB-P012-M008), Piperaquine phosphate (SIB-P012-M025), Primaquine diphosphate (SIB-P012-M026), Halofantrine hydrochloride (SIB-P012-M003) and Amodiaquine (SIB-P012-M027). Results, reported in figure 8 with respect to Chloroquine, showed a general increase in the ACE2 signal upon treatment with Chloroquine or related compounds, often accompanied by a decrease in ACE2 molecular weight, possibly confirming alterations in the glycosylation of the protein. However, based on the absence of ACE2- lowering effects, these molecules were not considered as promising candidates for further analyses.

Example 8: SARS-CoV-2 Antiviral Assay

To evaluate antiviral activity against SARS-CoV-2 (MEX-BC2/2020), a CPE- based antiviral assay was performed by infecting Vero E6 cells in the presence or absence of test-items. Infection of cells leads to significant cytopathic effect and cell death after 4 days of infection. In this assay, reduction of CPE (virus-induced cytopathic effect) in the presence of inhibitors was used as a surrogate marker to determine the antiviral activity of the tested items. Viability assays to determine test-item-induced loss of cell viability was monitored in parallel using the same readout (Neutral Red), but treating uninfected cells with the test-items.

Vero E6 cells were maintained in DMEM with 10% fetal bovine serum (FBS), hereby called DMEM10. Cells were seeded and incubated for 24 hours before being pre-incubated with test-items. Twenty-four hours post cell seeding, test samples were submitted to serial dilutions with DMEM2 in a different plate, cell culture was removed from cells, and serial dilutions of test-items were added to the cells and incubated for 4h at 37°C in a humidified incubator. After the pre-incubation of test-items with target cells, cells were challenged with the viral inoculum resuspended in DMEM with 2% FBS (DMEM2). The amount of viral inoculum was previously titrated to result in a linear response inhibited by antivirals with activity against SARS-CoV-2. Cell culture media with the virus inoculum was not removed, and the test- items and virus were maintained in the media for the duration of the assay (96h). After this period the extent of cell viability was monitored with the neutral red (NR) uptake assay.

The virus-induced CPE was monitored under the microscope after 3 days of infection and at day 4 cells were stained with neutral red to monitor cell viability. Viable cells incorporate neutral red in their lysosomes. The uptake relies on the ability of live cells to maintain the pH inside the lysosomes lower than in the cytoplasm. This process requires ATP. Inside the lysosome the dye becomes charged and is retained. After a 3h incubation with neutral red (0.033%), the extra dye was washed and the neutral red taken by lysosomes was then extracted for 15 minutes with a solution containing 50% ethanol and 1% acetic acid to monitor absorbance at 540nm.

Test-items were evaluated in triplicates (triplicates in two separate plates) using serial 3-fold dilutions. Controls included uninfected cells (“mock- infected”), and infected cells to which only vehicle was added. Some cells were treated with GS-441524. GS-441524 is the main metabolite of remdesivir, a broad spectrum antiviral that blocks the RNA polymerase of SARS-CoV-2.

The average absorbance at 540nm (A540) observed in infected cells (in the presence of vehicle alone) was calculated, and then subtracted from all samples to determine the inhibition of the virus induced CPE. Data points were then normalized to the average A540 signal observed in uninfected cells (“mock”) after subtraction of the absorbance signal observed in infected cells. In the neutral red CPE-based assay, uninfected cells remained viable and uptake the dye at higher levels than non-viable cells. In the absence of antiviral agents the virus-induced CPE kills infected cells and leads to lower A540 (this value equals 0% inhibition). By contrast, incubation with the antiviral agent (GS-441524) prevents the virus induced CPE and leads absorbance levels similar to those observed in uninfected cells. Full recovery of cell viability in infected cells represent 100% inhibition of virus replication. Uninfected cells were incubated with eight concentrations of test-items or control inhibitors dilutions using starting the same doses indicated for the antiviral assay. The incubation temperature and duration of the incubation period mirrored the conditions of the prevention of virus-induced CPE assay, and cell viability was evaluated with the neutral red uptake method but this time utilizing uninfected cells, otherwise the procedure was the same as the one used for the antiviral assays. The extent of viability was monitored by measuring absorbance at 540nm. When analyzing the data, background levels obtained from wells with no cells were subtracted from all data-points. Absorbance readout values were given as a percentage of the average signal observed in uninfected cells treated with vehicle alone.

The average signal obtained in wells with no cells was subtracted from all samples. Readout values were given as a percentage of the average signal observed in uninfected cells treated with vehicle alone (DMEM2). The signal- to-background (S/B) obtained was 21.7-fold. DMSO was used as a cytotoxic compound control in the viability assays. DMSO blocked cell viability by more than 99% when tested at 10% (Figure 9).

Artefenomel was effective in completely preventing the virus-induced cytopathic effect (CPE). Incubation of infected cells with 33mM and 100pM Artefenomel resulted in viability levels similar to those observed in uninfected cells (Figures 10-11). Of note, Artefenomel incubation at 33mM and 100pM demonstrated precipitation (Figure 12). The prevention of the virus-induced CPE with Artefenomel by microscopic evaluation of the monolayers incubated with concentrations of test-item at which the uptake of neutral red was increased (Figure 12).

The cell viability assay further proved that the antiviral activity displayed by Artefenomel was not due to cytotoxic. None of the concentrations evaluated of the test-item displayed any cytotoxicity.

By comparison, GS-441524 at concentrations of 0.74mM to 2.2mM or greater, completely prevented the virus-induced CPE (Figure 13).

In summary, Artefenomel prevented completely the virus-induced CPE at the concentration (33mM to 100pM), suggesting significant anti-SARS-CoV-2 antiviral activity. Of note, the antiviral activities observed in this study were reproduced in two assays performed in parallel in two different plates (Figure 11 ).

Claims

1. Artefenomel or its pharmaceutically acceptable salts for use in the treatment of pathologies related to overexpression and / or over ACE2 activity and / or to ACE2 receptor activity (angiotensin 2 converting enzyme).

2. Artefenomel or its pharmaceutically acceptable salts for use according to claim 1 , where said pathology is a SARS-related coronavirus infection.

3. Artefenomel or its pharmaceutically acceptable salts for use according to claim 2, where said virus is SARS-CoV.

4. Artefenomel or its pharmaceutically acceptable salts for use according to claim 2, where said virus is SARS-CoV-2.

5. Artefenomel or its pharmaceutically acceptable salts for use according to claim 2, where said virus is MERS-CoV.

6. Pharmaceutical composition comprising Artefenomel or its pharmaceutically acceptable salts as an active ingredient for use in the treatment of pathologies related to overexpression and / or over activity of ACE2 and / or to the receptor activity of ACE2.

7. Composition for use according to claim 6, where said pathology is caused by SARS-related coronavirus infections.

8. Pharmaceutical composition for use according to claim 7, wherein said composition also comprises one or more active ingredients selected in the group comprising antivirals, such as for example lopinavir / ritonavir, ribavirin, oseltamivir, umifenovir, remdesivir, favipiravir, immunomodulators such as baricitinib, imatinib, dasatinib, cyclosporine, interferon b, interferon a, chloroquine, hydrocloroquine, nitazoxanide, mesilate camostat, corticosteroids, monoclonal antibodies directed against inflammatory cytokines such as, for example, tocilizumab, sarilumab, bevacizumab, fingolimod, eculizumab.