Anti-fibrotic; PPT1

Here, researchers investigated the antiviral activity and associated mechanism of GNS561/Ezurpimtrostat, a small lysosomotropic molecule inhibitor of late-stage autophagy. Interestingly, GNS561 exhibited antiviral activity of 6-40 nM depending on the viral strain considered, currently positioning it as the most powerful molecule investigated in SARS-CoV-2 infection. It was then showed that GNS561 was located in lysosome-associated-membrane-protein-2-positive (LAMP2-positive) lysosomes, together with SARS-CoV-2. Moreover, GNS561 increased LC3-II spot size and caused the accumulation of autophagic vacuoles and the presence of multilamellar bodies, suggesting that GNS561 disrupted the autophagy mechanism. [2]

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GNS561 Exhibits Strong Antiviral Activity against SARS-CoV-2 Replication[2]
To assess the antiviral activity of GNS561, we investigated SARS-CoV-2 copies using Vero E6 cells. First, regarding GNS561-specific effects on autophagy in the Vero E6 cell model, we performed a protein expression analysis focused on the LC3-II and p62 protein levels, two well-known autophagy markers. Autophagy inhibition was evaluated at different drug concentrations applied for 24 h and in the presence or absence of bafilomycin A1 (Baf A1), a well-characterized inhibitor of the late stage of autophagy, added for the last 4 h of the experiment. Normalized LC3-II protein expression increased in a dose-dependent manner, without a further increase when Baf A1 treatment was added at the highest dose (Figure 1A), reflecting the accumulation of autophagosomes in cells. The same observation was made for the p62 protein level, suggesting that GNS561 blocks autophagic flux at the late stage in the Vero E6 cell line model.

It was revealed that inhibition of PPT1 using ezurpimtrostat decreased the liver tumor burden in a mouse model of hepatocellular carcinoma by inducing the penetration of lymphocytes into tumors when combined with anti-programmed death-1 (PD-1). Inhibition of PPT1 potentiates the effects of anti-PD-1 immunotherapy by increasing the expression of major histocompatibility complex (MHC)-I at the surface of liver cancer cells and modulates immunity through recolonization and activation of cytotoxic CD8+ lymphocytes. Ezurpimtrostat turns cold tumors into hot tumors and, thus, could improve T cell-mediated immunotherapies in liver cancer.[1]

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Resaerchers used the K18-hACE2 mouse model and highlighted that GNS561 treatment led to a decline in SARS-CoV-2 virions in the lungs associated with a disruption of the autophagy pathway. Overall, this study highlights GNS561 as a powerful drug in the treatment of SARS-CoV-2 infection and supports the hypothesis that autophagy blockers could be an alternative strategy for COVID-19.[2]

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In the DEN liver mass model, ezurpimtrostat hydrochloride (Compound 2-3) (15 mg/kg/day, PO) for 6 weeks decreased collagen fiber deposition levels (41.0% reduction) and the area of liver fibrosis [3].

In Vitro Antiviral Activity Assays[2]
Vero E6 (25,000 cells/well) and Calu-3 (12,000 cells/well) cell lines were plated into a 96-well tissue culture plate with 175 µL of appropriate medium and were allowed to adhere for a minimum of 24 h until the culture reached 90% confluence. Vero E6 and Calu-3 cells were treated with 25 µL of the tested drugs or vehicle control for 2 h and then infected with SARS-CoV-2 at 0.1 MOI and incubated for an additional 24 or 48 h, respectively. Viral extraction was performed on cell culture supernatants using a Macherey-Nagel™ NucleoSpin™ ARN viral kit. The yield of progeny virus production was assessed using a specific qRT–PCR operating with previously extracted viral RNA. Briefly, one-step qRT–PCR was conducted in a final volume of 25 μL containing 10 µL of extracted viral RNA and 15 µL of a mix containing the probe/primer mix targeting the E gene (Table S1) and Super Script master mix from the SuperScriptTM III PlatinumTM One-Step qRT–PCR Kit. Quantitative PCR measurement was performed using the Cobas z 480 PCR system, and data were analyzed with LightCycler 480 SW 1.5 software according to the manufacturer’s instructions. A melting curve analysis was performed after amplification to verify the accuracy of the amplicon. The yield of progeny virus production was calculated as the relative expression of the E gene of the considered condition normalized to both infected and untreated condition. Every experiment was performed in technical triplicates, and three independent experiments were performed.
Vero E6 and Vero E6-TMPRSS2 cells were plated for 24 h in a 24-well plate containing complete medium. Cells were infected at an MOI of 0.025 with increasing concentrations of GNS561 or with vehicle and incubated for an additional 16 h with SARS-CoV-2 (hCoV-19_IPL_France strain). Cells were rinsed with phosphate-buffered saline (PBS) and lysed in non-reducing Laemmli loading buffer. Proteins were then separated onto a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. SARS-CoV-2 nucleocapsid (N) protein was detected using a rabbit polyclonal antibody (1:1000) following a horseradish peroxidase-conjugated secondary antibody (1:10,000). Detection was carried out by chemoluminescence, and image quantification was performed using ImageJ software. Experiments were performed in duplicate.

In Vitro Cytotoxicity Assay[2]
Cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay or using the CellTiter 96® Non-Radioactive Cell Proliferation kit following the manufacturer’s instructions. Briefly, Vero E6 (25,000 cells/well) and Calu-3 (12,000 cells/well) cell lines were plated into a 96-well tissue culture plate with 175 µL of appropriate medium. Vero E6 and Calu-3 cells were treated for 2 h with 25 µL of increasing concentrations of tested drugs or with appropriate vehicle control and then infected with 50 µL of SARS-CoV-2 strain (IHUMI-6 or USA-WA1/2020) at 0.1 multiplicity of infection (MOI) and incubated for an additional 24 and 48 h, respectively. At the end of the treatment, CellTiter-Glo solution (IHUMI-6) or CellTiter 96® Non-Radioactive solution (USA-WA1/2020) was added to each well. Cells were briefly shaken and then incubated at room temperature (RT) for 10 min to allow stabilization of the luminescent signal, which was recorded using an Infinite F200 Pro plate reader. Cytotoxicity was expressed as the concentration of tested compounds that inhibited cell proliferation by 50% (CC50) and calculated using the Chou and Talalay method. Every experiment was performed in technical triplicates, and three independent experiments were performed.

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Immunofluorescence Assays and Analysis[2]
For cellular investigation, Vero E6 cells (500,000 cells/well) were cultured the day prior to infection in a 24-well plate containing a glass coverslip and 700 µL of medium. Cells were then treated with 100 µL of GNS561G, a fluorescent analog of GNS561, or vehicle for 2 h and thereafter infected by SARS-CoV-2 strain (IHUMI-6, MOI 0.1) for an additional 48 h.

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Cell and Mice Organs Electron Microscopy Imaging[2]
Vero E6 cells (2,100,000 cells) were cultured the day prior to infection in a T75 flask containing 7 mL of culture medium, reaching 90% confluency the day after. Cultivated cells were treated with 4 µM GNS561 or vehicle control for 2 h and thereafter infected for an additional 24 h with the SARS-CoV-2 strain (IHUMI-6, MOI 0.1).

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Western Blot Assay[2]
Vero E6 cells (2,000,000 cells) were cultured the day prior to infection in a T75 flask containing 7 mL of culture medium, reaching 90% confluency the day after. Cultivated cells were treated with GNS561 or vehicle control for 2 h and thereafter infected for an additional 24 h by SARS-CoV-2 strain (IHUMI-6, MOI 0.1) and incubated at 37 °C in the presence of 5% CO2 and 95% air in a humidified incubator.

The aim of this study was to determine the cellular and molecular activity of targeting PPT1 using ezurpimtrostat, in combination with an anti-PD-1 antibody. Methods: In this study, a transgenic immunocompetent mouse model of hepatocellular carcinoma was used. [1]

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Female K18-hACE C57BL/6J mice (strain: 2B6.Cg-Tg (K18-ACE2)2Prlmn/J, 8-9 weeks) were bred and housed in a ventilated cage system under SOPF requirements at the CIPHE animal facility with standard chow diets. Randomized mouse females were treated for 24 h with 50 mg/kg GNS561/ezurpimtrostat, 200 µL per os (i.e., oral gavage administration), or with vehicle for the control group, before intranasal infection with 1.1 × 105 plaque-forming units (PFUs) of the BetaCoV/France/IDF0372/2020 SARS-CoV-2 strain in a final volume of 30 μL, as illustrated in Figure S2. Twenty-four hours after treatment, virus inoculation was performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering. Mice were then treated daily with GNS561 compound or vehicle. Mice were humanely euthanized at 7 days post-infection, and lung tissues were harvested.[2]

[1]. Ezurpimtrostat, A Palmitoyl-Protein Thioesterase-1 Inhibitor, Combined with PD-1 Inhibition Provides CD8+ Lymphocyte Repopulation in Hepatocellular Carcinoma. Target Oncol . 2024 Jan;19(1):95-106.
[2]. GNS561 Exhibits Potent Antiviral Activity against SARS-CoV-2 through Autophagy Inhibition. Viruses . 2022 Jan 12;14(1):132.
[3]. Substituted 2,4 diamino-quinoline as new medicament for fibrosis, autophagy and cathepsins b (ctsb), l (ctsl) and d (ctsd) related diseases. EP3620164A1.

Ezurpimtrostat is an orally bioavailable, quinolone-derived, small molecule inhibitor of palmitoyl-protein thioesterase 1 (PPT1), with potential antineoplastic activity. Upon oral administration, ezurpimtrostat targets and inhibits the activity of PPT1 and induces lysosomal disruption, which results in the inhibition of autophagy and the induction of apoptosis via caspase activation. This may inhibit tumor cell proliferation and tumor growth. PPT1, a lysosomal thioesterase that plays an important role in lysosomal function and autophagy, is overexpressed in certain cancers.

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Mechanism of Action
GNS561, a potent chloroquine analog, is a lysosomotropic small molecule. It induces lysosomal dysregulation which causes cell death and has anti-cancer properties specifically targeting intrahepatic cholangiocarcinoma.

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Since December 2019, SARS-CoV-2 has spread quickly worldwide, leading to more than 280 million confirmed cases, including over 5,000,000 deaths. Interestingly, coronaviruses were found to subvert and hijack autophagic process to allow their viral replication. Autophagy-modulating compounds thus rapidly emerged as an attractive strategy to fight SARS-CoV-2 infection, including the well-known chloroquine (CQ). Here, we investigated the antiviral activity and associated mechanism of GNS561/Ezurpimtrostat, a small lysosomotropic molecule inhibitor of late-stage autophagy. Interestingly, GNS561 exhibited antiviral activity of 6-40 nM depending on the viral strain considered, currently positioning it as the most powerful molecule investigated in SARS-CoV-2 infection. We then showed that GNS561 was located in lysosome-associated-membrane-protein-2-positive (LAMP2-positive) lysosomes, together with SARS-CoV-2. Moreover, GNS561 increased LC3-II spot size and caused the accumulation of autophagic vacuoles and the presence of multilamellar bodies, suggesting that GNS561 disrupted the autophagy mechanism. To confirm our findings, we used the K18-hACE2 mouse model and highlighted that GNS561 treatment led to a decline in SARS-CoV-2 virions in the lungs associated with a disruption of the autophagy pathway. Overall, our study highlights GNS561 as a powerful drug in the treatment of SARS-CoV-2 infection and supports the hypothesis that autophagy blockers could be an alternative strategy for COVID-19.[2]

C25H31CLN4

422.9934

422.223

C, 70.99; H, 7.39; Cl, 8.38; N, 13.25

1914148-72-3

Ezurpimtrostat hydrochloride;1914148-73-4

121305180

Typically exists as solid at room temperature

5.9

2

4

6

30

520

0

QVXSJSXAVQJXOV-UHFFFAOYSA-N

InChI=1S/C25H31ClN4/c1-25(2,3)29-20-12-14-30(15-13-20)23-16-24(28-22-7-5-4-6-21(22)23)27-17-18-8-10-19(26)11-9-18/h4-11,16,20,29H,12-15,17H2,1-3H3,(H,27,28)

4-[4-(tert-butylamino)piperidin-1-yl]-N-[(4-chlorophenyl)methyl]quinolin-2-amine

Ezurpimtrostat; Ezurpimtrostat [INN]; MS6LGW5JFK; 1914148-72-3; GNS561; GNS-561; UNII-MS6LGW5JFK; Gns 561;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)