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. 2025 Feb;77(1):260-273.
doi: 10.1007/s43440-024-00650-0. Epub 2024 Sep 18.

JG26 attenuates ADAM17 metalloproteinase-mediated ACE2 receptor processing and SARS-CoV-2 infection in vitro

Affiliations

JG26 attenuates ADAM17 metalloproteinase-mediated ACE2 receptor processing and SARS-CoV-2 infection in vitro

Valentina Gentili et al. Pharmacol Rep. 2025 Feb.

Abstract

Background: ADAM17 is a metalloprotease implicated in the proteolysis of angiotensin-converting enzyme 2 (ACE2), known to play a critical role in the entry and spread of SARS-CoV-2. In this context, ADAM17 results as a potential novel target for controlling SARS-CoV-2 infection.

Methods: In this study, we investigated the impact on ACE2 surface expression and the antiviral efficacy against SARS-CoV-2 infection of the selective ADAM17 inhibitor JG26 and its dimeric (compound 1) and glycoconjugate (compound 2) derivatives using Calu-3 human lung cells.

Results: None of the compounds exhibited cytotoxic effects on Calu-3 cells up to a concentration of 25 µM. Treatment with JG26 resulted in partial inhibition of both ACE2 receptor shedding and SARS-CoV-2 infection, followed by compound 1.

Conclusion: JG26, an ADAM17 inhibitor, demonstrated promising antiviral activity against SARS-CoV-2 infection, likely attributed to reduced sACE2 availability, thus limiting viral dissemination.

Keywords: ADAM17; Antiviral activity; Arylsulfonamido-based hydroxamic acid; SARS-CoV-2; sACE2.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Chemical structures of ADAM17 inhibitors studied in the present work. JG26 [18], compound previously published by our research group which served as a starting point for the design of compound 1 and compound 2; GlcNAc (β-N-acetyl-D-glucosamine) moiety is highlighted in red
Fig. 2
Fig. 2
Synthesis of compound 2. Reagents and conditions: i) TFA, dry DCM, 0 °C, 1 h, 37%; ii) 5, dry DIPEA, dry DMF, 18 h, rt, 68%; iii) TFA, dry DCM, 0 °C, 1 h, 41%; iv) 7, Et3N, dry DCM, 18 h, rt, 63%; v) (1) NH3-MeOH 7 N, MeOH, 18 h, rt; (2) TFA, dry DCM, 8 h, rt (85% over two steps)
Fig. 3
Fig. 3
Cytotoxicity in Calu-3 cells evaluated by MTT assay. Cells were cultured in the presence of different concentrations of compound JG26, compound 1 and compound 2 for 24 (panels a-c) and 48 h (panels d-f). (g) CC50 (cytotoxic concentration 50%, µM) values reported for each compound. Data were analyzed by one-way ANOVA followed by Dunnett’s T3 multiple comparison post hoc test. The data are presented as mean ± SD (n = 3). NT, not treated; PMA, phorbol 12-myristate 13-acetate
Fig. 4
Fig. 4
ACE2 expression in SARS-CoV-2-infected Calu-3 cells after treatment with the tested compounds (25 µM) for 24 and 48 h using immunofluorescence staining. (a) Representative images of immunofluorescence for ACE2 (red); nuclei were stained with DAPI (blue). Images were taken with microscope Nikon Eclipse TE2000S, scale bars (low right) denote 25 μm. (b) Immunofluorescence levels of ACE2 expressed in RFU, quantified by QuPath software. Data were analyzed by two-way ANOVA followed by Dunnett’s multiple comparison post hoc test. The data are presented as mean ± SD (n = 3). NT, not treated; ACE2, angiotensin-converting enzyme 2; PMA, phorbol 12-myristate 13-acetate; RFU: relative fluorescence units
Fig. 5
Fig. 5
The antiviral activity of JG26, compound 1 and compound 2 on Calu-3 SARS-CoV-2-infected cell. (a) Quantification by plaque assay of SARS-CoV-2 infective particles in supernatants of treated cells 48 h post-infection. (b) Quantification by RT-qPCR of SARS-CoV-2 genomes in supernatants of treated cells 48 h post-infection. (c, d) Data of viral load logarithmic reduction (LR) in compounds-treated Calu-3 cells in comparison to DMSO-treated (vehicle) cells, infected with SARS-CoV-2, calculated with the results of plaque assay (c) or RT-qPCR (d). Data were analyzed by one-way ANOVA followed by Dunnett’s T3 multiple comparison post hoc test. The data are presented as mean ± SD (n = 3). NT, not treated; PMA, phorbol 12-myristate 13-acetate; PFU, plaque-forming unit; hpi, hours post-infection. *Logarithmic Reduction greater than 1 log, corresponding to a > 90% viral load decrease
Fig. 6
Fig. 6
The impact of the treatment with compound JG26 and its derivatives (compound 1, compound 2) on SARS-CoV-2 plaque morphology. (a) Representative images of SARS-CoV-2 plaques on VERO E6 cells stained with crystal violet 0.1%. Images were taken with NeXcope NE620 microscope, scale bar (low right) 0.1 mm. (b) The impact of the treatment on plaque diameter calculated as mean value of 10 plaques for each condition. Diameter sizes were measured using NeXcope visualization software (Capture V.2.0). Data were analyzed by one-way ANOVA followed by Dunnett’s T3 multiple comparison post hoc test. NT, not treated; PMA, phorbol 12-myristate 13-acetate

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