e64  (Tocris)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Cathepsin L as the Molecular Target of Hydroxychloroquine With Chemical Proteomics

doi: 10.1016/j.mcpro.2025.101314

Figure Lengend Snippet: HCQ targets endosomal virus entry mediated by CTSL. A , inhibition of CTSL activity by HCQ (50 μM), HCQ-P (50 μM), CQ (50 μM), quinine (50 μM), and mefloquine (4 μM). The known CTSL inhibitors, MG101 (4 μM) and E64 (30 μM), served as positive controls. Each group was calculated from three independent biological replicates. Statistical significance was measured by t test compared with dimethyl sulfoxide (DMSO) group. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. B , CTSL-mediated hydrolysis of the substrate AC-FR was quantified by monitoring the fluorescence of released 7-amino-4-trifluoromethylcoumarin (AFC). Reactions were conducted in pH 5.5 reaction buffer following overnight preincubation with HCQ at 4 °C. Each point was calculated from three independent biological replicates. C , inhibition of pseudovirus entry by HCQ. Huh7 CTSL-OE cells were treated with gradient dose of HCQ for 1 h before transduction with Omicron BA.1-S pseudoviruses. Pseudovirus entry was quantified by measuring the luciferase signal in the cell lysates 24 h after transduction. The inhibition (%) was normalized with 0 HCQ treatment group. D , inhibition of authentic Omicron BA.1 entry and replication by HCQ. Huh7 CTSL-OE cells were infected with authentic Omicron BA.1 at a multiplicity of infection (MOI) of 0.5. Viral gene copies in the cell lysate were determined by real-time quantitative PCR analysis at 24 hpi. The inhibition (%) was normalized to that of control group (0 HCQ). E and F , Huh7 cells were treated with CTSL gene knockdown (si-CTSL). The siRNA-treated cells were pretreated with HCQ for 1 h and then transduced with Omicron BA.1-S ( E ) or SARS-CoV-1-S ( F ) pseudoviruses. Pseudovirus entry was quantified by measuring the luciferase signal in cell lysates 24 h after transduction. The inhibition (%) was normalized to that of control group. The mean ± SEM from at least three independent experiments with technical triplicates is shown. Statistical significance was measured by t test between EV and CTSL-OE groups with the same HCQ concentration. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CQ, chloroquine; CTSL, cathepsin L; CTSL-OE, cathepsin L overexpression; EV, empty vector; HCQ, hydroxychloroquine; SARS-CoV, severe acute respiratory syndrome coronavirus.

Article Snippet: HCQ (HY-B1370), CQ (HY-17589A), E64 (HY-15282), quinine, mefloquine (HY-17437), promethazine (HY-B0781), azithromycin (HY-17506), and clemastine fumarate (HY-B0298A) are supplied by MCE.

Techniques: Virus, Inhibition, Activity Assay, Fluorescence, Transduction, Luciferase, Infection, Real-time Polymerase Chain Reaction, Control, Knockdown, Concentration Assay, Over Expression, Plasmid Preparation

Journal: Nature Communications

Article Title: Integrin beta 1 facilitates non-enveloped hepatitis E virus cell entry through the recycling endosome

doi: 10.1038/s41467-025-61071-y

Figure Lengend Snippet: ( A ) S10-3 LAMP-1-GFP cells were inoculated with eHEV (MOI = 20 GE/cell) or nHEV (MOI = 30 GE/cell) for 6 h or 2 h on ice and incubated at 37 °C for 10 h or 7 h, respectively. Genomes (magenta) were detected by RNA-FISH using the ORF1 probe. Scale bar = 5 μm. ( B ) and ( C ) S10-3 cells were treated with cathepsin inhibitor E64 or DMSO and infected with ( B ) nHEV (MOI = 30 GE/cell, n = 7) or ( C ) eHEV (MOI = 20 GE/cell, n = 6). E64 was added with virus for 24 h (“during”), or 24 h post-infection and throughout the course of infection (“after”). Infectivity was assessed 5 days post-infection. ( D ) and ( E ) S10-3 cells were treated with E64 or DMSO and infected with ( D ) nHEV (MOI = 30 GE/cell, n = 9) or ( E ) eHEV (MOI = 20 GE/cell, n = 7). 8 h later, the inoculum was replaced with fresh media containing drugs. 24 h post-inoculation, HEV capsid were detected by staining and genomes as in ( A ) and quantified using CellProfiler. ( F ) Maximum projections of E64-treated S10-3 LAMP-1-GFP cells inoculated with nHEV (MOI = 30 GE/cell). 24 h later, capsids (magenta) and genomes (yellow) were detected as in ( C ). Representative of n = 6 microscope fields. ( G ) S10-3 LAMP-1-GFP cells were treated with E64 or DMSO 30 min prior to inoculation with nHEV (MOI = 30 GE/cell). After 8 h at 37 °C, inoculum was replaced with fresh media containing drugs. Genomes were detected and analysed as in ( A ). ( H ) Proposed working model on HEV cell entry. The interaction of nHEV with ITGB1 triggers internalisation through Rab11+ recycling endosomes, while eHEV is routed into Rab5a+ early endosomes. Both particles traffic through Rab7+ late endosomes and reach Lamp1+ lysosomes. The capsid and envelope are degraded by lysosomal cathepsins, allowing the release of viral genomes into the cytosol through an unknown penetration mechanism. This figure was created in BioRender. Dao Thi, V. (2025). https://BioRender.com/f6k0uqw . All replicates are from three independent experiments. Statistical analysis was performed by unpaired two-tailed Student’s t test ( A , D , E ) or one-way ANOVA ( B , C , G ). ****: p < 0.0001; ns, non-significant.

Article Snippet: Bafilomycin A (Sigma), Concanamycin A (Biomol) and E64 (Selleckchem, 25 μM) were used at the indicated concentrations.

Techniques: Incubation, Infection, Virus, Staining, Microscopy, Two Tailed Test