Journal: Experimental & Molecular Medicine

Article Title: A dual inhibitor of PIP5K1C and PIKfyve prevents SARS-CoV-2 entry into cells

doi: 10.1038/s12276-024-01283-2

Figure Lengend Snippet: a Western blot analysis of p62, LC3B-I, LC3B-II and GAPDH in the presence of UNC3230, apilimod and UNI418. b Immunofluorescence staining with LC3B (green) and p62 (red) antibodies upon treatment with UNC3230, apilimod and UNI418, and images within the white rectangular boxes are magnified on the right side. Scale bar, 20 µm. c The area of fluorescence intensity from immunofluorescence images ( b ) was quantified after UNC3230, apilimod and UNI418 treatments. d, e Quantification of autophagic flux under normal conditions and under HBSS-induced starvation conditions with or without UNI418 treatment. RPE1 cells were treated with UNI418 (0.5 µM or 1 µM) for 2 h in the presence or absence of a lysosomal inhibitor (bafilomycin A 1 , BFA). Representative Western blot images ( d ) and the graph showing the average of 4 independent experiments ( e ). The error bars indicate the S.D. Statistical analysis was performed using two-way ANOVA; * p < 0.05, *** p < 0.001.

Article Snippet: The following chemicals were purchased from the indicated companies: bafilomycin A 1 (Cayman Chemical Company, Ann Arbor, MI, USA; 11038), dynasore (Sigma‒Aldrich, St. Louis, MO, USA; D7639), Hoechst (Cayman Chemical Company, 15547), UNC3230 (MedChem Express, NJ, USA; HY-110150), remdesivir (AdooQ, Irvine, CA, USA; A17170) and apilimod (MedChem Express, HY-14644).

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Experimental & Molecular Medicine

Article Title: A dual inhibitor of PIP5K1C and PIKfyve prevents SARS-CoV-2 entry into cells

doi: 10.1038/s12276-024-01283-2

Figure Lengend Snippet: a Live-cell imaging of HEK293T cells stably expressing mCherry-GFP-LC3 treated with 0.5 μM UNI418 at the indicated time points; the images within the white rectangular boxes are magnified on the right side. Scale bar, 30 µm. The number ( b ) and area ( c ) of GFP-positive dots per cell were quantified by ImageXpress. d Immunofluorescence images illustrating the inhibition of endolysosomal acidification by UNI418. Vero cells were treated with the mock control or 1 µM UNC3230, apilimod or UNI418 for 1 h at 37 °C. Acridine orange (4 µg/mL) was added to the compound-treated live cells, followed by additional incubation for 30 min. The cells were analyzed by confocal microscopy using two emission wavelengths at 493–560 (green) and 590–720 nm (red) after excitation at 488 nm. Merged images are presented, and images within the white rectangular boxes are magnified on the right side. Original magnification, 630×. The green-to-red fluorescence intensity ratio ( e ) and the area of fluorescent spots ( f ) were quantified. b , c , e , f The data are presented as the means ± SEMs, with a minimum of 200 spots. Statistical significance was determined by Student’s t test; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. n.s., not significant.

Article Snippet: The following chemicals were purchased from the indicated companies: bafilomycin A 1 (Cayman Chemical Company, Ann Arbor, MI, USA; 11038), dynasore (Sigma‒Aldrich, St. Louis, MO, USA; D7639), Hoechst (Cayman Chemical Company, 15547), UNC3230 (MedChem Express, NJ, USA; HY-110150), remdesivir (AdooQ, Irvine, CA, USA; A17170) and apilimod (MedChem Express, HY-14644).

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Immunofluorescence, Inhibition, Control, Incubation, Confocal Microscopy, Fluorescence

Journal: Experimental & Molecular Medicine

Article Title: A dual inhibitor of PIP5K1C and PIKfyve prevents SARS-CoV-2 entry into cells

doi: 10.1038/s12276-024-01283-2

Figure Lengend Snippet: a Representative Western blot image depicting pro- and mature cathepsin D after treatment with the indicated concentrations of UNC3230, apilimod and UNI418. GAPDH served as a loading control. b , c Evaluation of cathepsin D and LAMP1 colocalization in Vero cells after treatment with UNC3230, apilimod or UNI418 for 4 h. Immunofluorescence staining ( b ) and Pearson’s correlation coefficients ( c ) between LAMP1 and cathepsin D distribution. Scale bar, 15 µm. Images of individual cells, comprising at least 45 cells per sample, were randomly cropped and analyzed for colocalization using JACoP in ImageJ. The error bars indicate the SDs. Statistical significance was determined using Student’s t-test, where *** p < 0.001 and n.s., not significant. d , e Cathepsin L maturation in Vero cells was determined by Western blotting. d Representative Western blot images showing pro- and mature cathepsin L levels after treatment with UNI418 at the indicated concentrations. GAPDH was used as a loading control. e The ratio of cathepsin L to pro-cathepsin L was quantified and is presented as the mean ± SEM, with a sample size of n = 3. Statistical significance was determined by Student’s t test, where * p < 0.05 and ** p < 0.01. f Cleavage of the SARS-CoV-2 spike protein was determined with or without UNI418 treatment at specific time points. HEK293T cells stably expressing ACE2 (293T-ACE2) were treated with SARS-CoV-2 spike protein (Spike-HexaPro) in the absence or presence of 0.5 μM UNI418 and subjected to Western blotting for the SARS-CoV-2 spike protein, ACE2 and GAPDH. g The ratios of processed S2/GAPDH were quantified and are presented as the means ± SEMs, with n = 4. Statistical analysis was performed using two-way ANOVA; * p < 0.05.

Article Snippet: The following chemicals were purchased from the indicated companies: bafilomycin A 1 (Cayman Chemical Company, Ann Arbor, MI, USA; 11038), dynasore (Sigma‒Aldrich, St. Louis, MO, USA; D7639), Hoechst (Cayman Chemical Company, 15547), UNC3230 (MedChem Express, NJ, USA; HY-110150), remdesivir (AdooQ, Irvine, CA, USA; A17170) and apilimod (MedChem Express, HY-14644).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Stable Transfection, Expressing

Journal: Experimental & Molecular Medicine

Article Title: A dual inhibitor of PIP5K1C and PIKfyve prevents SARS-CoV-2 entry into cells

doi: 10.1038/s12276-024-01283-2

Figure Lengend Snippet: a Antiviral activity and cytotoxicity of UNI418. Vero cells mock-infected or infected with SARS-CoV-2 at an MOI of 0.01 or 0.001 were individually treated with increasing concentrations of UNI418, with apilimod, UNC3230 and remdesivir used as controls. On Day 2 (for UNI418, apilimod and remdesivir) or Day 1 (for UNC3230) postinfection, the number of viral spike protein-positive cells was determined by immunofluorescence microscopy using an anti-spike antibody. The antiviral activity (EC 50 ) was calculated as the concentration inhibiting the spike-positive cell number by 50% (red line). In parallel, the cytotoxicity (CC 50 ) was determined as the concentration that reduced the viability of mock-infected cells by 50% using MTT (black line). The selectivity index (SI) is the ratio of the CC 50 to the EC 50 . The values are expressed as the means ± SEMs from three independent experiments. b Reduction of SARS-CoV-2 genomic RNA as determined by quantitative RT‒PCR. Vero cells were infected with SARS-CoV-2 (MOI, 0.001) for 1 h at 37 °C. After removal of the unabsorbed virus, the cells were treated with increasing concentrations of UNI418 or apilimod for 24 h. Viral RNA was purified from culture supernatants and subjected to one-step quantitative RT‒PCR to measure relative amounts of the viral NP gene. The values are expressed as the means ± SEMs of three samples. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparison test; * p < 0.05; *** p < 0.001; **** p < 0.0001. c Reduction in SARS-CoV-2 NP expression by UNI418. SARS-CoV-2-infected Vero cells were treated with increasing concentrations of UNI418 or remdesivir (RDV). On Day 1 postinfection, cell lysates were harvested to measure viral nucleocapsid protein (NP) expression by Western blot analysis. β-Actin was used as a loading control. The marker size is labeled on the left side of the gels. d Blockade of the endocytic pathway by UNI418 in SARS-CoV-2-infected cells. Vero cells were pretreated with DMSO as a mock control (top), 1 µM UNI418 (middle) or 1 µM apilimod (bottom) for 1 h at 37 °C. To synchronize viral infection, the cells were infected with highly purified SARS-CoV-2 (MOI, 10) for 1 h at 4 °C in the presence of each compound. After shifting to 37 °C again, the cells were fixed at 1, 2, 4, and 8 h postinfection for immunolabeling with antibodies specific for viral NP (green) and cellular EEA1 (red). Nuclei were counterstained with DAPI (blue) for all samples. Original magnification, 630×.

Article Snippet: The following chemicals were purchased from the indicated companies: bafilomycin A 1 (Cayman Chemical Company, Ann Arbor, MI, USA; 11038), dynasore (Sigma‒Aldrich, St. Louis, MO, USA; D7639), Hoechst (Cayman Chemical Company, 15547), UNC3230 (MedChem Express, NJ, USA; HY-110150), remdesivir (AdooQ, Irvine, CA, USA; A17170) and apilimod (MedChem Express, HY-14644).

Techniques: Activity Assay, Infection, Immunofluorescence, Microscopy, Concentration Assay, Virus, Purification, Comparison, Expressing, Western Blot, Control, Marker, Labeling, Immunolabeling

Journal: Experimental & Molecular Medicine

Article Title: A dual inhibitor of PIP5K1C and PIKfyve prevents SARS-CoV-2 entry into cells

doi: 10.1038/s12276-024-01283-2

Figure Lengend Snippet: a SARS-CoV-2 infection-induced ACE2 downregulation was assessed in Vero cells. The cells were infected with SARS-CoV-2 at the indicated concentrations of UNI418 and subjected to Western blot analysis of ACE2, its cleaved ectodomain (ACE2 ecto ) and GAPDH at 1 and 3 h postinfection. b Endocytosis of ACE2 triggered by the interaction of ACE2 with the spike RBD protein (spike-RBD) was examined using 0.1 mM dynasore and 0.5 μM UNC3230, apilimod and UNI418. HEK293T cells were transfected with hACE2-GFP, treated with each compound and subjected to live cell imaging at the indicated times to visualize the hACE-GFP distribution. Membrane-bound ACE2 and internalized ACE2 are marked with yellow and white arrows, respectively. Scale bar, 10 µm. c The area ratio of internalized to membranous hACE2-GFP in individual cells was quantified and is presented as the mean ± SEM, n > = 20. Statistical significance was determined by one-way ANOVA; *** p < 0.001 and n.s., not significant.

Article Snippet: The following chemicals were purchased from the indicated companies: bafilomycin A 1 (Cayman Chemical Company, Ann Arbor, MI, USA; 11038), dynasore (Sigma‒Aldrich, St. Louis, MO, USA; D7639), Hoechst (Cayman Chemical Company, 15547), UNC3230 (MedChem Express, NJ, USA; HY-110150), remdesivir (AdooQ, Irvine, CA, USA; A17170) and apilimod (MedChem Express, HY-14644).

Techniques: Infection, Western Blot, Transfection, Live Cell Imaging, Membrane

Journal: Experimental & Molecular Medicine

Article Title: A dual inhibitor of PIP5K1C and PIKfyve prevents SARS-CoV-2 entry into cells

doi: 10.1038/s12276-024-01283-2

Figure Lengend Snippet: a Pseudotyped virus entry assay in the presence of the indicated concentrations of UNI418. Expression plasmids for VSV-G and the SARS-CoV-2 spike protein from WuHan-Hu-1, the Delta variant (B.1.617.2) and the Omicron variant (BA.1) were used to produce pseudotyped virus particles with a luciferase reporter. The 293T-ACE2 cells were infected with each pseudotyped virus under the indicated concentrations of UNI418. The relative viral entry efficiency was determined by the luciferase activity 24 h postinfection and is presented as the mean ± SEM, n = 3. Statistical significance was determined by two-way ANOVA, with *** p < 0.001. b , c Entry of Wuhan-Hu-1 spike-pseudotyped virus was assessed after PIKfyve and PIP5K1C knockdown in 293T-ACE2 cells. HEK293T-ACE2 cells were transfected with the indicated siRNAs and subjected to a luciferase-based virus entry assay (top panel) and Western blot analysis (bottom panel). The data are presented as the means ± SEMs, n = 3. Statistical significance was determined by Student’s t -test, with * p < 0.05 and ** p < 0.01. d Entry of Wuhan-Hu-1 spike-pseudotyped virus was measured by luciferase activity after treatment with apilimod or UNC3230 and their cotreatment in 293T-ACE2 cells. The data are presented as the means ± S.D.s, n = 3. Statistical significance was determined by Student’s t test, with * p < 0.05, ** p < 0.01 and *** p < 0.001. e , f Synergistic effect of UNC3230 and apilimod against SARS-CoV-2 infection. Vero cells infected with SARS-CoV-2 at an MOI of 0.01 were treated with 3-fold serial dilutions of different ratios of UNC3230 and apilimod: 5:0 (300 µM UNC3230 only), 4:1 (240 µM UNC3230 and 60 nM apilimod), 3:2 (180 µM UNC3230 and 120 nM apilimod), 2:3 (120 µM UNC3230 and 180 nM apilimod), 1:4 (60 µM UNC3230 and 240 nM apilimod), and 0:5 (300 nM apilimod only). e On Day 1 after infection, the antiviral efficacy of UNC3230 (red) and apilimod (blue) was individually determined in each combination. f Isobologram graph showing the sums of fractional EC 50 values (ΣFEC 50 ) for each combination. The mean value from the fixed dose ratios of 4:1, 3:2, 2:3 and 1:4 was 0.68. g The graphical model shows how UNI418 inhibits SARS-CoV-2 entry into cells by targeting PIP5K1C and PIKfyve.

Article Snippet: The following chemicals were purchased from the indicated companies: bafilomycin A 1 (Cayman Chemical Company, Ann Arbor, MI, USA; 11038), dynasore (Sigma‒Aldrich, St. Louis, MO, USA; D7639), Hoechst (Cayman Chemical Company, 15547), UNC3230 (MedChem Express, NJ, USA; HY-110150), remdesivir (AdooQ, Irvine, CA, USA; A17170) and apilimod (MedChem Express, HY-14644).

Techniques: Virus, Expressing, Variant Assay, Luciferase, Infection, Activity Assay, Knockdown, Transfection, Western Blot