Fig 2: A novel ACE2 peptide inhibitor.a Cell proliferation analysis of Caco-2 control (depicted in Fig. 3) and NACE2i (50 μM)-treated cells over a 72 h period. Proliferation was analyzed using WST-1 reagent and absorbance read after 2 h incubation. The graph depicts relative cell proliferation from triplicates expressed as a percentage of control cells (untreated, 0 h). Statistical significance was calculated using one-way ANOVA at each time point. b The ASI Digital pathology system was used to analyze ACE2 expression over time in Caco-2 cells treated with NACE2i (with FAM5 TAG). Dot plots displays the nuclear fluorescence intensity in Caco-2 cells for ACE2 and FAM5 tag as well as the % population of ACE2-expressing cells, >20 cells counted per group. Data represent mean ± SEM. Mann–Whitney test. ∗∗∗∗P < 0.0001 denote significant differences. c FACS analysis of cell surface and intracellular expression of ACE2 and LSD1 in Caco-2 cells treated with/without NACE2i for 48 h. The bar graph indicates the percentage ACE2+ or LSD1+ cells of the total Caco-2 population. Data are mean ± SEM (n = 3). Mann–Whitney test. ∗P < 0.05 denote significant differences. d Dot plot quantification of the fluorescence intensity (intracellular) of ACE2 and LSD1 in Caco-2 cells with NACE2i treatment. >50 cells were analyzed for each group. The PCC was calculated for colocalization (n = 20 cells were analyzed). Mann–Whitney-test: ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 denote significant differences. e qRT-PCR analysis to detect replicates of SARS-CoV-2 RNA in Caco-2 culture supernatants and infected cells at the indicated time points post-infection. The quantity of viral genome is expressed as TCID50 equivalents/mL. Data represent mean ± SEM, n = 3. One-way ANOVA, ∗P < 0.05, ∗∗P < 0.01 denote significant differences. f SARS-CoV-2 levels in infected Caco-2 cells. The percentage of virus-aligned reads (over total reads) is indicated for each sample. Error bars represent standard deviation from three independent biological replicates. g Dot plot quantification of the fluorescence intensity (intracellular) of SARS-CoV-2 spike, ACE2, LSD1, and TMPRSS2 in SARS-CoV-2-infected Caco-2 cells with phenelzine, GSK, or NACE2i treatment. >50 cells were analyzed for each group. The PCC was calculated to assess colocalization (n = 20 cells were analyzed). Data are mean ± SEM. Mann–Whitney-test: ∗∗∗∗P < 0.0001 denote significant differences. h Dot plot quantification of the fluorescence intensity (intracellular) of SARS-CoV-2 nucleocapsid, ACE2, and LSD1 in SARS-CoV-2-infected Caco-2 cells with phenelzine, GSK, or NACE2i treatment. >50 cells were analyzed for each group. The PCC was calculated to assess colocalization (n = 20 cells were analyzed). Data are mean ± SEM. Mann–Whitney-test: ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 denote significant differences. i Representative image of uninfected or SARS-CoV-2 infected Caco-2 cells using the Andor WD Revolution Inverted Spinning Disk microscopy system. SARS-CoV-2-infected Caco-2 cells with phenelzine, GSK, or NACE2i treatment, followed by permeabilization (intracellular) for immunostaining for SARS-CoV-2 nucleocapsid, ACE2, and IMPα3. DAPI (blue) was used to visualize nuclei. Scale bar, 12 mm (inset). Dot plot quantification of the fluorescence intensity of IMPα3 in SARS-CoV-2-infected Caco-2 cells. >50 cells were analyzed for each group. The PCC was calculated to assess colocalization (n = 20 cells were analyzed). Data are mean ± SEM. Mann–Whitney test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 denote significant differences.

Fig 3: LSD1–ACE2 interactions and the effect of LSD1 on the spike protein.a Inhibition of ACE2me2 peptide demethylation by LSD1 inhibitors. LSD1 enzyme was incubated with tranylcypromine, GSK, SP2509, or DMSO for 30 min. The LSD1 activity in each sample was measured by a demethylation assay with ACE2me2 peptide. Fluorescence was measured at 40 s intervals on a Synergy H4 multi-mode plate reader. Statistical significance was calculated using one-way ANOVA, ∗∗∗∗P < 0.0001. b Structure of ACE2 bound to the SARS-CoV-2 spike domain (PDB 6M17). Binding of ACE2 and the spike domain involves a Lys31 (ACE2) and Gln493 (spike) interaction. ACE2 is shown in yellow in cartoon mode, and spike domain in gray. Residues are shown in stick format. Methylation of ACE2 Lys31 (right panel) would disrupt this interaction. c Western blot analysis of ACE2 IP samples. Following ACE2 IP of Caco-2 cell membrane extract lysates, samples were analyzed by SDS-PAGE and blotted for pan-methylation lysine. d Schematic of SARS-CoV-2 infection assays. Caco-2 cells were seeded 24 h before the experiment. Then, cells were treated with each drug component for 48 h followed by SARS-CoV-2 infection (MOI 1.0). After 1 h viral adsorption incubation, the virus inoculum was removed and drug-containing medium was added. Then, cell culture supernatants were harvested at 0, 24, or 48 hpi and infected cells were collected at 48 hpi. Detection of viral genomes in the extracted RNA was performed by qRT-PCR, and viral spike proteins were quantified by a digital pathology assay (ASI system). e Duolink® proximity ligation assay measurements of protein interactions were performed on unpermeabilized Caco-2 cells infected with SASR-CoV-2 and treated with control or GSK. The Duolink assay produces a single bright dot per interaction within the cell. Representative images (left) are shown for ACE2 and pan-methylation lysine antibody. PLA signal intensity of the Duolink® assay (right) is shown for average dot intensity (single Duolink dot) or overall cell intensity for each cell. Data represent n = 20 cells, with significant differences calculated with the unpaired t-test (∗∗∗∗P < 0.0001). Representative images are shown with 10 µM scale bar in orange. f Duolink® proximity ligation assay measurements of protein interactions were performed on unpermeabilized Caco-2 cells transfected with VO, ACE2 WT, or ACE2 F31 plasmids and treated with 50 ng of SARS-CoV-2 spike protein. Samples were then probed with antibodies specific for ACE2 and SARS-CoV-2 spike protein. The Duolink assay produces a single bright dot per interaction within the cell. Representative images (left) are shown for ACE2 and SARS-CoV-2 Spike Duolink®. PLA signal intensity of the Duolink® assay (right) is shown for average dot intensity (single Duolink dot). Data represent n = 20 cells, with significant differences calculated with Kruskal–Wallis ANOVA (∗∗∗∗P < 0.0001). Representative images are shown with 10 µM scale bar in orange. g Dot plot quantification of the fluorescence intensity (cell surface) of SARS-CoV-2 spike protein in SARS-CoV-2-infected Caco-2 cells with phenelzine or GSK treatment. >50 cells were analyzed for each group and were quantified using the digital pathology assay (ASI system). Mann–Whitney test: ∗P < 0.05, ∗∗∗∗P < 0.0001 denote significant differences. h Principal component analysis (PCA) depicting transcriptional profiles for control, GSK, and phenelzine groups after batch effect removal. Experimental batches represented by different shapes (circles, batch 1; triangles, batch 2). i Heatmap of DEGs belonging to Reactome pathways: R-HSA-913531 interferon signaling (left); MAPK signaling-related pathways (R-HSA-5683057 MAPK family signaling cascades, R-HSA-5673001 RAF/MAP kinase cascade, and R-HSA-5684996 MAPK1/MAPK3 signaling; middle); and translation related pathways (R-HSA-70614 amino acid synthesis and interconversion (transamination), R-HSA-8957275 post-translational protein phosphorylation; right). The heatmap values depict the log2-fold change (logFC) of DEGs from treated cells compared with control cells (GSK vs. control and phenelzine vs. control). j Dot plot visualization of the top enriched Reactome pathways in treated cells compared to control cells. The dot color represents the false discovery rate (FDR) value for each enriched Reactome pathway and size represents the gene ratio. k Structure of ACE2 bound to the SARS-CoV-2 spike protein as depicted in a. Also depicted are the two peptide inhibitors targeting this region (ACE2-01, ACE2-02) and the interaction with the SARS-CoV-2 spike protein. l Schematic of SARS-CoV-2 infection. Caco-2 cells were seeded for 24 h and then infected with SARS-CoV-2 at MOI 1.0 in the presence of ACE2 peptide inhibitors (ACE2-01 or ACE2-02) for 1 h. The virus inoculum was removed and inhibitor-containing medium was added. Then, cell culture supernatants were collected at 0 or 48 hpi and infected cells were harvested at 48 hpi. Antiviral activity was assessed with three viral assays: SARS-CoV-2 qRT-PCR, median tissue culture infective dose assay (TCID50), and viral SPIKE protein quantified by digital pathology (ASI system). m Cell proliferation analysis of Caco-2 control and ACE2-01/ACE2-02-treated cells over a 96-h period. Proliferation was analyzed using WST-1 reagent and absorbance read after 2 h incubation. The graph depicts relative cell proliferation from three replicates expressed as a percentage of control cells (untreated, 0 h). Statistical significance was calculated using one-way ANOVA at each time point. n qRT-PCR analysis to detect replicates of SARS-CoV-2 RNA in Caco-2 culture supernatants and infected cells at the indicated time points post-infection. Relative infection was normalized to the uninfected control. Data represent mean ± SEM, n = 3. One-way ANOVA, ∗∗∗∗P < 0.0001 denotes significant differences. o TCID50 assay to measure infectious viral titers in the culture supernatants of infected cells. Data represent mean ± SEM, n = 3. One-way ANOVA, ∗∗P < 0.01 denotes significant differences. p Dot plot quantification of the fluorescence intensity (cell surface) of SARS-CoV-2 spike and ACE2 in SARS-CoV-2-infected Caco-2 cells with ACE2-01 or ACE2-02 treatment. >50 cells were analyzed in each group and were quantified by digital pathology (ASI system). The PCC was calculated for colocalization (n = 20 cells were analyzed). Mann–Whitney test: ∗∗∗∗P < 0.0001 denote significant differences. q Duolink® proximity ligation assay measurements of protein interactions were performed on unpermeabilized Caco-2 cells infected with SARS-CoV-2 and treated with control, GSK, or ACE2-01 or ACE2-01 peptide inhibitors. The Duolink assay produces a single bright dot per interaction within the cell. Representative images (left) are shown for ACE2 and SARS-CoV-2 Spike Duolink®. PLA signal intensity of the Duolink® assay (right) is shown for average dot intensity (single Duolink dot). Data represent n = 20 cells, with significant differences calculated with Kruskal–Wallis ANOVA (∗P < 0.05, ∗∗∗∗P < 0.0001). Representative images are shown with 10 µM scale bar in orange.

Fig 4: LSD1 co-localizes with ACE2 in SARS-CoV-2-infected primary cells.a FACS analysis of cell surface and intracellular expression of ACE2 and LSD1 and the cell subset composition of T cells in COVID-19 patient PBMCs (n = 3). The bar graph indicates the percentage ACE2+LSD1+ cells in the total Caco-2 population and each cell subset frequency of the total CD8+ T cells. b Representative image of PBMCs derived from COVID-19 patients using the ASI digital pathology system. Cells were not permeabilized (surface) for immunostaining for CD8, LSD1, and ACE2. DAPI (blue) was used to visualize nuclei. Scale bar, 12 μm (inset). Dot plot quantification of the fluorescence intensity (cell surface) of ACE2 and LSD1 in COVID-19 patient PBMCs (n = 3). >50 cells were analyzed for each patient sample. c Representative image of PBMCs derived from COVID-19 patients using the ASI digital pathology system. Cells were permeabilized (intracellular) for immunostaining for CD8, LSD1, and ACE2. DAPI (blue) was used to visualize nuclei. Scale bar, 12 μm (inset). Dot plot quantification of the fluorescence intensity (cytoplasmic and nuclear) of ACE2 and LSD1 in COVID-19 patient PBMCs (n = 3). >50 cells were analyzed for each patient sample. d Dot plot quantification of the fluorescence intensity (nuclear) of ACE2 in uninfected or SARS-CoV-2-infected Caco-2 cells. >50 cells were analyzed for each group. Mann–Whitney-test: ∗∗∗∗P < 0.0001 denote significant differences. e The ASI digital pathology system was used to analyze ACE2 expression in Caco-2 cells treated with the SARS-CoV-2 spike protein. Dot plots displays the nuclear fluorescence intensity in Caco-2 cells for ACE2 in control cells or cells positive for the spike protein. n > 1000 cells counted per group. Data represent mean ± SEM. Mann–Whitney test. ∗∗∗∗P < 0.0001 denotes significant differences. n.s. non-significant.

Fig 5: A summary model of SARS-CoV-2/ACE2/LSD1 interactions.Our data support a model in which: (Left) at the cell surface, SARS-CoV-2 entry relies on LSD1-dependent demethylation of ACE2, which is required for ACE2/SARS-CoV-2 spike binding. LSD1 inhibition therefore reduces viral entry via ACE2, especially LSD1 catalytic inhibitors such as GSK. In addition, our novel ACE2 peptide inhibitors, which compete with binding to SARS-CoV-2 spike protein, also block viral replication. (Right) Intracellularly, NACE2i blocks LSD1/ACE2 binding at cytoplasmic tail, and this change in protein–protein interactions might consequently inhibit LSD1’s demethylation activity. IMPα binds to the cytoplasmic tail of non-methylated ACE2 to mediate nuclear translocation and Pol II association to regulate gene expression. Therefore, NACE2i blocking IMPα/ACE2 binding impact ACE2-Pol II nuclear co-localization, which is required for SARS-CoV-2-related transcription, to reduce viral replication.