Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 reduces cholesterol abundance in the plasma membrane in general and lipid rafts in particular, and decreases lipid raft area more efficiently than atorvastatin (ATO). Control HEK293T (A) and Calu-3 (B) cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were labeled with cholesterol-binding mCherry-conjugated D4H*, the D434S mutant of domain 4 (D4) of C. perfringens theta-toxin. Fluorescence intensities correlating with plasma membrane cholesterol levels of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The average intensity values obtained in n = 9 independent biological replicates, and their average values (± SEM) are plotted in the figure. (C) To examine changes in the cholesterol content of raft and non-raft microdomains of the plasma membrane, control HEK/ACE2 + TMPRSS2 cells and those treated as above were labeled with Alexa Fluor 647-conjugated cholera toxin subunit B (CTX-AF647), a lipid raft marker, and F66. F66 is a fluorescent indicator with spectral properties depending on the cholesterol-dependent local molecular order (dipole potential) of the membrane; therefore, this dye, combined with CTX-AF647, can provide information about the extent of cholesterol reduction separately in raft and non-raft membrane regions. Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverglass show F66 intensities detected in 2 wavelength ranges of emission (“F66 N*” and “F66 T*”), their ratio (“F66 emission ratio” calculated as T*/N* pixel by pixel), and CTX-AF647 intensities. Cell “membrane masks” selected manually in CTX images were segmented using the maxentropy algorithm to CTX-high “rafts” and CTX-low “non-rafts” corresponding to high- and low-intensity regions, respectively, as shown by the representative images. Violin plots were generated from median F66 emission ratio values determined separately for the CTX-high “raft” (D) and CTX-low “non-raft” (E) masks of n = 54 to 73 individual cells, which also display median values with quartiles. (F) Pixelwise distributions of the F66 emission ratio in CTX-high “rafts” and CTX-low “non-rafts” of control cells are displayed. For the quantification of the relative area of lipid rafts, as an alternative definition for raft regions, a threshold value of the F66 emission ratio was determined (green dashed line) and membrane pixels were considered as “F66 raft” and “F66 non-raft” regions when being above and below the threshold, respectively. (G) Violin plots were generated from the relative fraction of F66 raft pixels (“F66 raft area”) of individual cells, which also display median values with quartiles. (H) Representative images show changes in the lateral distribution of the F66 emission ratio on a color-scale image and reduction in the relative F66 raft area induced by 1 μM SH42. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Clinical Proteomics, Membrane, Control, Labeling, Binding Assay, Mutagenesis, Fluorescence, Flow Cytometry, Marker, Generated

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 decreases ACE2 binding of SARS-CoV-2 spike receptor-binding domains (RBDs) more efficiently than ATO. (A) ACE2-expressing HEK/ACE2 + TMPRSS2 and Calu-3 control cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated with the GFP-conjugated RBDs of the Wuhan-Hu-1 strain (WT), Delta, and Omicron BA.1 variants for 4 min. RBDs were applied at 0.2 and 1.0 μg/ml for HEK/ACE2 + TMPRSS2 and Calu-3 cells, respectively. Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. (B) Representative RBD-GFP versus forward-scattered light intensity (FSC) density plots demonstrate decreases in the bound WT RBD-GFP in response to 1 μM SH42 in HEK/ACE2 + TMPRSS2 cells. Dashed lines represent average values of the fluorescence intensity obtained in the displayed representative samples. The average intensities obtained in n = 9 independent biological replicates and normalized to the mean value determined in untreated control samples, and their average values (± SEM) are plotted for WT, Delta, and Omicron BA.1 variants in HEK/ACE2 + TMPRSS2 (C) and Calu-3 (D) cells. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, **** P < 0.0001), while hashes show those between samples treated with ATO and SH42 at identical concentrations ( ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Binding Assay, Expressing, Control, Incubation, Fluorescence, Flow Cytometry

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42-induced reduction in ACE2 binding of WT SARS-CoV-2 spike RBDs negatively correlates with the applied RBD concentration. ACE2-expressing HEK/ACE2 + TMPRSS2 (A) and Calu-3 (B) control cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated with different concentrations of the GFP-conjugated RBDs of the Wuhan-Hu-1 strain (WT) for 4 min. Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The extents of inhibition (calculated as 1 − average of treated/average of control) were determined in n = 9 independent biological replicates, and their average values (± SEM) are plotted as a function of the applied RBD concentration ranging between 0.1 and 5 μg/ml for HEK/ACE2 + TMPRSS2 and between 1 and 10 μg/ml for Calu-3 cells. Asterisks indicate significant differences between samples treated with the lowest versus highest RBD concentrations for each treatment (** P < 0.01, **** P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Binding Assay, Concentration Assay, Expressing, Control, Incubation, Fluorescence, Flow Cytometry, Inhibition

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 inhibits the cellular entry of SARS-CoV-2 spike trimers more efficiently than ATO. Control HEK/ACE2 + TMPRSS2 and Calu-3 cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated for 4 h in the presence of WT, Delta, or Omicron BA.1 SARS-CoV-2 spike trimers conjugated with Alexa Fluor 488 (AF488-trimers) and labeled with F66. (A) Representative orthogonal views of confocal Z-stack images of F66 for the visualization of the plasma membrane and AF488-trimers to estimate entry demonstrate notable trimer accumulation in the intracellular space of untreated control HEK/ACE2 + TMPRSS2 cells. During image analysis, pixels corresponding to plasma membrane and intracellular pixels were segmented based on F66 Z-stack images. Markers were manually placed inside cells (green circles in the grayscale orthogonal view), and a MATLAB implementation of the 3D watershed algorithm identified the intracellular space of cells and their membrane (colored regions and red lines in the orthogonal view in the middle, respectively, and their overlay image displayed on the right). (B) Representative 3D reconstruction images displaying AF488 fluorescence intensities on a green-red color scale above a threshold intensity overlaid on intracellular pixels of individual cells (in transparent blue) demonstrate decreases in the amount of intracellular WT trimers in response to 1 μM SH42. Subsequently, the average fluorescence intensity values emitted by AF488-trimers were calculated exclusively from data of intracellular pixels for individual cells. The average intensities obtained in n = 400 to 600 HEK/ACE2 + TMPRSS2 (C) and Calu-3 (D) cells and normalized to the median value determined in untreated control samples are plotted along with median values with quartiles for WT, Delta, and Omicron BA.1 trimer variants. Asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA. Lognormal functions fitted to normalized mean intracellular AF488-trimer fluorescence intensity histograms of individual HEK/ACE2 + TMPRSS2 (E) and Calu-3 (F) cells also demonstrate the effects of ATO and SH42 on the internalization of WT, Delta, and Omicron BA.1 trimer variants.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Control, Incubation, Labeling, Clinical Proteomics, Membrane, Fluorescence

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 decreases cell surface ACE2 expression and its colocalization with lipid rafts more efficiently than ATO. (A) Control HEK/ACE2 + TMPRSS2 and Calu-3 cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were labeled with Alexa Fluor 488-conjugated anti-ACE2 antibodies (AF488-anti-ACE2). Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The average intensities obtained in n = 10 independent biological replicates and normalized to the mean value determined in untreated control samples, and their average values (± SEM) are plotted in the panel. (B) Control cells and those treated as above were labeled with AF488-anti-ACE2 and Alexa Fluor 647-conjugated cholera toxin subunit B (AF647-CTX). Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverglass show AF488-anti-ACE2 and AF647-CTX intensities, and their overlay, while the colocalization of the 2 signals and its changes in response to 1 μM SH42 are displayed in representative dot plots obtained from pixelwise fluorescence intensities. (C) Violin plots were generated from Pearson correlation coefficient values between fluorescence intensities of the 2 applied fluorophores determined from pixelwise data of n = 81 to 90 individual cells, which also display median values with quartiles. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Expressing, Control, Labeling, Fluorescence, Flow Cytometry, Membrane, Generated

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: DHCR24 inhibition by its selective blocker SH42 is a potential novel therapeutic approach to suppress initial membrane-coupled events of SARS-CoV-2 infection. SH42, a novel steroidal highly selective and potent DHCR24 inhibitor, interferes with ACE2 binding of SARS-CoV-2 spike RBDs and cellular uptake of spike proteins. By efficiently decreasing cholesterol levels of the host cell plasma membrane and causing the concomitant disruption of lipid raft microdomains, SH42 decreases cell surface levels of ACE2 and, in addition, reduces raft partitioning of the receptor protein, thereby altering its local microenvironment required for an efficient ACE2-mediated cellular binding and uptake of the virus. As a result, early membrane-coupled events of SARS-CoV-2 infection are inhibited as mirrored by the decreased binding of spike RBDs to host membrane and decreased cellular uptake of spike trimers, and culminate in decreased cellular infection with replication-competent SARS-CoV-2 virions.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Inhibition, Membrane, Infection, Binding Assay, Clinical Proteomics, Disruption, Virus

Journal: bioRxiv

Article Title: Staged heavy-chain filtering enables Fab discovery from combinatorially intractable library spaces

doi: 10.64898/2026.05.10.724059

Figure Lengend Snippet: (a, b) Binding ELISA curves and apparent half-maximal effective concentration (EC₅₀) values of CLR101 and reference antibodies (CR3022, P2B-2F6, and S309) against the SARS-CoV-2 D614G spike protein (a) and wild-type RBD (b) (n = 2 independent experiments, mean ± s.d.). (c) Apparent EC₅₀ values of CLR101, CR3022, P2B-2F6, and S309 against RBDs of six SARS-CoV-2 variants—wild-type (Wuhan-Hu-1), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and BA.5 (B.1.1.529.5)—determined by ELISA (n = 2 independent experiments, mean ± s.d.). (d) Epitope binning analysis of CLR101 by competitive ELISA. The heatmap displays the mean percent inhibition of CLR101 binding to wild-type RBD in the presence of excess competitor proteins (ACE2, CLR101, CR3022, P2B-2F6, and S309). Self-competition by CLR101 was included as a positive control for binding inhibition. The color scale indicates the degree of inhibition from 0% to 100% (n = 3 independent experiments). (e, f) Evaluation of in vitro neutralizing activity against D614G spike-pseudotyped lentiviral particles using hACE2-293T cells. (e) Dose-response neutralization curve of CLR101, showing neutralizing activity with an apparent EC₅₀ of 11.1 ± 2.6 nM (n = 2 independent experiments, mean ± s.d.). (f) Percent neutralizing activity of CLR101 alongside benchmark antibodies at a fixed antibody concentration of 100 nM (n = 2 independent experiments, mean ± s.d.).

Article Snippet: Virus–antibody mixtures were then added to monolayers of hACE2-expressing 293T cells (hACE2-293T; Takara Bio Inc., Kusatsu, Shiga, Japan) in 96-well plates.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Competitive ELISA, Inhibition, Positive Control, In Vitro, Activity Assay, Neutralization

Journal: iScience

Article Title: CGM23 corresponds to a pan-coronavirus lipopeptide inhibitor potently inhibiting virion fusion

doi: 10.1016/j.isci.2026.115334

Figure Lengend Snippet: Peptide design and evaluation (A) Schematic representation of SARS-CoV-2 spike protein and amino acid sequences of HR2 peptides from SARS-CoV-2 and EK1 (HCoV-OC43 HR2 derived peptide). N , N-terminus ; C , C-terminus ; S1/S2 , cleavage site at S1/S2 boundary ; RBD , receptor-binding domain ; HR1 , heptad repeat 1 ; HR2 , heptad repeat 2 ; HR2P , heptad repeat 2 peptide. Syncytia assay. Left: GFP- and spike-expressing 293T cells were co-cultured with RFP- and ACE2-expressing Calu-6 cells for 16 h in the presence or absence of peptides. Center: double-positive cells (white arrows) indicative of syncytia formation are frequent in the presence of the scrambled EK1 peptide (Ⅰ) but not in the presence of CGM23 (Ⅱ) (100 nM). Right: quantification of syncytia formation in the presence of the scrambled EK1 peptide (top) or CGM23 (bottom) relative to mock treatment. (B) Sequence, N- and C-terminal modifications and IC 50 of the 15 peptides with IC 50 s < 10 nM in the syncytia assay. CoV-2, SARS-CoV-2; N-term, N-terminus; C-term, C-terminus. IC 50 data are means of samples from a representative experiment. Ac, acetylation; PPA, 4-phenylpropanoic acid; PBA, 4-phenylbutanonic acid. (C) Correlation between IC 50 values in the pseudotyped SARS-CoV-2 spike virion assay and live SARS-CoV2 virus infection assay for the 15 peptides with IC 50 values below 10 nM in the syncytia assay. Statistical analysis was performed using Spearman’s rank test. (D and E) Dose-dependent inhibitory activity of CGM23 and EK1C4 relative to CG167 (EK1 scrambled peptide with EK1C4 lipidation) in the pseudotyped SARS-CoV2 spike virion assay (D) and live SARS-CoV2 infection assay (E). IC 50, half-maximal inhibitory concentration.

Article Snippet: Live virus experiments were performed with Calu-6 epithelial cells (ATCC HTB-56) stably expressing human Angiotensin Converting Enzyme 2 (ACE2) (OriGene, RC08442) as target cells.

Techniques: Derivative Assay, Binding Assay, Expressing, Cell Culture, Sequencing, Virus, Infection, Activity Assay, Concentration Assay

Journal: iScience

Article Title: CGM23 corresponds to a pan-coronavirus lipopeptide inhibitor potently inhibiting virion fusion

doi: 10.1016/j.isci.2026.115334

Figure Lengend Snippet: In vivo prophylactic and therapeutic efficacy of CGM23 against SARS-CoV-2 live virus in mice (A) The SARS-CoV-2 outgrowth assay. Lung homogenates were collected 2 days post viral infection (DPI) combined with intranasal administration of CGM23 and EK1C4 (12.5 μg, 0.865 mg/kg). (B) Diluted lung homogenates were added to Calu-6-ACE2 cells and infection titers were measured by the luciferase assay 48 h later. CG167, EK1 scrambled peptide with EK1C4 lipidation. Data presented correspond to mean ± SD. ∗ p < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (C) Histopathological findings of mouse lungs at 2 days after virus inoculation. Images are shown clockwise starting from the top left: CG167 group, EK1C4 group, non-infected group, and CGM23 group. Lung sections were stained with anti-spike antibody (green) and anti-MAC-2 antibody (magenta) and DAPI (blue). Scale bars, 100μm. (D and E) Quantitative analysis of lung histopathological findings for each group. Data shown represent means ± SD. (F) Therapeutic treatment. CGM23 and EK1C4 were administered intranasally (12.5 μg, 0.865 mg/kg; 25 μg, 1.73 mg/kg) 8 h after SARS-CoV-2 inoculation, and lung homogenates were collected 24 h later for plaque assay analysis. Data presented correspond to mean ± SD. ∗ p < 0.05, ∗∗∗ p < 0.001. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (G) Prophylactic treatment. SARS-CoV-2 was administered intranasally 30 min after intranasal administration of CGM23 or EK1C4 (12.5 μg, 0.865 mg/kg; 25 μg, 1.73 mg/kg). Lung tissues were collected 24 h later for plaque assay analysis.

Article Snippet: Live virus experiments were performed with Calu-6 epithelial cells (ATCC HTB-56) stably expressing human Angiotensin Converting Enzyme 2 (ACE2) (OriGene, RC08442) as target cells.

Techniques: In Vivo, Drug discovery, Virus, Infection, Luciferase, Staining, Plaque Assay