Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: Summary of sero-negative, acute, and convalescent-phase of COVID-19 patients from which the plasma ACE2 + EVs and RBD-IgG levels were measured.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Sampling

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of the cell-based neutralization assay. b Representative flow profiles showing the percentage (fluorescence mean intensity) of RBD-AF647 binding (at 16 and 3.3 nmol/L) to ACE2 + HEK-293 cells, inhibited by rhACE2 and ACE2 + EVs (evACE2) isolated from HEK-293 and HeLa cells (HEK-EV1 and HeLa-EV2, respectively) whereas ACE2 − EVs (evCon) had no neutralization effects (no RBD in black, PBS in dark blue, rhACE2 in orange, evCon in light blue, and evACE2 in green). c IC 50 of rhACE2 (orange line) and ACE2 in the EVs from ACE2 + HEK (ev1ACE2) and HeLa (ev2ACE2) cells (green lines) on 16 nM RBD-host cell binding (%). GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. d IC 50 of evACE2, ev1 from HEK and ev2 from HeLa cells (green lines), and rhACE2 (orange line) neutralizing infections by wild-type (WT) S + pseudotyped SARS-CoV-2. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. e IC 50 (nM) of ACE2 in ev1ACE2 (HEK) (green line) and rhACE2 (orange line) upon wild-type SARS-CoV-2 infection. GraphPad Prism 9.0.2 was used to calculate the IC 50 with three biological replicates. Data are presented as mean values ± SD. f Distinct effects of ACE2 + EVs (green lines) and ACE2 − control EVs (light blue line) on inhibiting Vero-6 cell death caused by SARS-CoV-2. N = 2 experiments with three biological replicates each. Data are presented as mean values ± SD. g The IC 50 of ev1ACE2 (HEK) neutralizing infections by pseudotyped SARS-CoV-2 expressing WT (black), B.1.1.7 (α) variant (red), B1.351 (β) variant (dark blue) and B.1.617.2 (δ) (light green) S protein. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates each. Data are presented as mean values ± SD. h Effects of ev1ACE2 (HEK) on protecting Vero-6 cell viability against infections of SARS-CoV-2 WT (black), B.1.1.7 (α) variant (red) and B1.351 (β) variant (dark blue) ( n = 3 biological replicates). Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Neutralization, Fluorescence, Binding Assay, Isolation, Infection, Control, Expressing, Variant Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of plasma EV ultracentrifugation and RBD-bead based depletion. b Cryo-EM images of human EV pellets isolated from acute phase COVID-19 plasma (bar = 100 nm). c Immunoblots of plasma EV pellets (sero-negative and COVID-19 acute phase patients CBB-005 and -013) for ACE2 and loading control of protein staining with Ponceau). Laemmli buffer was used for lysis ( N = 1 experiment). d ACE2 + EV pellets from acute phase patients 007, 008, 009, 012, and 013 (CBB) ( n = 2 biological replicates each) blocked SARS-CoV-2 infection-induced death of Vero-6 cells whereas the sero-negative control ( n = 2 biological replicates) and CBB-005 (no detectable ACE2) ( n = 2 biological replicates) did not show neutralization effects. One-tail t test, **** p = 2.24E−08 shown as compared to sero-negative. e , f Levels of ACE2 + EV counts ( n = 3 biological replicates) in plasma EVs (green) and bead-depleted EVs (light blue). One-tail paired t test, * p = 0.011 and ** p = 0.0063 (data are presented as mean values ± SD) ( e ) and altered neutralization effects on RBD–host cell binding ( f ) of the COVID-19 plasma EV pellets prior to and after RBD-bead depletion (convalescent phase CSB-012 and -024; acute phase CBB-008, 009, and 013). One-tail paired t test **** p = 5.11E−05.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Cryo-EM Sample Prep, Isolation, Western Blot, Control, Staining, Lysis, Infection, Negative Control, Neutralization, Binding Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Probability of severe disease-free survival in B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) mice receiving SARS-CoV-2 infection (10,000 pfu) and intranasal EVs (130 µg as measured on Nanodrop) per mouse (evCon in light blue and evACE2 in green). Log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests **** p = 2.27E−07. b Viral loads in mouse lungs on day 5/6 after receiving SARS-CoV-2 infection and administration of evCon ( N = 5 mice) (light blue) or evACE2 ( N = 10 mice) (green). T -test–nonparametric-one tailed, * p = 0.013. Data are presented as mean values ± SD. c . Representative H&E images of mouse lung sections at day 5 or 6 post virus inoculation and EV treatment (evCon and evACE2) intranasally. d , e Acute and chronic inflammation scores ( d ), and alveolar hemorrhage and necrosis scores ( e ) in mouse lungs on day 5/6 after receiving evCon ( N = 5 mice) (light blue) or evACE2 ( N = 7 mice) (green). T -test–nonparametric-one tailed, ** p = 0.005, ** p = 0.003 and *** p = 0.0004. Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Infection, One-tailed Test, Virus

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) On top, cartoon diagram of the SARS-CoV-2 spike with highlighted coldspot areas at the fusion peptide (FP, red), heptad repeat 2 region (HR2, blue), and subdomain 1 (SD1, green). Thick horizontal lines indicate the location of all coldspots (see also fig. S1A). At the bottom, amino acid changes in SARS-CoV-2 variants. Each circle represents a single aa substitution over ancestral virus. (B) Structure of the SARS-CoV-2 spike; FP (aa 814–838) and HR2 (aa 1142–1161) coldspots are in red and blue, respectively (PDB: 6XM4). (C) ELISA measurements of convalescent plasma IgG reactivity to FP (top) or HR2 (bottom) peptides. Optical density units at 450 nm (OD, Y axis) and reciprocal plasma dilutions (X axis). Non-infected controls in black; samples selected for cell sorting by flow cytometry are in red or blue. Two independent experiments. (D) Representative flow cytometry plots of B cells binding to fluorescently labeled FP (top) or HR2 (bottom) peptides. Numbers indicate percentage of double-positive cells in the gate. (E) Number of heavy and light chain V gene somatic mutations of antibodies to the FP (top) or HR2 (bottom) peptides. (F) Heatmaps with ELISA EC 50 values of monoclonal antibodies binding to the S of CoVs (top) and to the FP and HR2 peptides (bottom) corresponding to the CoV species, whose genus is indicated by Greek letters. The monoclonal antibodies to the HR2 region S2P6 and CV3–25 were assayed alongside for comparison. Cross indicates not tested. Two experiments. (G) Graph with IC 50 values of monoclonal antibodies neutralizing pseudoviruses corresponding to the indicated VOC. Two experiments. (H) ACE2 binding to ancestral S in ELISA in the presence of select FP and HR2 antibodies. Dotted line represents the limit of detection. Two experiments. (I) Inhibition of cell fusion by FP and HR2 antibodies. (J and K) fp.006 and hr2.016 antibodies protect in vivo. Top, diagram of the experiment’s timeline. Middle, mouse weight over time after challenge with ancestral SARS-CoV-2 of AAV-hACE2 mice treated with antibodies either 24 hours before ( (J) ; n = 6 per group, p = 0.0022 for both fp.006 and hr2.016 versus isotype at day 7), or 2 hours after ( (K) ; n = 5 per group, p = 0.0079 for both fp.006 and hr2.016 versus isotype at day 7) the infection. Mann-Whitney U test, standard deviation is shown. At the bottom, representative lung images at day 7.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, FACS, Flow Cytometry, Binding Assay, Labeling, Inhibition, In Vivo, MANN-WHITNEY, Standard Deviation

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) Overview of the complex structure of fp.006 Fab (surface representation; heavy chain in teal, light chain in light teal) bound to the SARS-CoV-2 FP (orange cartoon) with interacting side chains represented as sticks. (B) Visualization of FP residues F 823 , E 819 , and R 815 resting in a deep groove formed at the antibody paratope, with coloring as in ( A ). (C) Overlay of the fp.006-FP crystal structure with a cryo-EM structure of the SARS-CoV-2 prefusion S trimer (PDB: 6VXX). Models were aligned on Cα atoms of FP residues 818–822 (helical in both structures) with a root mean square deviation of 0.97 Å. (D) Residue-level interactions between FP residue R 815 and the antibody heavy chain include hydrogen bond formation with N 31 and a cation-π interaction with Y 52A . (E) Water-mediated interactions between FP residue E 819 and heavy chain residues Y 52A , N 56 , and F 97 . Water molecules are shown as red spheres. (F) van der Waals contacts between FP residue F 823 (orange stick) and residues that comprise a groove at the heavy and light chain interface (teal surfaces). (G) Interactions between FP residue D 820 and fp.006 CDRH2 residues include a salt bridge with R 55 and additional hydrogen bond formation with N 56 . Hydrogen bonds, salt bridges, and cation-π interactions are shown as dashed blue lines. (H) Flow cytometry detection of anti-FP and anti-HR2 antibody binding to SARS-CoV-2 S expressed on 293 T cells. Left, representative FACS plots (pre-gated on live-singlets-GFP + cells). Black lines indicate isotype control in the presence (continuous line) or absence (dotted line) of soluble ACE2. Right, quantification of the geometric mean fluorescent intensity (gMFI; n = 3). Two-tailed paired t-test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; standard deviation is shown.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Cryo-EM Sample Prep, Flow Cytometry, Binding Assay, Two Tailed Test, Standard Deviation

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) Structure of the SARS-CoV-2 S. S protomer with RBD up (left) or down (middle) and S trimer with two down and one up (right; PDB: 6XM4). SD1 and RBD are in green and yellow, respectively. (B) Graph shows ELISAs measuring plasma IgG reactivity to SD1-RBD. Negative controls in black; samples selected for sorting in green. Mean of two independent experiments. 82.1% of the plasma samples were positive (4SD higher than the average AUC of the controls) (C) Representative flow cytometry plot of B cells binding to fluorescently labeled SD1-RBD. Percentage refers to gated cells. (D and E) ELISAs measuring the reactivity of monoclonal antibodies to SD1-RBD ( D ) and to RBD ( E ). Mean of two independent experiments. (F) Heatmaps with the binding (EC 50 ) of SD1 monoclonal antibodies to S (top) or SD1-RBD (bottom) proteins corresponding to SARS-CoV-2 VOC. Two experiments. (G) Graph shows normalized relative luminescence values in cell lysates of 293T ACE2 cells after infection with ancestral SARS-CoV-2 pseudovirus in the presence of increasing concentrations of broadly cross-reactive SD1 monoclonal antibodies. At least two independent experiments.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Flow Cytometry, Binding Assay, Labeling, Infection

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) ACE2 binding to ancestral S in ELISA in the presence of sd1.040 or C121 control antibody. Representative of two experiments. (B) Structure of the sd1.040-S complex. Spike SD1 and RBD regions are shown as surface representation and colored wheat and gray, respectively. The sd1.040 Fab heavy chain (dark green) and light chain (light green) are shown as cartoon. The S N331-glycan that interacts with the sd1.040 Fab is shown as teal spheres. Inset: sd1.040 binding orientation on trimeric S shows clashes. (C and D) Surface rendering of sd1.040 epitope is highlighted on the SD1 and RBD surfaces, with sd1.040 CDR loops shown (ribbon). The majority of sd1.040 contacts are mediated by CDRH2, CDRL1 and CDRL3 loops. (E) Surface plasmon resonance (SPR) experiment showing the binding of sd1.040 Fab to ancestral SD1-RBD or S. (F) Antibody sd1.040 prevents ACE2-induced rearrangements. Flow cytometry detection of fp.006 binding to ancestral SARS-CoV-2 S expressed on 293 T cells. Left, representative FACS plots. Black lines indicate isotype control in the presence (continuous line) or absence (dotted line) of soluble ACE2. Right, quantification of the geometric mean fluorescent intensity (gMFI; n = 4). Two-tailed unpaired t-test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Flow Cytometry, Two Tailed Test

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) SPR assay of the sequential binding of immobilized antibodies to SD1-RBD protein followed by either sd1.040, rbd.042 or CoV-X4042. (B) SPR analysis showing that both arms of CoV-X4042 bind simultaneously to the same SD1-RBD molecule, since avidity is retained at decreasing SD1-RBD concentrations. Increasing normalized kd values indicate loss of avidity. Solid lines, IgG; dotted lines, Fab (see also fig. S8A). (C) Normalized relative luminescence values in cell lysates of 293T ACE2 cells after infection with ancestral SARS-CoV-2 pseudovirus in the presence of increasing concentrations of CoV-X4042 or its parental monoclonal antibodies individually or as a cocktail. Isotype control in black. On the right: mean IC 50 values and significance (P) when parental antibodies are compared to CoV-X4042 (n = 4; Welch’s t-test, two-tailed). (D) Graph with IC 50 values of bispecific and parental monoclonal antibodies neutralizing pseudoviruses corresponding to the indicated VOC. Mean of two independent experiments. (E) In vitro neutralization of SARS-CoV-2 by CoV-X4042. (F) CoV-X4042 protects in vivo. Top, diagram of the experiment’s timeline. Bottom, mouse weight over time after challenge, with ancestral SARS-CoV-2, of AAV-hACE2 mice treated with antibodies either 24 hours before (PRE; n = 5 per group, p = 0.0079), or 2 hours after (POST; n = 5 per group, p = 0.0079; day 7) the infection. Mann-Whitney U test, standard deviation is shown. (G) CoV-X4042 reduces viral titers in the lungs. Mice were treated with antibodies 24 hours before infection and virus titers evaluated on day 3 (n = 5 per group p = 0.0079 with both ancestral and Omicron BA.1; Mann-Whitney U test, standard deviation is shown).

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: SPR Assay, Binding Assay, Infection, Two Tailed Test, In Vitro, Neutralization, In Vivo, MANN-WHITNEY, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein–Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System

doi: 10.3390/ijms25010166

Figure Lengend Snippet: Staple screening performed on a short hACE2-derived α-helical sequence (His34-Gln42) (shown in pine green) via i , i + 4 side-to-side chain cyclizations by lactamization, olefin ring-closing metathesis (RCM), S-alkylation, S-arylation and copper(I)-catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Ball-and-stick model staple representations are shown in the insets and macrocycle sizes are indicated in the upper-righthand corner. Residues from hACE2 involved in the SARS-CoV-2 S RBD/hACE2 interaction are shown as yellow sticks labeled with three-letter codes.

Article Snippet: HEK-293T-hACE2 cells were chemically transfected with 3 μg of pCMV3 hTMPRSS2 (Sino Biological HG13070-UT) plasmid.

Techniques: Derivative Assay, Sequencing, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein–Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System

doi: 10.3390/ijms25010166

Figure Lengend Snippet: In silico alanine scan (BudeAlaScan a ) results for α1-helix (Ser19-Thr52) of hACE2 taken from the crystal structure of SARS-CoV-2 S RBD bound with hACE2 (PDB 6M0J). Residues from hACE2 involved in the SARS-CoV-2 S RBD/hACE2 interface are shown as yellow sticks labeled with three-letter codes. Selected hACE2-derived sequences (Asp30-Asp38) and (His34-Gln42) are outlined in light green and blue, respectively. a BudeAlaScan is an online software (version 1.0) available at https://pragmaticproteindesign.bio.ed.ac.uk/balas/ (accessed on 7 June 2023); it is only applicable to proteins consisting of natural amino acids.

Article Snippet: HEK-293T-hACE2 cells were chemically transfected with 3 μg of pCMV3 hTMPRSS2 (Sino Biological HG13070-UT) plasmid.

Techniques: In Silico, Labeling, Derivative Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein–Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System

doi: 10.3390/ijms25010166

Figure Lengend Snippet: Data generated from Far-UV CD spectra for hACE2-derived peptides measured at a concentration of 100 µM in 10 mM sodium phosphate buffer at pH 7.4 and 25 °C. ( A ) Far-UV CD spectra of hACE2 (Asp30-Asp38)-derived linear and i , i + 4 staple peptides in molar ellipticity per residue. ( B ) Table listing the derivatives of 1 with a focus on the macrocyclization technique and measure-based calculated helicity (%) via CD. ( C ) CD spectra of hACE2 (His34-Gln42)-derived linear and i , i + 4 staple peptides in molar ellipticity per residue. ( D ) Table listing the derivatives of 2 with a focus on the macrocyclization technique and measure-based calculated helicity (%) via CD. HDY 1,5-Hexadiyne; BMB 1,4-Bis(bromomethyl)benzene; HFB hexafluorobenzene. a Predicted helicity (%) using AGADIR online software available at http://agadir.crg.es (accessed on 17 April 2023); it is only applicable to peptides consisting of natural amino acids.

Article Snippet: HEK-293T-hACE2 cells were chemically transfected with 3 μg of pCMV3 hTMPRSS2 (Sino Biological HG13070-UT) plasmid.

Techniques: Generated, Circular Dichroism, Derivative Assay, Concentration Assay, Residue, Software

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein–Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System

doi: 10.3390/ijms25010166

Figure Lengend Snippet: The SARS-CoV-2 Spike RBD/hACE2 inhibition assessment involving a bioluminescence-based bioreporter assay and a pseudovirus-based entry inhibition assay. ( A ) Split-luciferase bioreporter assay demonstrating the disruptor capacity of peptides 1 , 2 , linear 4 , 4 , linear 11 and 11 . Asterisks indicate a statistically significant difference between the RLUs measured for SmBiT-ACE2 + LgBiT-RBD and the neutralizing Ab control or an individual peptide. ( B ) Antiviral activity of peptides linear 4 , 4 , linear 11 and 11 was assessed using a SARS-CoV-2 pseudovirus carrying a fluorescent reporter gene and HEK-293T-hACE2 cells transfected with TMPRSS2. The fluorescence data (RFUs) were converted into percentages via normalization with the infection-free control (Dulbecco’s Eagle Medium; DMEM) set as 0%; we furthermore considered that the pseudovirus-only samples represented maximum infection (100%) using GraphPad Prism software (version 9.3.1). Both experiments were repeated in triplicate three times, and the data are expressed as means ± standard error of the mean (SEM) (error bars). The means of more than two groups were compared using one-way ANOVA with Tukey’s multiple comparison correction. For all analyses, **** p < 0.0001; ** 0.0017 ≤ p ≤ 0.0025; n.s., not significant.

Article Snippet: HEK-293T-hACE2 cells were chemically transfected with 3 μg of pCMV3 hTMPRSS2 (Sino Biological HG13070-UT) plasmid.

Techniques: Inhibition, Luciferase, Control, Activity Assay, Transfection, Fluorescence, Infection, Software, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein–Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System

doi: 10.3390/ijms25010166

Figure Lengend Snippet: Plasma stability measured in rat plasma over 24 h of incubation at 37 °C. ( A ) The proteolytic stability of hACE2 (Asp30-Asp38)-derived peptides recorded as a function of degraded peptide over 24 h. ( B ) The proteolytic stability of hACE2 (His34-Gln42)-derived peptides recorded as a function of degraded peptide over 24 h. The data are plotted as means and SEMs of duplicate independent experiments. The percentage of residual peptide was monitored using UPLC-MS. All of the experiments were repeated three times.

Article Snippet: HEK-293T-hACE2 cells were chemically transfected with 3 μg of pCMV3 hTMPRSS2 (Sino Biological HG13070-UT) plasmid.

Techniques: Incubation, Derivative Assay