Chemical Synthesis
General Procedures. All commercial reagents were used as provided unless otherwise indicated. An anhydrous solvent dispensing system (J. C. Meyer) using 2 packed columns of neutral alumina was used for drying THF, Et2O, and CH2Cl2, whereas 2 packed columns of molecular sieves were used to dry DMF. Solvents were dispensed under argon. Flash chromatography was performed with RediSep Rf silica gel columns on a Teledyne ISCO CombiFlash® Rf system using the solvents as indicated. Nuclear magnetic resonance spectra were recorded on a Varian 600 MHz or Bruker 400 MHz spectrometer with Me4Si or signals from residual solvent as the internal standard for 1H or 13C. Chemical shifts are reported in ppm, and signals are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br s (broad singlet). Values given for coupling constants are first order. High resolution mass spectra were recorded on an Agilent TOF II TOF/MS instrument equipped with either an ESI or APCI interface at the Center for Drug Design, University of Minnesota (Minneapolis, MN, USA).
LC-1541 (PIXN). To compound 6,9-difluorobenzo[g]isoquinoline-5,10-dione (220 mg, 0.89 mmol, 1.0 eq.) in THF (20 mL) under argon atmosphere was added ethane-1,2-diamine (0.6 mL, 8.97 mmol, 10.0 eq.). The resultant solution was stirred at 50 ℃ for 24 h. The mixture was diluted with methanol (10 mL) and the solvents were removed under vacuum. The crude product was purified by column chromatography (30% MeOH: NH4OH (9:1) in CH2Cl2) to give compound LC-1541 as a blue gum (95 mg, 56%). 1H NMR (600 MHz, CD3OD) δ 9.44 (s, 1H), 8.88 (d, J = 5.2 Hz, 1H), 8.14 (d, J = 5.2 Hz, 1H), 7.52 (d, J = 4.2 Hz, 2H), 3.71 (td, J = 6.4, 2.0 Hz, 4H), 3.13 (q, J = 6.0 Hz, 4H). HRMS (ESI+): m/z calcd for C17H20N5O2 [M+H]+ 326.1612, found 326.1602.
The following compounds were prepared in a fashion similar to the one described for LC-1541.
LC1539. 1H NMR (600 MHz, DMSO-d6) δ 13.49 (s, 2H), 10.45 (t, J = 6.5 Hz, 2H), 7.67 (s, 2H), 7.22 (s, 2H), 3.82 (q, J = 6.5 Hz, 4H), 3.06 (d, J = 8.8 Hz, 4H). HRMS (ESI+): m/z calcd for C18H21N4O4 [M+H]+ 357.1557, found 357.1559.
LC1540. 1H NMR (600 MHz, CD3OD) δ 9.04 (d, J = 1.8 Hz, 1H), 8.67 (d, J = 5.0 Hz, 1H), 7.74 (d, J = 4.9 Hz, 1H), 6.99 (dd, J = 4.2, 2.3 Hz, 2H), 3.75 (q, J = 3.5 Hz, 4H), 3.45 (dt, J = 13.0, 6.5 Hz, 4H), 2.95 (td, J = 6.5, 3.6 Hz, 4H), 2.87 (td, J = 5.5, 1.6 Hz, 4H). HRMS (ESI+): m/z calcd for C21H28N5O4 [M+H]+ 414.2136, found 414.2132.
LC1542. 1H NMR (600 MHz, CD3OD) δ 7.38 (s, 2H), 7.32 (s, 2H), 3.92 (s, 6H), 3.70 (t, J = 5.5 Hz, 4H), 3.55 (t, J = 6.4 Hz, 4H), 2.98 (t, J = 6.5 Hz, 4H), 2.83 (t, J = 5.5 Hz, 4H). HRMS (ESI+): m/z calcd for C24H33N4O6 [M+H]+ 473.2395, found 473.2395.
LC1553. 1H NMR (600 MHz, CD3OD) δ 7.27 (d, J = 9.2 Hz, 1H), 7.17 (d, J = 9.7 Hz, 1H), 7.07 (d, J = 9.8 Hz, 1H), 7.02 (d, J = 9.1 Hz, 1H), 3.87 (s, 3H), 3.71 (dt, J = 7.7, 5.5 Hz, 4H), 3.49 (t, J = 6.5 Hz, 2H), 3.44 (t, J = 6.5 Hz, 2H), 2.97 (t, J = 6.5 Hz, 2H), 2.93 (t, J = 6.5 Hz, 2H), 2.83 (dt, J = 11.6, 5.6 Hz, 4H). HRMS (ESI+): m/z calcd for C23H31N4O6 [M+H]+ 459.2238, found 459.2233.
LC1519. 1H NMR (600 MHz, CD3OD) δ 6.81 (s, 2H), 6.75 (s, 2H), 3.70 (t, J = 4.5 Hz, 4H), 3.65 (t, J = 5.1 Hz, 4H), 3.58 (t, J = 4.5 Hz, 4H), 3.34–3.26 (m, 4H), 2.91–2.83 (m, 8H). HRMS (ESI+): m/z calcd for C26H37N4O8 [M+H]+ 533.2606, found 533.2601.
LC1520. 1H NMR (600 MHz, CD3OD) δ 6.91 (s, 2H), 6.90 (s, 2H), 3.69–3.61 (m, 16H), 3.54 (t, J = 4.8 Hz, 4H), 3.37 (t, J = 6.6 Hz, 4H), 2.92–2.84 (m, 8H). HRMS (ESI+): m/z calcd for C30H45N4O10 [M+H]+ 621.3130, found 621.3136.
LC1521. 1H NMR (600 MHz, CD3OD) δ 7.02 (s, 2H), 6.95 (s, 2H), 3.68–3.62 (m, 16H), 3.62–3.57 (m, 8H), 3.52 (t, J = 4.5 Hz, 4H), 3.44 (t, J = 6.9 Hz, 4H), 2.92 (t, J = 6.6 Hz, 4H), 2.88 (t, J = 5.1 Hz, 4H). HRMS (ESI+): m/z calcd for C34H53N4O12 [M+H]+ 709.3654, found 709.3648.
Chemoenzymatic synthesis of HS 6-mers
A total of seven 6-mers were synthesized in this study using the chemoenzymatic synthetic approach 44. These 6-mers are differed in the number of sulfo groups as well the presence or absences of 2-O-sulfated iduronic acid (IdoA2S) residue. All synthesis was initiated from glucuronide para-nitrophenyl (GlcA-pNP), which is commercially available (Carbosyn). To synthesize the 6-mers without an IdoA2S residue, the synthesis involved the use of heparosan synthase 2 from Pasteurella multocida (pmHS2) and UDP-GlcNAc (or UDP-GlcNTFA, NTFA represents N-trifluoroacetylated glucosamine was incubated with pmHS2 (30 mg) and UDP-GlcNAc (3 mM) in a 100 mL of the reaction buffer containing 25 mM Tris-HCl (pH 7.2), 5 mM MnCl2. The reaction was incubated at 37 °C overnight, and the 2-mer product was purified by a C-18 reverse phase column. The 2-mer product was further elongated to 3-mer in the 100 mL reaction buffer (pH 7.2) containing UDP-GlcA and purified on a C-18 column. The elongation and purification steps were repeated until the desired 6-mer was achieved. The final compound was confirmed for structural identity with Mass Spec and purity was checked with analytical HPLC. The pmHS2 enzyme, UDP-GlcA and UDP-GlcNTFA were made according to the protocol described in a prior publication 45,46. Additional modification steps, including N-sulfation, 6-O-sulfation were completed using N-sulfotransferase and 6-O-sulfotransferase isoform 3, respectively 46. To install an IdoA2S residue, 2-O-sulfotransferase and C5-pimerase were employed. The 3-O-sulfation to prepare NS2S6S3S 6-mer, 3-O-sulfotransferanse was used. The purity of the products was confirmed by high resolution anion exchange HPLC, and the molecular weight (MW) was determined by electrospray ionization mass spectrometry. As shown below, the purity of the 6-mers was in the range of 92% to 99%, and the measured MW was very close to the calculated MW (Calc MW).
ID
|
Abbreviated sequence
|
Calc MW
|
Measured MW
|
Purity by HPLC
|
0S
|
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-pNP*
|
1277
|
1277
|
99%
|
NS
|
GlcNS-GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP
|
1391
|
1391
|
99%
|
2S
|
GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP
|
1471
|
1470
|
97%
|
NS6S
|
GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-pNP
|
1631
|
1630
|
94%
|
NS2S6S3S
|
GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S-GlcA-pNP
|
1791
|
1791
|
92%
|
NS6S(6)
|
GlcNAc6S-GlcA-GlcNAc6S-GlcA-GlcNS6S-GlcA-pNP
|
1555
|
1554
|
91%
|
NS6S(7)
|
GlcNS6S-GlcA-GlcNAc6S-GlcA-GlcNAc6S-GlcA-pNP
|
1555
|
1554
|
99%
|
*pNP refers to para-nitrophenyl group
Cell culture and virus infection
293T cells stably expressing GFP-tagged human ACE2 (ACE2-GFP) were reported previously 9. To make SLC35B2 KO 293T cells expressing ACE2-GFP, SLC35B2 CRISPR KO cells 30 were transfecting cells with pCMV-ACE2-GFP (Codex). GFP-positive cells were sorted by FACS after neomycin (1 mg/mL) selection for 1 week. These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin.
Vero TMPRSS2-E6 (TE6), a Vero E6-based cell line stably transfected with human TMPRSS2 was obtained from BPS Bioscience (San Diego, CA) and was maintained in Gibco high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 10 mM HEPES pH 7.3, 1% Na pyruvate plus 3 μg/mL of Puromycin in a 37 °C incubator supplemented with 5% CO2. Vero E6-TMPRSS2-T2A-ACE2 (TA6), a Vero E6 cell line overexpressing both human TMPRSS2 and human ACE2 was obtained from BEI Resources (Manassas, VA) and was maintained in Gibco high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 10 mM HEPES pH 7.3 plus 10 μg/mL of Puromycin in a 37 °C incubator supplemented with 5% CO2.
The seeds of SARS-CoV-2 clinical isolates — USA-WA1/2020 and the Delta variant were obtained through BEI Resources (Manassas, VA). All seed viruses were amplified in TE6 cells in the infection medium (DMEM supplemented with 3% FBS) at 37 °C, 5% CO2 for 3 days. Amplified viruses were aliquoted and stored in a secured −80 °C freezer until use. Virus titers were titrated in TE6 cells using an ELISA-based 50% tissue culture infectious dose (TCID50) method 47,48.
Infection with live SARS-CoV-2 in cultured cells
Vero TA6 cells were pre-seeded into 8-chamber slides (ibidi #80800) overnight. The next day after the aspiration of the growth media, cells were pre-treated with PIXN, MTAN, or DMSO diluted in the infection medium at 37°C for 30 mins. Then, without removing the compounds, cells were infected with USA-WA1/2020 or Delta variant (MOI of 0.1 or 0.5) at 37 °C, 5% CO2 for another four 4 h before fixation and staining. For heparinase I/III treatment, cells were treated with a Heparinase I and III mixture that contains 180 μL DMEM medium, 20 μL 10x digestion buffer (NEB), 1.6 μL heparinase I, 4.0 μL heparinase II at 37 °C for 3 hours. Treated cells were washed once with the medium and then subject to infection or spike-induced cell-cell fusion. Cells were fixed with 4% paraformaldehyde in Phosphate Buffer Saline (PBS) for 10 min followed by 4 washes with PBS before antibody staining. To confirm the removal of HS, we incubated cells with recombinant CFP+ at ~100 nM on ice for 10 min before fixation and imaging.
Infection with live SARS-CoV-2 in mice
B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) transgenic mice (JAX Stock No. 034860) 33 were bred at FDA White Oak Vivarium and were individually genotyping-confirmed (Transnetyx) before experiments. Age-matched male and female K18-hACE2 adult mice (the male and female ratio was approximately 1:1) at 12-16 weeks of age were injected intraperitoneally with 0.1 mL/mouse of PIXN at a final dose of 50 or 100 mg/kg, respectively. PIXN was dissolved in sterile PBS (pH 7.4) containing 1% of Penicillin/Streptomycin. K18-hACE2 mice receiving 0.1 mL/mouse of sterile PBS (pH7.4) containing 1% of Penicillin/Streptomycin served as controls. Two hours after injection, mice were inoculated intranasally with 1000 TCID50/50 µL/mouse of live infectious USA-WA1/2020. Three days after infection, mice were euthanized, and whole lungs were harvested for viral load determination. Lungs were homogenized, and total RNA was extracted using the RNeasy Plus Mini Kit. The copies of the viral nucleocapsid (N) gene in homogenized lung tissues were amplified using the High-Capacity cDNA Reverse Transcription Kit and QuantiNova SYBR Green PCR kit in combination with 500 nM of 2019-nCoV RUO Kit according to the following program: 95 °C for 120 s, 95 °C for 5 s and 60 °C for 18 s (50 cycles) 1,3. A value of 1 was assigned if gene copies were below the detection limits. The mouse infection experiments with live SARS-CoV-2 were performed in an FDA Animal Biosafety Level-3 (ABSL-3) laboratory equipped with advanced access control devices and by trained personnel equipped with powered air-purifying respirators.
Pseudoviral particle entry assay
HEK293T-ACE2-GFP cells were seeded in white, transparent bottom 96-well microplates at 20,000 cells per well in 100 µL growth medium and incubated at 37 °C with 5% CO2 overnight (~16 h). The growth medium was carefully removed, and 50 µL PP or PP-containing compounds were added to each well. The plates were then spinoculated by centrifugation at 1500 rpm (453× g) for 45 min and incubated for 24 h (48 h for Calu-3 cells) at 37 °C, 5% CO2 to allow cell entry of PP and the expression of luciferase. After incubation, the supernatant was carefully removed. Then 50 µL/well of Bright-Glo luciferase detection reagent (Promega) was added to assay plates and incubated for 5 min at room temperature. The luminescence signal was measured by a Victor 1420 plate reader (PerkinElmer). For ACE2-GFP cells, the GFP signal was also determined by the plate reader. Data were normalized with wells containing PP but no compound as 100%, wells mock-treated with phosphate buffer saline (PBS) as 0%, and the ratio of luciferase to the corresponding GFP intensity was calculated.
SARS-CoV-2 infection in a 3D EpiAirway model
Human bronchial epithelial cells (HBEC’s 3D-EpiAirway™) were seeded into culture inserts for 6-well plates one day before viral infection. Before adding drugs or virus, accumulated mucus from the tissue surface was removed by gently rinsing the apical surface twice with 400 μL TEER buffer. All fluids from the tissue surface were carefully removed to leave the apical surface exposed to the air. MTAN was diluted into the assay medium and placed at room temperature before co-treatment with a virus (MOI of 0.1) onto the apical and basal layers for one h. Following one h treatment, the virus was removed from the apical layer. The basolateral medium was replaced with fresh maintenance medium and compound at 24 h, 48 h, and 72 h post-infection.
At 24 hours and 96 hours post-infection, the apical layer was washed with 0.4 mL of the TEER buffer (PBS with Mg2+ and Ca2+). The washes were combined and aliquoted into separate microfuge tubes (1.5 mL). Eight-fold serial dilutions of apical layer supernatant sample concentrations were added to 96-well assay plates containing Vero E6 cells (20,000/well). The plates were incubated at 37 °C, 5% CO2, and 95% relative humidity. Following three days (72 ± 4 h) incubation, the plates were stained with crystal violet to measure the cytopathic effect (CPE). Virus titers were calculated using the method of Reed and Muench (Reed et al., 1938). The TCID50 values were determined from duplicate samples.
LDH assay
Medium from the basolateral layer of the 3-D tissue culture inserts was removed 24- and 96-h post-infection and diluted in an LDH Storage Buffer per the manufacturer’s instructions (LDH-Glo Cytotoxicity Assay, Promega). Samples (5 μL) were further diluted with the LDH Buffer (95 μL) and incubated with an equal volume of LDH Detection Reagent. Luminescence was recorded after 60 min incubation at room temperature. As a negative control, we included a no-cell sample in determining the culture medium background. We used tissues treated with the apoptosis-inducing drug bleomycin as a positive control.
ATP-based cytotoxicity assay
HEK293T-ACE2-GFP cells were seeded in a white, transparent bottom 96-well microplate (Thermo Fisher Scientific) at 20,000 cells per well in 100 µL growth medium and incubated at 37 °C with 5% CO2 overnight (~16 h). The growth medium was carefully removed, and 100 µL medium with compounds was added into each well. The plates were then incubated at 37 °C for 24 h (48 h for Calu-3 cells) at 37 °C 5% CO2. After incubation, 50 µL/well of ATPLite (PerkinElmer) was added to assay plates and incubated for 15 min at room temperature. The luminescence signal was measured using a Victor plate reader (PerkinElmer). Data were normalized with wells containing cells but no compound as 100% and wells containing media-only as 0 %.
NMR study
NMR spectra were recorded at 313 K with a Bruker Avance III spectrometer operating at 850 MHz and equipped with a high sensitivity 5 mm TCI cryoprobe. Samples were lyophilized twice to remove residual solvents and were then dissolved in D2O (99.996%, Sigma, Co.) and placed in 5 mm NMR tubes. For the experiments involving free ligands, the samples were prepared to obtain a final concentration of 1.2x10-3 M by dissolving hexasaccharide (1 mg) and Pixantrone (0.2 mg) in D2O. Proton spectra were recorded using water presaturation with a recycle delay of 10 seconds and 24 scans.
HSQC (heteronuclear single quantum coherence) experiments were performed in phase-sensitive mode with Echo/Antiecho-TPPI gradient selection using decoupling during acquisition and multiplicity editing during the selection step.
Thirty-two dummy scans and 20 scans with decoupling during the acquisition period with 1.5 seconds relaxation delay were accumulated, using a matrix size of 2048x256 datapoints.
Bidimensional HSQC-TOCSY data were acquired by using 20 scans per increment using a 2048x256 datapoints matrix with zero-filling in F1 to 2048x2048 points. 1H-13C HSQC-TOCSY were performed using 1.5 seconds relaxation delay and 100 msec mixing time.
For NOE experiments, the samples were prepared by dissolving hexasaccharide (1 mg) and Pixantrone (0.1 mg) in D2O, reaching a molar ratio of hexasaccharide/Pixantrone 2:1. NOESY experiments were performed at 313 K. A total of 24 scans was collected for each free induction decay (matrix 2048x256 points), the data were zero-filled to 2048x2048 points before Fourier transformation, and mixing time values of 120 ms was used.
Drug and protein binding studies
HS or HS 6-mers of different concentrations were added to MTAN or PIXN (50uM). After a brief incubation, absorbance was measured from 500 nm to 700 nm by Nanodrop 2000 (Thermo Fisher Scientific). The change in OD650 was determined and fitted into a binding curve using GraphPrism 9.0.
To study the interaction of spike with ACE2 in vitro, we used spike (200 ng/well) in phosphate buffer saline (PBS) to treat a high binding 96 well plate at 4 °C for 15 h. The spike-coated plate was washed with PBS once and then incubated with the TMS buffer containing 4% bovine serum albumin (BSA), 20 mM Tris-HCl, 7.4, 150 mM NaCl, 2mM MgCl2 2 mM CaCl2 for three h at 37 °C. The plate was washed once with a BSA-free TMS buffer and then incubated for one h with heparin (5 µM) together with PIXN (20 µM) in the TMLS buffer containing 20 mM Tris-HCl, 7.4, 50 mM NaCl, 2mM MgCl2 2 mM CaCl2, 1% BSA to allow heparin binding to the spike. The plate was washed with the TMLS buffer once and then incubated with ACE2 recombinant protein bearing an hFC tag at various concentrations at room temperature for 1 h in the TMLS buffer. The plate was washed three times with the TMS buffer and then incubated with HRP-conjugated protein A in the TMS buffer for 1 h. The plate was washed four times and then developed with TMB turbo substrate for 5-10min before the addition of 1 M sulfuric acid to quench the reactions. The absorbance was measured at 450 nm.
Spike- and ACE2-mediated cell fusion assay
293T cells expressing spike protein and mCherry were generated by transfecting cells in a 3.5 mm culture dish with 2.0 µg of pcDNA3.1-SARS-CoV-2-Spike (From the Delta variant) and pLV-mCherry at 10:1 ratio for 24 h. Acceptor cells are 293T cells stably expressing ACE2-GFP reported previously (Zhang et al., 2020a). ACE2-GFP cells were seeded in fibronectin-coated 8-well Ibidi glass-bottom chambers at 50,000/well on day 1. Cell-cell fusion was conducted 48 hours later. For each fusion reaction, 50,000 spike/mCherry-transfected cells (effector cells) were added to the imaging chamber. Live cell 4D imaging was initiated ~1 minute after adding the effector cells. The fusion reactions were stopped for immunostaining experiments by incubating cells with a fixing buffer containing 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were then stained with a Hoechst 33,342 staining solution to label the nuclei or with primary and secondary antibodies diluted in a PBS-based staining buffer containing 10% FBS and 0.2% Saponin.
Transmission electron microscopy
Spike/mCherry donor cells and ACE2-GFP acceptor cells were mixed at 1:1 ratio and incubated in suspension at 37 °C for 12 min to form spike- and ACE2-induced membrane synapses. Cells were then fixed in a mixture of 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, for 90 minutes at room temperature. Subsequently, samples were washed three times for ten minutes each in 0.1M sodium cacodylate buffer, pH 7.4, before being post-fixed in 1% OsO4 and 1.5% K3Fe(CN)6 in 0.1M cacodylate buffer for 60 minutes on ice. Next, samples were rinsed and washed two times for ten minutes each in water and incubated with 1% uranyl acetate overnight at 4 °C. The following day samples were rinsed and washed in water for ten minutes and gradually dehydrated through a graded ethanol series followed by propylene oxide. Samples were then infiltrated in a gradient mix of propylene oxide and resin (Embed 812 resin) before being infiltrated with three changes of pure resin and embedded in 100% resin and baked at 60ºC for 48 hours. Ultrathin sections (65 nm) were cut on an ultramicrotome (Leica EM UT7), and digital micrographs were acquired on JOEL JEM 1200 EXII operating at 80 Kv and equipped with an AMT XR-60 digital camera.
In vitro ACE2 clustering assay
To prepare small unilateral vesicles (SUV), we used glass syringes to prepare a DOGS-NTA lipid mixture in a glass vial as follows: rinse a glass vial with chloroform, and then add ~1 mL of chloroform plus individual lipids (POPC, 2.98mg; DOGS-NTA, 0.085mg, PEG-5000 PE, 0.023 mg). The lipid mixture was dried with a stable flow of nitrogen and then in a vacuum desiccator for 2 h. Resuspend the dried lipids in 1.5 mL of PBS and vortex. Transfer the resuspension into two 1.5 mL conical microcentrifuge tubes. Freeze the lipid resuspension in liquid nitrogen and thaw immediately in a water bath at room temperature. Repeat the freeze-thaw for 30 cycles. The cloudy solution will become clear over the freeze-thaw cycles. The lipid resuspensions were centrifuged at 22,000 × g for 45 min at 4 °C. The SUV-containing supernatant was collected in a clean tube.
Ibidi u-Slide 8 well Glass Bottom chambers were soaked in 5% Hellmanex III for 24 h (pre-heated to 50 °C) overnight, rinsed extensively with ultrapure water, and air dried. The glass surface was then treated with NaOH 1M for 1 h at 50 °C. Treated chambers were rinsed with ultrapure water and washed 3 times with the Basic buffer (HEPES pH 7.3 50mM, NaCl 150mM). Each well was treated with 200 µL Basic buffer containing 7uL SUV at 37 °C for 1 h followed by three washes with the Basic buffer. After the last wash, we blocked the lipid-coated surface with 200 µL Clustering Buffer (Basic buffer plus BSA 1 mg/mL) at 37 °C for 20 min. Add His8-tagged, Alexa555-labeled ACE2 1-740 (~50 nM) in the Clustering Buffer to each well and incubate the chamber at 29 °C for 1 h to allow His-tagged ACE2 to attach to the lipid bilayer. Wash each well three times with the Signaling Buffer (HEPES pH 7.3 50mM, NaCl 50mM). At this point, determine the mobility of ACE2 by Fluorescence Recovery After Photobleaching (FRAP). Add the spike in the Clustering Buffer at a final concentration of 14 nM in the presence or absence of HS. ACE2 clustering was imaged at the indicated time point by a Nikon CSU-W1 SoRa microscope equipped with a temperature control enclosure.
Imaging processing and statistical data analysis
Fluorescence confocal images were acquired by a Nikon CSU-W1 SoRa microscope equipped with a temperature control enclosure and a CO2 control. 3D or 4D image reconstructions and analyses were done by Imaris software (Licensed to NIH). Fluorescence intensity was analyzed by open-source Fiji software. To this end, images were converted to individual channels, and regions of interest were drawn for measurement. Statistical analyses were performed using either Excel or GraphPad Prism 9.0. P values were calculated by Student’s t-test using Excel or one-way ANOVA by GraphPad Prism 9.0. Linear curve fitting, nonlinear curve fitting, and IC50 calculation were done with GraphPad Prism 9.0. For nonlinear fitting, the inhibitor vs. response -variable slope model or the exponential decay model was used. Images were prepared by Photoshop and Illustrator (Adobe). Data processing and reporting are adherent to the community standards.