Materials
Vero E6 (Cercopithecus aethiops; kidney epithelial; ATCC: CRL-1586) and A549 cells with ACE2 overexpression (A549ACE2+)32 were used in the study. For all cultures Dulbecco’s MEM (ThermoFisher Scientific, Poland) supplemented with 3% foetal bovine serum (heat-inactivated; ThermoFisher Scientific, Poland) and antibiotics: penicillin (100 U/ml), streptomycin (100 μg/mL), and ciprofloxacin (5 μg/mL) were used. Commercially available MucilAir HAE cultures were used for the ex vivo analysis (Epithelix Sarl, Switzerland). All cultures were carried out at 37 °C under 5% CO2.
SARS-CoV2 was isolate 026 V-03883 (Charité—Universitätsmedizin Berlin, Germany, European Virus Archive—Global—EVAG, https://www.european-virus-archive.com) for the in vitro work. SARS-CoV-2 was isolate Munchen-1.2 2020/98435 for the in vivo work. All experiments with the infectious agents were carried out in the ABSL3 + facility approved for work with the airborne BSL3 pathogens, including the SARS-CoV-2 virus.
The XTT cell viability kit (Biological Industries, Israel) was used for the cell viability assays. The following reagents were also used: viral DNA/RNA isolation kit (A&A Biotechnology, Poland), High-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Poland), Real-time qPCR kit (RT-HS-PCR mix probe, A&A Biotechnology, Poland) and the real-time qPCR oligonucleotides are listed in Table 1.
The GPCQ compounds are listed in Table 2. The compounds were suspended in 1 × PBS to the final concentration of 5 mg/mL. All stocks were stored at 4 °C until use.
Methods
Tissue culture
The cytotoxicity of compounds was assessed by incubating confluent monolayers of Vero E6 and A549ACE2+ cells with a range of GCPQ compound concentrations. The XTT assay was carried out 48 h later, according to the manufacturer’s protocol using 200,000 cells per well and DMEM supplemented with foetal bovine serum, penicillin and streptomycin (please see above under materials).
The ability of each compound to inhibit the virus replication was determined by infecting confluent Vero E6 and A549ACE2+ monolayers with the SARS-CoV-2 virus at 400 TCID50/mL in the presence of test compounds or phosphate buffered saline—PBS (TCID50 = 50% Tissue Culture Infectious Dose). Mock controls (cell lysate without the virus) and medium (supplemented DMEM—please see above) controls were included. Each compound was present during and after the infection. The cells were then incubated for 2 h at 37 °C and 5% CO2. Afterward, the cells were washed three times with PBS, and each compound was re-applied onto the cell monolayer. 100μL of the cell culture supernatants were subsequently collected from each designated well after two days of culture. The experiments were carried out in triplicate.
Virus replication inhibition in HAE was evaluated by infecting MucilAir™ (Epithelics Sarl, Switzerland) with SARS-CoV-2 virus at 5000 TCID50/mL in the presence of GCPQa or PBS. GCPQa diluted in PBS was added to the apical side of the insert (200 μg/ml or 500 μg/ml) and incubated at 37 °C for 30 min before the infection. After the pre-incubation was completed, the compound was removed and fresh dilutions of the compound with the virus were added and incubated for 2 h at 37 °C. Next, the apical side of the HAE was washed thrice with PBS and each compound was re-applied and incubated again for 30 min at 37 °C. After the last incubation with the GCPQ, the samples (50 μL) were collected and the HAE cultures were left in an air–liquid interphase. Every 24 h the HAE apical surface was incubated for 30 min with the GCPQ or PBS and the samples were collected for virus yield evaluation. Viral RNA was isolated from the apical washings or cell culture supernatant, RNA was isolated (Viral DNA/RNA; A&A Biotechnology, Poland), reverse-transcribed into cDNA (High Capacity cDNA Reverse Transcription Kit; ThermoScientific, Poland), and subjected to the qPCR analysis. Briefly, cDNA was amplified in a reaction mixture containing 1 × qPCR Master Mix (A&A Biotechnology, Poland), in the presence of probe (100 nM) and primers (450 nM each), sequences provided in Table 1.
The reaction was carried out using the 7500 Fast Real-Time PCR machine (Life Technologies, Poland) according to the scheme: 2 min at 50 °C and 10 min at 92 °C, followed by 40 cycles of 15 s at 92 °C and 1 min at 60 °C. In order to assess the copy number for N gene, DNA standards were prepared, as described before36. The obtained data is presented as virus yield and as the log removal value (LRV), showing the relative decrease in the amount of virus in cell culture media compared to the control.
Intranasal delivery in a healthy animal model
GCPQ (molecular weight = 10 kDa, mole% palmitoyl groups = 16 and mole% quaternary ammonium groups = 13) was radiolabelled using a two stage strategy: first an acylating reagent [N-succinimidyl-3[4-hydroxyphenyl]propionate—the Bolton and Hunter reagent (BH)] was initially covalently coupled to GCPQ and then the GCPQ-BH complex was iodinated with 125I. Briefly, GCPQ (90 mg) was dissolved in DMSO (3 mL). To this solution was added 200 µL of triethylamine and 0.05 molar equivalents (10 mg) of BH reagent and the reaction allowed to proceed overnight at room temperature with stirring. The next day, the GCPQ-BH conjugate was precipitated using an acetone: diethyl ether mixture (1:2, v/v) and the pellet was washed 3 times with the same acetone: diethyl ether mixture. The washed pellet was dissolved in methanol (2 mL) and dialyzed against water overnight. The dialysed GCPQ-BH was then freeze dried and collected. Labelling of GCPQ-BH with 125I was performed using iodination beads ® (Thermo Scientific Pierce, UK). Briefly, GCPQ-BH (20 mg) and 100 mg GCPQ were dissolved in methanol with stirring then the methanol was removed under vacuum and Tris–HCL buffer (25 mM, pH 4.8, 1.8 mL) was added to the dry film to produce a final concentration of 66.7 mg/mL. This solution was then added to a tube containing the I125 (1 mCi, 17 Ci/mg, 0.392 nmol, Perkin-Elmer, USA) and four iodination beads® (Thermo Scientific Pierce, UK). The reaction was incubated for 1.5 h at room temperature, after which the reaction was terminated by separating the solution from the beads. PD Spin Trap G-25 Columns (GE Healthcare Life Sciences, UK), that are prepared by vortexing and discarding of the eluting storage buffer by centrifuging (2800 rpm for 1 min), were used in order to remove the free iodine (with the free iodine removed through the addition of 50 μL of the reaction per column and centrifuging at 2,800 rpm for 2 min). The eluent was placed in Amicon ultra centrifugal filters (3 kDa, Millipore, USA) with 200 μL H2O, and was subject to repeated washes (through centrifuging at 10,000 rpm for 10 min), until the washed out water produced negligible counts.
All animal experiments were performed under a UK Home Office licence (PPL 70/8224) and were approved by the local ethics committee—the UCL Animal Welfare and Ethical Review Body. The animal experiments were carried out in accordance with the guidelines contained in the licence and ARRIVE guidelines were followed, however there was no blinding or randomisation carried out. An exploratory study on a Male Balb/C mouse weighing 25 g (Charles River, UK), allowed free access to standard rodent chow and water, was intranasally administered radiolabelled GCPQ-BH (10 mg/kg, 1.2 MBq) by using a pipette to place 5uL of the radiolabelled material into the mouse nares and allowing the mouse to sniff in the dose. At various time points after the administration of the radiolabelled GCPQ-BH, animals were anaesthetised using isofluorane (1–2%v/v in oxygen), maintained at 37 ºC and submitted for NanoSPECT/CT analysis (Mediso, USA).
In vivo SPECT/CT imaging and analysis
SPECT/CT scans of the mouse head at 30 min, 2 h 30 min and 24 h after nasal administration were acquired using a NanoSPECT/CT scanner (Mediso, Hungary). The mouse was anaesthetised using isoflurane (1–2%v/v in oxygen) and maintained at 37ºC. SPECT images were obtained over 30 min using a 4-head scanner with nine 1.4 mm pinhole apertures in helical scan mode with a time per view of 60 s. CT images were subsequently acquired using a 45 kilo volt peak (kVp) X-ray source, 500 ms exposure time in 180 projections, a pitch of 0.5 with an acquisition time of 4:30 min. Body temperature was maintained by a warm air blower and the respiration and core body temperature was monitored throughout. CT images were reconstructed using Bioscan InVivoScope (Bioscan, USA) software in voxel size 124 × 124 × 124 μm, whereas SPECT images were reconstructed using HiSPECT (ScivisGmbH, Bioscan) in a 256 × 256 matrix. Images were fused and analysed using VivoQuant (Invicro, A Konica Minolta Company) software. 3D Regions of Interest (ROIs) were created for the uptake within the nares for each time point and the activity calculated as the percentage of administered dose. Representative images are scaled the same (same min and max). After the final scan the mouse was sacrificed and the entire head of the mouse analysed using a curimeter (Capintech, Mirion Technologies, UK) for ex vivo validation of 125I concentration.
In vivo viral inhibition in transgenic mice expressing the ACE2 receptor
All animal experiments were approved by the local ethics committee. Transgenic mice expressing the human ACE2 protein under the human cytokeratin 18 promoter were purchased from the Jackson Laboratory, USA. Mice were quarantined for at least 7 days prior to the experiment. Each experimental group consisted of 10 animals (14 animals in the control group). GPCQa was administered once daily intranasally (20 mg/kg per day). The treatment control group received remdesivir intramuscularly (25 mg/kg per day). Mice had free, permanent access to the water during the experiment (from day − 1 to 6 post-infection).
Mice received GPCQ or remdesivir every 24 h from day − 1 until day 6 post-infection. No adverse effects were observed during the experiment. On day 0 the animals were infected intranasally with the SARS-CoV-2 virus (Munchen-1.2 2020/984; 5 μl to each nostril) at 426,000 TCID50/ml, which corresponded to ~ 3 × 105 pfu. The virus was propagated and titrated on Vero cells prior to infection. Infected mice were examined and weighed daily. On day 6 post-infection, animals were killed by an anesthetic overdose. Nasal swabs were taken and brains were collected. Tissues were homogenized using a bead homogenizer (TissueLyser II, Qiagen, Poland). Viral RNA was isolated using mirVana™ miRNA Isolation kit (ThermoFisher Scientific, Poland) according the manufacturer’s instructions. The viral infection was quantified using the RT-qPCR method described above.
Statistical analyses
Statistical analyses was carried out using one way ANOVA plus Tukey’s post tests. Statistical significance was set at a p < 0.05.
