Anti‐Coronaviral Nanocluster Restrain Infections of SARS‐CoV‐2 and Associated Mutants through Virucidal Inhibition and 3CL Protease Inactivation

Abstract Antivirals that can combat coronaviruses, including SARS‐CoV‐2 and associated mutants, are urgently needed but lacking. Simultaneously targeting the viral physical structure and replication cycle can endow antivirals with sustainable and broad‐spectrum anti‐coronavirus efficacy, which is difficult to achieve using a single small‐molecule antiviral. Thus, a library of nanomaterials on GX_P2V, a SARS‐CoV‐2‐like coronavirus of pangolin origin, is screened and a surface‐functionalized gold nanocluster (TMA‐GNC) is identified as the top hit. TMA‐GNC inhibits transcription‐ and replication‐competent SARS‐CoV‐2 virus‐like particles and all tested pseudoviruses of SARS‐CoV‐2 variants. TMA‐GNC prevents viral dissemination through destroying membrane integrity physically to enable a virucidal effect, interfering with viral replication by inactivating 3CL protease and priming the innate immune system against coronavirus infection. TMA‐GNC exhibits biocompatibility and significantly reduces viral titers, inflammation, and pathological injury in lungs and tracheas of GX_P2V‐infected hamsters. TMA‐GNC may have a role in controlling the COVID‐19 pandemic and inhibiting future emerging coronaviruses or variants.

TMA-GNC was synthesised through a hydrothermal reduction approach. Briefly, the aqueous solution of TMA (25 mM, 2 mL) and GSH (100 mM, 0.5 mL) were mixed into a roundbottom flask that contained 6.5 mL Milli-Q water. The flask was then placed into an oil bath (70 °C) under vigorous stirring (600 rpm). Then, a 1 mL aqueous solution of auric acid (25 mM, 1mL) was added to the mixture to start the reaction, and the speed of stirring was increased to 1000 rpm. After reaction for 24 h, the GNC was purified by dialysing (molecular weight cut-off: 3500 Da) against Milli-Q water to remove excess TMA and GSH. The obtained GNC was condensed by ultrafiltration (Pall corporation, cat no. FG8207) and stored at 4 °C for further study.
The hydrodynamic diameter and zeta potential of TMA-GNC were measured by the Nano-ZS Zetasizer (Malvern Instruments). The high angle annular dark-field STEM (HAADF-STEM) images and elemental analysis of TMA-GNC were conducted with the Themis G2 (FEI) at the accelerating voltage of 300 kV. The ratio of TMA and GSH on the surface of GNC was determined by 1 H NMR analysis after etching the gold core with iodine. X-ray photoelectron spectroscopy (XPS) analysis was conducted on the ESCALAB 250Xi instrument (Thermo Scientific) with an excitation source of Al Kα X-ray radiation (1486.6 eV). The concentration of gold element was determined by the inductively coupled plasma mass spectrometry ICP-MS (7700X, Agilent).

Viral infection assay in vitro and determination of EC 50
To test the anti-GX_P2V ability of TMA-GNC, Vero E6 cells were inoculated into 96-well plate (4×10 4 cells/well) and incubated overnight. GX_P2V at the MOI of 0.01 was mixed with TMA-GNC in gradient dilutions (final concentrations ranged from 0.0078 μM to 2 μM) and were then added to the cells in duplicate. After 2 h incubation, the inoculum was removed and replaced by fresh media with the same concentration of TMA-GNC contained. At 48 h postinfection in the presence of TMA-GNC, cytopathic effects were observed by microscope.
Cells were collected for the RT-qPCR analysis, and the EC 50 was determined according to the RT-qPCR results. Antiviral assays of Nirmatrelvir (PF-07321332), Remdesivir (GS-5734), and Aluvia were performed as control. In the SARS-CoV-2 trVLP assay, SARS-CoV-2 trVLP (MOI of 0.01) and TMA-GNC (final concentrations ranged from 0.0078 μM to 2 μM) were mixed and directly added into the Caco-2-N cells without incubation. The following procedures remained the same to that of GX-P2V assays.
To measure the viral gene copies number, the PCR product was inserted into a vector to synthesize standard plasmid. After determining the copy number, the plasmid was serially diluted (10 -3 ~ 10 -9 ) for RT-qPCR analysis. The standard curve was generated according to the copy numbers and cycle threshold values.
TMA-GNC with different dilutions (0.0078-2 μM) were introduced to the cells to incubate for 48 h. After incubation, each well was treated with 20 μL of resazurin (Promega) for 2 h and the absorbance at 570 nm was measured using microplate reader. The cytotoxity was calculated by: Inhibition (%) = 1 -(OD TMA-GNC /OD control ) × 100 %. The calculation of CC 50 was based on the results of inhibition.

Pseudovirus Inhibition Assay
SARS-CoV-2 pseudoviruses were generated using the Vesicular Stomatitis Virus (VSV) pseudovirus packaging system 50 . Plasmids expressing various S protein variants of SARS-CoV-2 were synthesized by RuiBiotech (Beijing, China). Briefly, TMA-GNC was mixed with pseudoviruses of SARS-CoV-2 wild type or mutant strains for a co-incubation in the cell incubator. The concentration of TMA-GNC was 3 μM and 6 μM. After 90 min incubation, resuspended BHK21-ACE2 cells (5×10 4 cells/well) were added into the each well of the TMA-GNC/pseudoviruses mixtures. Cells were collected at 24 h post-incubation, washed with PBS, and fully lysed. The activity of firefly luciferase was measured using the firefly luciferase reporter gene assay kit (Yeasen, cat no. 11401ES76) for the determination of inhibition rate of TMA-GNC against the pseudoviruses. The relative light unit (RLU) was recorded by a microplate reader (Bio-Rad, USA).

Plaque assay
To perform the plaque assay, Vero E6 cells were pre-seeded in a 6-well plate. The supernatant of infected cells with 10-fold dilution (10 -1 ~ 10 -6 ) was added to the plate (1 mL per well) and incubated for 1 h. After the free viruses were discarded, the cells were overlaid with medium containing 1% agarose to prevent cross infection. At 3 days, cells were fixed with 4% paraformaldehyde for 2 h and stained with crystal violet for 10 minutes. The plaques were count after the crystal violet was washed with water. The titer was calculated as follow: Titer (PFU/mL) = the number of plaques × dilution multiple.

Western blotting
Western blotting was performed to detect the production of GX_P2V nucleocapsid protein.
After protein quantification, 20 μL of the samples with the same protein concentration were loaded on a 12% SDS-PAGE gel for electrophoresis (80 V for 30 min and 120 V for 60 min).
After electrophoresis, proteins were transferred to a polyvinylidene (PVDF) fluoride membrane (15 V for 60 min). The PVDF membrane was blocked by 5% skim milk (TBST) for 1 h at room temperature. After washing out of the blocking reagent, the antibody against nucleocapsid protein of anti-SARS-CoV-2 N protein (Genscript, USA) and GAPDH of anti-GAPDH (Proteintech, USA) were used at 1:200 and 1:2000 dilutions, respectively. After 2 h incubation at room temperature, the antibody was removed thoroughly, and the second antibody of HRP-conjugated AffiniPure Goat anti-mouse IgG (H+L) diluted at 1:10000 was used for another 2 h incubation. Then SuperSignal® West Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Scientific, USA) was used for imaging.

Time-of-addition assay
Time-of-addition assay was performed to determine at which stage of life cycle TMA-GNC exerted its inhibition effect. Vero E6 cells were seeded into 48-well plate (1×10 5 cells/well) and incubated overnight. GX_P2V at the MOI of 0.01 and TMA-GNC at concentrations of 1.5 μM and 3.0 μM were mixed respectively. The mixtures were added to the cells for 2 h incubation to allow viral attachment and internalization. Then the viruses were removed, and the cells were washed with PBS. The cells were re-supplemented with fresh media and cultured for another 48 h. For post-entry experiment, the cells were first infected by GX_P2V at the MOI of 0.01 for 2 h. After the infection, viruses were removed, and the cells were wash with PBS. The cells were then cultured with the media containing TMA-GNC at 1.5 μM and 3.0 μM for 48 h. The cells were collected for RT-qPCR analysis to measure the intracellular viral production.

Viral attachment assay
Vero E6, Calu 3 and BGM cells were seeded into a 24-well plate (5 × 10 5 cells/well) and incubated overnight. 3 μM of TMA-GNC was incubated with GX_P2V (MOI=10) at 4 o C for 2 h to allow interaction, then cells were treated with the mixture for 2 h incubation at 4 o C.
After GX_P2V had been attached to cells, the unbind GX_P2V was removed, and cells were washed with PBS for 3 times and collected for RT-qPCR analysis.

In vitro FRET assay of SARS-CoV-2 3CL pro inhibition
The TMA-GNC was first diluted to successive concentrations, and each TMA-GNC solution was thoroughly mixed with SARS-CoV-2 3CL pro . The mixtures were incubated at 4 °C for 2 h.

Bio-layer interferometry (BLI) analysis
The BLI experiments were performed on the OctetRed384 (ForteBio) to determine the binding kinetics between SARS-CoV-2 3CL pro and TMA-GNC. Briefly, the alginate coated biosensors (AR2G, cat no. 18-5092) were first dipped into the aqueous solution of TMA-GNC (1.2 μM) to attach GNC onto the sensors through electrostatic interaction. The loaded TMA-GNC did not detach from the biosensors in buffers we used in this study except in the glycine solution (pH = 2.0, Cytiva, cat no. BR100355). The biosensors were then dipped into PBS solutions of SARS-CoV-2 3CL pro (62.5, 125, 250, 500, and 1000 nM) for association (100 sec) and dissociation (150 sec). The data were corrected with blank curves and fitted with Octet evaluation software using the 1:1 binding mode. To determine the binding affinities under different pH conditions, SARS-CoV-2 3CL pro was diluted with buffers with a pH value ranging from 7.4 to 4.5. To determine the binding sites of TMA-GNC towards SARS-CoV-2 3CL pro , the 3CL pro (1000 nM) got trypsinisation overnight at 37 °C before the BLI experiments. Enriched peptides were detached from the surface of TMA-GNC by using iodine to dissolve gold and further desalted through StageTip C18 (Thermo) before MS analysis. The sequence identification by MS was performed with the Orbitrap Fusion (Thermo Fisher Scientific). Proteome Discovery (Version 2.4) software (Thermo Fisher Scientific) was applied to search the raw data against SARS-CoV-2 3CL pro fasta files (downloaded on Dec 13, 2021). 1% of the false discovery rate was set for peptide identification.

Biodistribution and toxicity evaluation in vivo
To investigate the biodistribution and clearance of TMA-GNC post-injection, the GNC was administrated into BLAB/c mice at 10 mg/kg intraperitoneally. At designed time points (24, 48, and 72 h), mice were sacrificed, and major organs (heart, liver, spleen, lung, and kidney) were collected. Urine and faeces of the mice were collected using the metabolism cages. All biological samples were lysed with freshly prepared aqua regia overnight and followed by heating for efficient nitrolysis and complete removal of aqua regia. Samples were re-dissolved with 2% (v/v) HNO 3 and applied to the ICP-MS measurements for the qualitative determination of gold. Blood was sampled from the eyes of mice. The whole blood (~50 μL) was applied to routine tests, and the rest of the blood samples were centrifuged at 3000 rpm and 4 °C for 10 min to obtain the serum. Biochemical indicators in all serum samples were measured with an automatic biochemistry analyser MS480 (MedicalSystem, China). Tissues of major organs were also subjected to H&E staining for pathological analysis. To determine the potential toxicity of TMA-GNC at a high dosage, mice were treated with TMA-GNC with a concentration of 3.2, 16, 32, and 64 mg/kg (n = 10), and the survival rate was recorded.

Antiviral evaluation in the golden hamster model
Fifteen golden hamsters (7 females and 8 males, 8-9-week-old) were divided into four groups. Golden hamsters in the groups above were challenged by 2 × 10 5 pfu GX_P2V on 0 dpi with a nasal infection. The TMA-GNC treatment group consisted of two females and three males, which were intraperitoneally administered with TMA-GNC diluted in distilled water at a dose of 3.15 mg/kg on 0, 1, 2, 3 dpi. Remdesivir group, comprising two males and two females, was designed as a positive drug control. In this group, remdesivir was injected intraperitoneally at a dose of 25 mg/kg on 0, 1, 2, and 3 dpi. The dosage of remdesivir was referred to in previous research 12 . The negative control group was performed by intraperitoneal injection with the same amount of PBS per day. Animals in each group were sacrificed and anatomised at 4 dpi. The lung tissues were obtained by anatomy and photographed. Partial lung tissues were collected to quantify the viral yield by TCID 50 assay and RT-qPCR analysis. Lung tissues soaked in PBS were ground to harvest homogenate using a freeze grinder (JingN·9548R; Hoder, Beijing, China), then centrifuged at 12000 ×g for 3 min at 4 °C. For the TCID 50 assay, the supernatant was serially diluted (10 -1 -10 -3 ), and dilutions were inoculated to a 96-well plate pre-seeded with Vero E6 cells in duplicate. After 72 h incubation, the CPE of each well was observed to calculate TCID 50 . For RT-qPCR analysis, viral RNA was extracted from supernatant using Flying Shark ® Tissue& Cell RNA Kit (Cat No. RNE11,Nobelab Biotech,Beijing,China). The following treatments were as described above. Tissues of the lung and trachea were subjected to H&E staining for pathology analysis. Technology, Beijing, China). Sequences were corrected and then mapped using TopHat2 and bowtie2. Each gene was counted using HTSeq-count. Pairwise comparisons were performed, and the differentially expressed genes were analysed using an R packaged cluster profiler.

Statistical analysis
Statistical analyses were conducted using GraphPad Prism 8. Unpaired t tests were used to evaluate the changes in viral titres and RNA content after drug treatments. P values ≤ 0.05 was regarded as significant for the analysis and marked with asterisks: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. For the determination of EC 50 , CC 50 and IC 50 value, nonlinear fit analysis was performed.