In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2

FB2001 is a drug candidate that targets the main protease of SARS-CoV-2 via covalently binding to cysteine 145. In this study, we evaluated the inhibitory activities of FB2001 against several SARS-CoV-2 variants in vitro and in vivo (in mice), and we also evaluated the histopathological analysis and immunostaining of FB2001 on lung and brain which have been rarely reported. The results showed that FB2001 exhibited potent antiviral efficacy against several current SARS-CoV-2 variants in Vero E6 cells, namely, B.1.1.7 (Alpha): EC50 = 0.39 ± 0.01 μM, EC90 = 0.75 ± 0.01 μM; B.1.351 (Beta): EC50 = 0.28 ± 0.11 μM, EC90 = 0.57 ± 0.21 μM; B.1.617.2 (Delta): EC50 = 0.27 ± 0.05 μM, EC90 = 0.81 ± 0.20 μM; B.1.1.529 (Omicron): EC50 = 0.26 ± 0.06 μM and EC50 = 0.042 ± 0.007 μM (in the presence of a P-glycoprotein inhibitor). FB2001 remained potent against SARS-CoV-2 replication in the presence of high concentrations of human serum, which indicating that human serum had no significant effect on the in vitro inhibitory activity. Additionally, this inhibitor exhibited an additive effect against SARS-CoV-2 when combined with Remdesivir. Furthermore, FB2001 significantly reduced the SARS-CoV-2 copy numbers and titers in the lungs and brains in vivo, and alleviated the pathological symptoms. In addition, FB2001 could alleviated local bleeding, erythrocyte overflow, edema, and inflammatory cell infiltration in brain tissue, and inhibitors reducing viral titers and improving inflammation in the brain have been rarely reported. A physiologically based pharmacokinetic model was established and verified to predict the FB2001 concentration in human lungs. When FB2001 was administered at 200 mg twice a day for 5 days, the observed Ctrough ss in plasma and predicted Ctrough ss of lung total concentration were 0.163 and 2.5 μg/mL, which were approximately 9 and 132-fold higher than the EC50 of 0.019 μg/mL (0.042 μM) against Omicron variant. Taken together, our study suggests that FB2001 is a promising therapeutic agent in COVID-19 treatment and can be combined with remdesivir to achieve improved clinical outcomes. Owing to its good safety and tolerability in healthy human (NCT05197179 and NCT04766931), FB2001 has been approved for Phase II/III clinical trial (NCT05445934).


Introduction
The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and related COVID-19 (coronavirus disease 2019) have caused a large outbreak worldwide and become an increasingly serious concern to public health. The frequent emergence of SARS-CoV-2 variants with high infectivity, such as B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron), have reduced the effectiveness of the existing vaccines (Tregoning et al., 2021). In addition, some recent studies showed that SARS-CoV-2 not only causes lung inflammation, but also causes severe brain-related abnormalities, for example, a reduction in global brain size and grey matter thickness and olfactory dysfunction. However, the deleterious impacts in the brain remains to be further investigated (de Erausquin et al., 2021;Douaud et al., 2022).
Since the beginning of the pandemic, numerous studies and clinical trials have been conducted to evaluate the effects of anti-SARS-CoV-2 therapeutics. Currently, there are two main drug groups against SARS-CoV-2: virus-directed drugs targeting proteins of the virus, and hostdirected drugs targeting proteins or biological processes in the host that supports the virus (Simsek-Yavuz and Komsuoglu Celikyurt, 2021). Most of these drugs are based on repurposing existing antiviral agents, such as the nucleotide analogues Remdesivir and Galidesivir, protease inhibitors Lopinavir and Ritonavir, antimalarial drug Chloroquine, and host natural immunity booster interferon (Marei et al., 2021). However, the Solidarity clinical trial initiated by the World Health Organization has concluded that some of these treatments had little or no effect on mortality, initiation of ventilation, and duration of hospital stay in hospitalized patients. Although the disclosed data suggest that Molnupiravir (Jayk Bernal et al., 2022), Paxlovid (PF-07321332/ritonavir) (Mahase, 2021), and Ensitrelvir (S-217622) show some efficacy in clinical studies on nonhospitalized patients with mild-to-moderate COVID-19, the development of a novel specific effective treatment for COVID-19 and viral clearance of SARS-CoV-2 in the brain remains important.
Virus-encoded proteases play vital roles in the life cycle of coronaviruses. The main protease (M pro , also named 3CL protease) of SARS-CoV-2 is a cysteine protease that cleaves the viral polyproteins at more than 11 sites, releasing the vital proteins required for viral replication (Thiel et al., 2003). M pro is highly conserved among all coronaviruses and has no human homolog, representing an ideal antiviral target for SARS-CoV-2 and several variants (Yang et al., 2006). Some specific effective treatments that inhibit M pro have entered clinical trials for treating COVID-19, including PF-07321332, and S-217622 (Anirudhan et al., 2021;Boras et al., 2021;Owen et al., 2021;Unoh et al., 2022). Furthermore, potential M pro inhibitors, such as GC373, GC376, and compounds 11r and 13b, have also exhibited potent anti-SARS-CoV-2 efficacy in vitro, and the representative main protease inhibitors were shown in Scheme S1 (Hoffman et al., 2020;Vuong et al., 2020;Zhang et al., 2020).
Previously, we reported the drug candidate FB2001 (also named compound 11a) with the design, synthesis, and analysis of the crystal structure of the coronavirus M pro ; FB2001 exhibited potent inhibitory activity against SARS-CoV-2 in vitro (M pro : IC 50 = 0.053 ± 0.005 μM; EC 50 = 0.53 ± 0.01 μM) (Dai et al., 2020). In this study, we further evaluated the anti-SARS-CoV-2 activity of FB2001 in vitro and in vivo (in mice), and the histopathological analysis and immunostaining of FB2001 in the lung and brain of mice were also evaluated. Our results suggested that FB2001 could potently inhibit the replication of SARS-CoV-2 and several variants and reduce the copy numbers and virus titers in the lungs and brains of mice. FB2001 could potentially alleviate the pathological symptoms in the lungs and brains. Considering the seriously brain-related abnormalities caused by SARS-CoV-2, the ability of FB2001 to reduce viral load in the brain has important implications. In addition, according to the established physiologically based pharmacokinetic (PBPK) model, the trough concentration of FB2001 in the human lung was predicted to be more than 132-fold higher than the EC 50 value, and the observed C trough ss in plasma was 9-fold higher than the EC 50 . Therefore, FB2001 is a promising therapeutic agent for the treatment of SARS-CoV-2 infection and has an additive antiviral effect when combined with Remdesivir.
One day prior to testing, the cell culture medium was removed and 2 mL trypsin solution was added for 1-2 min of digestion at 37 • C after the cells were rinsed once with the extra cell fluid. Medium was added, and cells were counted prior to their transfer to a 48-well plate at 50,000 cells per well.

Compound preparation
FB2001 (Batch #: 20190714) was dissolved in DMSO to form 40 mM stock solution. On the day of testing, the stock solution was subject to 10-fold serial dilutions with DMEM, i.e., 1 μL of stock solution was added to 9 μL of DMEM. Ten-fold dilution was performed twice to obtain 0.4 mM solution. Further diluted to obtain 20 μM solution with DMEM, followed by 3-fold serial dilution to obtain the lowest concentrations.
Positive control PF-07321332 was dissolved in DMSO to form 40 mM stock solution. On the day of testing, the stock solution was subject to 10-fold serial dilutions with DMEM, i.e., 1 μL of stock solution was added to 9 μL of DMEM. Ten-fold dilution was performed thrice to obtain 0.04 mM solution. Further diluted to obtain 20 μM solution with DMEM, followed by 3-fold serial dilution to obtain the lowest concentrations.
The content of DMSO in the final testing concentration did not exceed 0.2%. This concentration of DMSO had no effect on the replication of SARS-CoV-2.

Effect of compound on viral replication
After supernatant was removed from the 48-well plate, the diluted compound was added to each well and incubated for 1 h. Cells were then inoculated with SARS-CoV-2 in biosafety level 3 (BSL-3) laboratory at multiplicity of infection (MOI) of 0.01 or 0.005, and incubated for 1 h. Thereafter, the supernatant was removed, cells were washed with PBS, and diluted compound FB2001 with or without P-gp inhibitor CP-100356 was added. At 24 h after infection, the supernatant was collected. The viral RNA in the supernatant was extracted and used for reverse transcription. Viral RNA copy number was then determined using real-time fluorescence quantitative PCR.

Effect of compound on cell viability
After the supernatant has been removed from the 96-well plate, the diluted compound was added to each well, and cell viability was determined using the CCK8 assay kit after cells were incubated for 24 h.

Data processing and statistics analysis
Data analysis and processing were performed using GraphPad Prism 6 and Microsoft Excel. The extent of inhibition of SARS-CoV-2 by compounds at different concentrations can be calculated with the following formula: Inhibition % = [1 -(I/Io)] × 100%Where, Inhibition % represents the percentage of inhibition of SARS-CoV-2 replication by the compound, I and Io represent the copy numbers of SARS-CoV-2 viral RNA in the compound and control (DMSO) wells, respectively. The EC 50 of the compound was calculated by fitting the following equation using GraphPad Prism 6: Y = Bottom + (Top-Bottom)/(1 + 10^(LogIC 50 -X) * HillSlope). Where, X is the Log value of the testing concentration of the sample, Y is the percentage of inhibition at the corresponding concentration, and Bottom and Top are the minimum and maximum percentage of inhibition, respectively. The content of DMSO in the final testing concentration did not exceed 0.2%. This concentration of DMSO had no effect on the replication of SARS-CoV-2.
One day prior to testing, the cell culture medium was removed and the cells were rinsed once with the extracellular fluid, before 2 mL of Trypsin solution was added for 1-2 min of digestion at 37 • C. Culture medium was added and cells were counted prior to their transfer to a 96well plate, at 20,000 cells per well.

Compound preparation
FB2001 (Batch #: 20190714) was dissolved in DMSO to form 40 mM stock solution. On the day of testing, the stock solution was subject to 10-fold serial dilutions with DMEM, i.e., 1 μL of stock solution was added to 9 μL of DMEM Ten-fold dilution was performed twice to obtain 0.4 mM solution. Eight-fold dilution was performed to obtain 50 μM solution, followed by DMEM gradient dilution to obtain concentrations of 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 μM. The final concentration solution of FB2001 was mixed with different concentrations of remdesivir (GS-5734).
Remdesivir was dissolved in DMSO to form 40 mM stock solution. On the day of testing, the stock solution was subject to 10-fold serial dilutions with DMEM, i.e., 1 μL of stock solution was added to 9 μL of DMEM ten-fold dilution was performed twice to obtain 0.4 mM solution.
Eight-fold dilution was performed to obtain 50 μM solution, followed by DMEM gradient dilution to obtain concentrations of 20, 10, 5, 2.5, 1.25 and 0.625 μM. The final concentration solution of remdesivir was mixed with different concentrations of FB2001.
The content of DMSO in the final testing concentration did not exceed 0.2%. This concentration of DMSO had no effect on the replication of SARS-CoV-2.

Effect of compound on viral replication by IFA
After supernatant was removed from the 96-well plate, the diluted compound (dissolved in DMEM containing different concentrations of human serum) was added to each well and incubated for 1 h. Cells were then inoculated with SARS-CoV-2 (2019-nCoV-WIV04) in biosafety level 3 (BSL-3) laboratory at multiplicity of infection (MOI) of 0.01. At 24 h after infection, the supernatant was removed, cells were fixed, and the 96-well plate was washed with PBS. After permeabilization and blocking, cells were incubated with primary and secondary antibodies, and NP content was determined using fluorescence imaging. The DMSO group was used as the control well to calculate the inhibitory rate of viral replication by the compound.

Data processing and statistical analysis
Data analysis and processing were performed using GraphPad Prism 6 and Microsoft Excel. The extent of inhibition of SARS-CoV-2 by compounds at different concentrations can be calculated with the following formula: Inhibition % = [1 -(I/Io)] × 100%Where, Inhibition % represents the percentage of inhibition of SARS-CoV-2 replication by the compound, I and Io represent the fluorescence intensity of SARS-CoV-2 NP protein in the compound and control (DMSO) wells, respectively.

Antiviral testing against SARS-CoV-2 in vivo
7-8 weeks K18-hACE2 mice were purchased from Jiangsu Gem-Pharmatech Biotechnology Co., Ltd. (Jiangsu, China). All the K18-hACE2 mice were cared following the recommendations of National Institutes of Health Guidelines for the Care and Use of Experimental Animals. Viral infections were performed in biosafety level 3 (BSL-3) facility. (Ethics number: WIVA25202202).

Preparation of FB2001 for injection
0.9% sterile normal saline was added to one vial of FB2001 for injection, then shaken to completely dissolve and filtered with 0.22 μm membrane to obtain FB2001 for injection solution.
The dosage in FB2001 for injection groups were 100 mpk and 200 mpk: According to the average body weight (23 g) of mice, the administration volume of each mouse was 0.2 mL. The concentration of FB2001 for injection were 11.5 mg/mL and 23 mg/mL, respectively. Animals were intraperitoneally injected twice daily.

Preparation of vehicle negative control
0.9% sterile normal saline were used as a negative control. Dosage: According to the average body weight (23 g) of mice, the administration volume of each mouse was 0.2 mL. Animals were intraperitoneally injected twice daily.

Virus challenge, drug treatment and sample collection
K18-hACE2 mice aged 7-8 weeks were transferred to BSL-3 laboratory in Wuhan Institute of Virology. Each mouse was infected with 1 × 10 3 PFU SARS-CoV-2 Delta variant by nasal drip, which was designated as Day 0. After 2 h of SARS-CoV-2 challenge, mice were intraperitoneally injected with FB2001 for injection or vehicle control. A total of 21 mice were divided into 3 groups: 7 in Vehicle group (BID, intraperitoneal injection), 7 in FB2001-100 mpk group (BID, intraperitoneal injection) and 7 in FB2001-200 mpk group (BID, intraperitoneal injection). Mice were administered with FB2001 for injection or vehicle control once daily on Day 0 and twice daily at 9-h intervals on Day 1, 2 and 3. The body weight of the mice were recorded once daily.
Four mice per group were sacrificed on Day 2, and 3 mice per group were sacrificed on Day 4. Lung and brain tissues were then removed from mice for detection of viral RNA. The right lung was ground with DMEM, part of the homogenate was taken for RNA extraction, and the rest was stored at − 80 • C; The left lung was fixed with paraformaldehyde for histopathological examination.

Detection of viral RNA
Viral RNA from the lung and brain tissues were extracted with the kit and reverse transcribed according to the operation instruction, then the absolute viral RNA copy in the tissue was detected quantitatively by real-time quantitative PCR. The viral RNA copy was calculated by standard plasmid concentration according to the kit instruction.

Histological analysis
For histologic examination, mouse lungs and brains were collected directly after euthanasia and placed in 4% paraformaldehyde for >5 days after which tissues were embedded in 3.5-mm paraffin. Fixed tissue samples were used for hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) for the detection of the SARS-CoV-2 antigen (SARS-CoV-2 Nucleocapsid Protein (HL344) Rabbit mAb #26369, CST). The image information was collected using a Pannoramic MIDI system (3DHISTECH, Budapest) and FV1200 confocal microscopy (Olympus). The slices were browsed under the microscope or used CaseViewer 2.4 to view the digital slices, observed the tissue structure in detail at different magnifications, described the typical pathological changes in the slices such as inflammation, necrosis, degeneration, hyperplasia, and fibrosis, and reflected the differences between each group of mice. Representative images were selected based on the prevalent trend for a given treatment group, showing images representative of the mean pathological score.

PBPK model establishment and validation method
The physical and chemical properties were actually measured or predicted according to FB2001 drug chemical structures, including acidbase dissociation equilibrium constant (pKa), oil-water partition coefficient (logP), log 10 of the distribution coefficient of an ionized form of a drug molecule (logD) at a particular pH, permeability and blood/plasma concentration ratio (Rbp) and fraction unbound in plasma (Fu), see Table S4. PBPK model in SD rat and Beagle dogs was established, validated and optimized according to the concentration of FB2001 in plasma and lung tissue concentration. The human PBPK model is established and verified according to the plasma drug concentration. After the predicted and measured values fit well, the drug concentration in human lung was predicted. The process of model building and lung concentration prediction is shown in Fig. S5A. The EC 50 (0.042 μM = 0.019 μg/mL) of FB2001 in Vero E6 will be used in the PBPK model simulation and estimation for effective human dose at different administration intervals. Gastroplus (version 9.8, simulations plus, USA) was used for PBPK modeling and simulation. R software (version 4.0.2) was used for drawing.

SD rat PBPK model and validation
The intravenous PBPK model of FB2001 in SD rats was established using the physical and chemical properties. Then the measured plasma concentration of FB2001 in SD rats after intravenous administration was used to optimize and validate the rat PBPK model. The optimized parameters included tissue partition coefficient (Kp) values of skin, muscle, adipose and other tissues, as shown in Figs. S5B and C. When the simulated plasma concentration of FB2001 in SD rats was well consistent with the measured data in the rat PBPK model, the lung tissue concentration of FB2001 in rats was predicted, optimized and validated according to the measured lung tissue drug concentration in SD rats. The key optimization parameters include permeability-surface area product for tissue (PStc), tissue partition coefficient (Kp), intracellular drug fraction unbound (Fu Int) and extracellular drug fraction unbound (Fu Ext), so that the predicted values of FB2001 in lung tissue were consistent with the measured values. Predicted and observed PK parameters of plasma drug concentration in rats were shown in Table S5, and simulated and observed drug concentration in plasma and lung total were shown in Fig. S6A.

Beagle dogs PBPK model and validation
The PBPK model of FB2001 after intravenous administration in beagle dogs was established using physical and chemical properties. Then the measured plasma concentration of FB2001 after intravenous administration was used to optimize and validate the beagle dogs PBPK model. The optimized parameters include Kp values of skin, muscle, adipose and other tissues. When the simulated plasma concentration of FB2001 in beagle dogs was well consistent with the measured data through intravenous administration in the beagle dog PBPK model, the lung tissue drug concentration in beagle dogs was predicted, and then optimized and validated according to the measured lung tissue concentration of FB2001 in beagle dogs. The key parameters optimized include PSTC, KP, Fu Int and Fu Ext. The adjustment ratio of parameters was the same as that in SD rats PBPK model. The predicted values were considered reasonable if the ratio of predicted to observed data was within a predefined 2-fold range (0.5 ≤ ratio ≤2.0). Predicted and observed PK parameters of plasma drug concentration in dogs were shown in Table S5, and simulated and observed drug concentration in plasma and lung total were shown in Fig. S6B.

Human PBPK model and validation
The human intravenous PBPK model of FB2001 was established using physical and chemical properties. The optimized parameters include KP values of skin, muscle, adipose and other tissues. When the simulated plasma concentration of FB2001 in human was well consistent with the measured data in the human PBPK model, the lung tissue concentration of FB2001 in human was predicted. The adjusted key parameters included PSTC, KP, Fu Int and Fu Ext, the adjustment ratio of the parameters was the same as that of the SD rat and beagle dogs PBPK model. Predicted lung total concentration of FB2001 in human were shown in Table 2.

FB2001 shows antiviral activity against several current SARS-CoV-2 variants in vitro
The antiviral effect of FB2001 and PF-07321332 against SARS-CoV-2 and several current variants were investigated in vitro using by measuring viral copy number, as shown in Table 1 and Fig. S1. FB2001 Table 1 In vitro activity of FB2001 against SARS-CoV-2 and variants. The time of addition assay also were conducted, FB2001 (2 μM, 10 μM, 1 μM + P-gp inhibitor) were incubated with the cells for different time according to the following conditions: entry (− 2 to 2 h), post entry (2-24 h) and full-time (− 2 to 24h). The FB2001 showed potent antiviral activities under all conditions, especially when administrated under two conditions: post entry and full-time (Fig. S2), and those results were in agreement with its antiviral mechanism as a main protease inhibitor. In addition, it also exhibited inhibitory effect on the process of virus entry.
Previously, crystal complexes showed that the aldehyde group of FB2001 formed covalent bond with Cys145 of SARS-CoV-2 M pro (PDB: 6LZE), and the binding modes was also proved to be reversible in this manuscript. Reversibility of M pro inhibition was demonstrated upon incubation of SARS-CoV-2 M pro (2 mM) with 2 μM of FB2001, reversible M pro inhibitor (PF-07321332) or an irreversible M pro inhibitor (N3) for 60 min and monitoring M pro activity after 50-fold and 200-fold dilution of the incubation mixtures. The results showed that the inhibitory activity was not recovered after M pro incubation with N3, and M pro activity were recovered after incubation with FB2001 and PF-07321332 which indicated that the binding model of FB2001 with M pro was reversible. In addition, compared with PF-07321332, FB2001 was more difficult to dissociate when combined with SARS-CoV-2 M pro (Fig. 1A).
We compared the amino acid sequence of M pro isolates from wildtype with that from four other variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron), using multiple sequence alignment (Fig. 1B). Two mutations, K90R and P132H, were found in the M pro of B.1.351 (Beta) and B.1.1.529 (Omicron), respectively, while the sequences of the M pro from the other three variants wild-type, B.1.1.7 (Alpha) and B.1.617.2 (Delta) were identical. The Xray crystal structure of the M pro with FB2001 shows that the K90R and P132H mutants were far from the ligand binding site (Fig. 1C), indicating that these mutants hardly affected the binding of FB2001 to the M pro . In other words, the K90R and P132H mutants would not affect the inhibitory activity of FB2001 against the M pro of B.1.351 (Beta) and B.1.1.529 (Omicron). Those results accord with the antiviral activity data presented in Table 1.

FB2001 inhibits SARS-CoV-2 replication in vero E6 cells in the presence of different concentrations of human serum
Because the high plasma protein binding rate of these compounds as well as binding to albumin and/or α1-acid glycoprotein in human serum may attenuate the potency of protease inhibitors (Gulati et al., 2009;Islam et al., 2016), we tested FB2001 for anti-SARS-CoV-2 (2019-nCoV-WIV04) activity in the presence of different concentrations of human serum in Vero E6 cells (Table S1). The results showed that although the EC 50 values of FB2001 against SARS-CoV-2 tended to increase with the increasing concentrations of human serum (1.10 μM, 1.91 μM, 2.57 μM, 2.15 μM, and 2.40 μM in 10%, 20%, 30%, 40%, and 50% of human serum, respectively), FB2001 remained potent in inhibiting SARS-CoV-2 replication in the presence of high concentrations of human serum. At 20-50% human serum present, the inhibitory activity of FB2001 against SARS-CoV-2 did not change significantly, and the EC 50 value of FB2001 was about 2 times higher of that at 10% human serum, indicating that human serum had no significant effect on the in vitro inhibitory activity of FB2001.

Combined treatment with remdesivir and FB2001 exhibits an additive effect against SARS-CoV-2 in vitro
Remdesivir is an adenosine analogue that has shown inhibitory effects on the replication of multiple coronaviruses, including human coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, by incorporating into nascent viral RNA (Brown et al., 2019;de Wit et al., 2020;Sheahan et al., 2017). Recent studies have reported that remdesivir inhibits SARS-CoV-2 replication in vitro with an EC 50 of 0.77-26.9 μM (Choy et al., 2020;Kotaki et al., 2021;Pizzorno et al., 2020;Wang et al., 2020), depending on the virus strain, assay type, and calculation method. As remdesivir is the first approved treatment for COVID-19 in the United States and Europe (Lamb, 2020), and the targets of FB2001 and remdesivir are different, the synergistic effect of combined treatment with FB2001 and remdesivir against SARS-CoV-2 was investigated. Schloer et al. (Pizzorno et al., 2020;Schloer et al., 2021) have reported that the combination of remdesivir with the repurposed antifungal drug itraconazole or the antidepressant fluoxetine achieved a 90% reduction in infectious SARS-CoV-2 particles in Calu-3 cells with lower doses of the individual drugs. Furthermore, Kalil et al. (2021) reported that cotreatment with remdesivir and the anti-inflammatory drug baricitinib was safe and outperformed remdesivir alone in improving clinical status and reducing the time to recovery of hospitalized patients with COVID-19. These findings suggest that combining two antiviral compounds with different viral or cellular targets may achieve enhanced therapeutic effects with lower doses of the individual drugs compared with monotherapy. Thus, the synergistic effect of FB2001 and remdesivir was explored. The inhibitory activity of the combination of FB2001 and remdesivir at different concentrations against SARS-CoV-2 was determined in vitro, and the detailed study results are shown in Table S2 and Fig. 2. The inhibition rate of the combination of FB2001 and remdesivir was determined using the Synergyfinder package (Ianevski et al., 2020). The Zero interaction potency (ZIP) model was used to calculate the synergy score of FB2001 combined with remdesivir, which was 6.947 (Fig. 2). It was found that the inhibitory activity against SARS-CoV-2 increased after administration of the combination of both compounds, and the combined effect was mainly an additive effect rather than a synergistic effect.

In vivo inhibitory activity of FB2001 in K18-hACE2 mice model of SARS-CoV-2 delta variant infection
Next, we investigated the efficacy of FB2001 in K18-hACE2 mice challenged with SARS-CoV-2 (Delta variant). After 2 h of challenge, the mice were injected intraperitoneally, once daily on day 0 and twice daily on day 1, 2 and 3, with FB2001 or vehicle control for 4 consecutive days (Fig. 3A). Mice were euthanized and dissected on day 2 and 4. Lung and brain tissues were removed for the detection of viral RNA and viral titer, and histopathological analysis and immunostaining of lungs and brains were also evaluated.
The weights of mice on day 0 were used as control. There is no significant change in the body weight of the mice in control group and treatment groups (FB2001-100 mpk (mg/kg) group and FB2001-200 mpk group). It was also discovered that the virus titers of the FB2001 groups in the lung presented a dose-dependent efficacy, and the virus titers of the FB2001-200 mpk group showed a 3.57 log 10 PFU/g reduction on day 2, which was then decreased to below the detection limit at day 4 ( Fig. 3B and C). And the viral titers of the FB2001-200 mpk group were reduced to below the detection limit on day 4 in the brain (Fig. 3D). Compared with the vehicle group, the lung viral loads in the FB2001-100 mpk group and FB2001-200 mpk group were reduced by 1.02 and 1.60 log 10 copies/g on day 2, respectively, and 0.67 and 1.14 log 10 copies/g on day 4, respectively (Figs. S3A and S3B). In addition, compared with the vehicle group, the brain viral loads were significantly reduced by 2.37 and 5.26 log 10 copies/g in the FB2001-100 mpk group and FB2001-200 mpk group on day 4, respectively ( Fig. S3C, S3D). The detailed data are summarized in Table S3. The antiviral activity of FB2001 against the SARS-CoV-2 Delta variant was dose-dependent, and both 100 mpk FB2001 and 200 mpk FB2001 could significantly reduce viral load in mouse lung and brain. Considering the serious brain-related abnormalities caused by SARS-CoV-2, that FB2001 could reduce viral load in the brain has important implications.

Histopathology and immunohistochemical results of FB2001 in K18-hACE2 mice model of SARS-CoV-2 delta variant infection
Representative images from hematoxylin and eosin stains of tissues  of infected K18-hACE2 transgenic mice showed that FB2001 treatment significantly improved the histopathology in both the lung and brain compared with that of the control group. The lungs of the mice in the control group showed moderate incremental lymphoplasmacytic, histiocytic perivascular infiltrates and alveolar septal thickening observed at day 2 and day 4 (black arrows, Fig. 4B and H). In contrast, the lungs in the FB2001-100 mpk group showed mild interstitial inflammatory cell infiltration (red arrows, Fig. 4D and J), and decreased inflammation and normal alveolar spaces were observed in lungs of the FB2001-200 mpk group (Fig. 4F, L). Immunohistochemistry staining targeting viral NP demonstrated that FB2001 suppressed viral titer in mice lungs in a dosedependent manner (Fig. S4A-L), and the virus was almost cleared at day 4 in both the FB2001-100 mpk and FB2001-200 mpk groups. Histopathological analysis and immunostaining of brains from the SARS-CoV-2 Delta variant-infected K18-hACE2 transgenic mice showed that FB2001 limited cellular infiltration (Fig. 4M-R) and protected brain tissue from damage caused by virus replication at day 4 ( Fig. S4M-R). Neuronal cells were neatly arranged and tightly arranged, some neurons were degenerated, the cell body was solidified and deeply stained, and the nissl body disappeared in the control group (black arrow, Fig. 4N). The arrangement of neuronal cells in the cerebral cortex was more disordered, some neurons were denatured, and the staining was deepened in the FB2001-100 mpk group (black arrow, Fig. 4P). Neuronal cells were neatly arranged and abundant, and the nissl body was normal in the FB2001-200 mpk group (Fig. 4R). Immunohistochemistry staining targeting viral NP demonstrated that FB2001 suppressed viral titer in mice brains in a dose-dependent manner, and the virus was nearly cleared at day 4 in both the FB2001-100 mpk and FB2001-200 mpk groups (Fig. S4).

Projected effective dose
SARS-CoV-2 is rarely detected in blood, and the main viral target organs are the respiratory tract and lungs. We established a PBPK model to predict total lung drug concentration (Figs. S5 and S6 and Table S4). The model was validated using plasma and lung FB2001 concentration in rat and dogs (Table S5). For 200 mg BID FB2001 administered for 5 consecutive days in human, the plasma trough concentration (C trough ss ) of FB2001 was observed to be 0.163 μg/mL, and the total lung trough concentration (C trough, SS ) of FB2001 was predicted to be 2.5 μg/mL (Table 2). In a Vero E6 cell-based in vitro antiviral assay, in the presence of the P-gp efflux inhibitor, the EC 50 of FB2001 against SARS-CoV-2 Omicron (B1.1.529) variant was 0.019 μg/mL. Thus, the above C trough ss of FB2001 in plasma and lung were 9 and 132-fold higher than the EC 50 values, respectively. The molecule also showed similar antiviral activity against the SARS-CoV-2 Delta variant.
Based on this analysis, dosage of 200 mg FB2001 BID may offer a highest trough lung concentration, which could become an effective dosage in future clinical studies.

Discussion
In the past two decades, three coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) have seriously affected human life and social stability. The COVID-19 pandemic, especially, has caused the death of millions of lives worldwide. Furthermore, multiple SARS-CoV-2 variants are emerging and reducing the effectiveness of vaccines. SARS-CoV-2 not only causes pneumonia, but recent studies have shown that it can also cause brain-related abnormalities, for example, the reduction in global brain size and grey matter thickness; thus, the development of effective drugs remains of paramount importance. In this study, we found FB2001 showed potent inhibitory activity against SARS-CoV-2 and its variants. FB2001 used in combination with remdesivir exhibited an additive effect against SARS-CoV-2 in vitro. In addition, FB2001 decreased the viral loads and viral titers of the lung and brain in a SARS-CoV-2 Delta variant model, and FB2001 treatment significantly improved the histopathology in both the lung and brain. Notably, FB2001 significantly reduced viral titers and alleviated brain damage in the brain of mice, which were rarely reported previously. SARS-CoV-2 is mainly detected in the respiratory tract and lungs, hence, a PBPK model was established to predict total lung drug concentration. For 200 mg BID FB2001 administered for 5 consecutive days, the total lung C trough ss of FB2001 was predicted to be 132-fold higher than the EC 50 values, and the observed plasma C trough ss of FB2001 was observed to be 9-fold higher than the EC 50 values. Based on the PBPK results, 200 mg FB2001 BID could become an effective dosage in future clinical studies.
In summary, FB2001 exhibited broad-spectrum activity against several coronaviruses, and also showed additive antiviral effect when combined with remdesivir. In addition, FB2001 could significantly reduce the viral loads and titers in the lungs and brain of mice infected with SARS-CoV-2. Moreover, FB2001 has been approved for Phase II/III clinical trials, and presents a potentially good treatment for COVID-19.

Funding
This work was supported by grants from National Natural Science

Declaration of competing interest
The author(s) declared no potential conflicts of interest with respect to the research.

Data availability
Data will be made available on request.