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

Abstract

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 C_{trough ss} in plasma and predicted C_{trough 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).

Keywords: Anti-SARS-CoV-2 in vivo; FB2001; Main protease; PBPK; SARS-CoV-2; SARS-CoV-2 variants.

Graphical abstract

Fig. 1

Comparing the M^proof SARS-CoV-2 with four SARS-CoV-2 variants of concern (VOCs). (A) FB2001 is a reversible inhibitor of SARS-CoV-2 M^pro as demonstrated by recovery of enzymatic activity. (B) Sequence alignment of M^pro in SARS-CoV-2 and several variants, wild-type, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). (C) An X-ray crystal structure of SARS-CoV-2 M^pro in complex with FB2001 (PDB code: 6LZE). Compound FB2001 is shown as pink sticks. K90, P132 and FB2001 are indicated by red spheres, pink spheres and pink sticks, respectively.

Fig. 2

Synergistic analysis of FB2001 combined with remdesivir. Where −10 < synergy score <10 indicates an additive effect, synergy score < −10 indicates an antagonistic effect, and synergy score >10 indicates a synergistic effect.

Fig. 3

In vivo efficacy of FB2001 in K18-hACE2 mice infected with SARS-CoV-2 delta variant. The transgenic K18-hACE2 mice were divided into 3 groups: Vehicle control group, FB2001-100 mg/kg (100 mpk) group and FB2001-200 mg/kg (200 mpk) group. 7 mice per group. Transgenic mice in treatment group were challenged with SARS-CoV-2 delta variant by nasal drip. (A) Experiment flow diagram. (B, C) Viral titer in lung on Day 2 and 4 after SARS-CoV-2 challenge (Log₁₀ PFU/g). Dotted line represents the limit of detection of virus titration. (D) Viral titer in brain on Day 4 after SARS-CoV-2 challenge (Log₁₀ PFU/g). Dotted line represents the limit of detection of virus titration.

Fig. 4

FB2001 protects lung and brain tissue from damage caused by SARS-CoV-2 delta variant. (A-L) Representative H&E images of lung histopathology from vehicle-and FB2001-treated K18-hACE2 mice at 2 dpi and 4 dpi. Vehicle group (A-B: day 2 and G-H: day 4); FB2001 group treated with 100 mpk drug (C-D: day 2 and I-J: day 4); FB2001 group treated with 200 mpk drug (E-F: day 2 and K-L: day 4). (M-R) Representative H&E images of brain histopathology from vehicle-and FB2001-treated K18-hACE2 mice at day 4. Vehicle group (M-N); FB2001 group treated with 100 mpk drug (O-P); FB2001 group treated with 200 mpk drug (Q-R). Scale bar: A, C, E, G, I, K, M, O, Q = 1000 μm; B, D, F, H, J, L, N, P, R = 50 μm.