A new generation Mpro inhibitor with potent activity against SARS-CoV-2 Omicron variants

Abstract

Emerging SARS-CoV-2 variants, particularly the Omicron variant and its sublineages, continually threaten the global public health. Small molecule antivirals are an effective treatment strategy to fight against the virus. However, the first-generation antivirals either show limited clinical efficacy and/or have some defects in pharmacokinetic (PK) properties. Moreover, with increased use of these drugs across the globe, they face great pressure of drug resistance. We herein present the discovery and characterization of a new generation antiviral drug candidate (SY110), which is a potent and selective inhibitor of SARS-CoV-2 main protease (M^pro). This compound displayed potent in vitro antiviral activity against not only the predominant SARS-CoV-2 Omicron sublineage BA.5, but also other highly pathogenic human coronaviruses including SARS-CoV-1 and MERS-CoV. In the Omicron-infected K18-hACE2 mouse model, oral treatment with SY110 significantly lowered the viral burdens in lung and alleviated the virus-induced pathology. Importantly, SY110 possesses favorable PK properties with high oral drug exposure and oral bioavailability, and also an outstanding safety profile. Furthermore, SY110 exhibited sensitivity to several drug-resistance M^pro mutations. Collectively, this investigation provides a promising new drug candidate against Omicron and other variants of SARS-CoV-2.

Fig. 1

Discovery of a hit compound against SARS-CoV-2 M^pro. a The chemical structure of Hit-1. The P1′, P1, and P2 moieties of Hit-1 are labeled. The warhead carbon is marked with a black asterisk. b Dose-activity curve of Hit-1 against SARS-CoV-2 M^pro in the FRET assay. Data shown are the mean ± standard deviation (SD) from three independent experiments. c Differential scanning fluorimetry analysis of the effect of Hit-1 on SARS-CoV-2 M^pro stability. Exposure of hydrophobic residues monitored by an increase in relative fluorescence units (RFUs). Curves represent the average of three experiments. d Hit-1 (orange) is located at the substrate-binding pocket of M^pro (cyan). His41 of M^pro is in blue, Cys145 is yellow. Pockets (S1′, S1, S2, and S4) of M^pro and moieties (P1′, P1 and P2) of Hit-1 are both labeled. e Interactions between Hit-1 and M^pro. Fo – Fc density map is shown for Hit-1 (gray mesh, σ = 2.5) and Cys145. Covalent bond is shown by a black arrow and hydrogen bonds are displayed by red dashed lines. f Regions of Hit-1 for structural optimization. Images in d, e were processed by using PyMOL (https://pymol.org)

Fig. 2

The stepwise structural optimization process of Hit-1. Bioactivities were measured by the FRET assay and are represented as the mean ± SD from three experiments. Molecules with IC₅₀ < 30 nM are shown in red color

Fig. 3

In vitro cellular antiviral activity of SY110. a Cells were infected with Omicron BA.1 sublineage at 0.1 MOI and treated with 3r, 3 u, 3t, 3 v, 3w (SY110), 3x, Nirmatrelvir or DMSO at 1 hpi.. At 24 hpi., cell lysates were harvested. Viral genome copies were quantified by probe-based RT-qPCR (n = 4). b Cells were infected and treated with 3w (SY110) or Nirmatrelvir at 2 hpi. (n = 3). Cells were fixed at 72 hpi. for plaque formation units quantification by visualization with 0.5% crystal violet staining. Plaque formation units were normalized to that of the vehicle group. Statistical significance is evaluated by one-way ANOVA in a compared to vehicle group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, not statistically significant

Fig. 4

Crystal structure of SARS-CoV-2 Omicron variant M^pro in complex with SY110. a The chemical structure of SY110. The warhead carbon is marked with a black asterisk. b Close-up view of SY110 with the substrate-binding pocket of M^pro. SY110 is in orange, M^pro is aquamarine, His41 of M^pro blue, Cys145 yellow. Four sites S1′, S1, S2, and S4 of M^pro, and four moieties P1′, P1, P2, and P3 of SY110 are labeled, respectively. Fo – Fc density map of SY110 is shown (gray mesh, σ = 2.5). c Interactions between M^pro and SY110. The residues of M^pro involved in SY110 binding are displayed by sticks. The hydrogen bonds are displayed as red dashed lines. d Comparison of the binding modes of SY110 (orange, PDB ID: 8HHU), 11a (blue, PDB ID: 6LZE), MI-23 (wheat, PDB ID: 7D3I) and Nirmatrelvir (green, PDB ID: 7RFW). e Comparison of the binding modes between M^pro-SY110 and M^pro-calpain inhibitor XII (white, PDB: 6XFN). Images b–e were prepared using PyMOL (https://pymol.org)

Fig. 5

In vivo pharmacokinetics and safety data of SY110.SY110 was administered by intravenous injection (i.v.) or oral gavage (p.o.) route, then the blood samples of the dogs (a), monkeys (b), and mice (c) were analyzed. The two dash lines in (c) indicate the EC₅₀ and EC₉₀ values for SY110 against BA.2 variant (Fig. 3b). d The weight change (percentage of initial weight) of mice treated with SY110 by oral administration for two weeks (n = 6)

Fig. 6

In vivo antiviral activity of SY110. a The schematic illustration of experiment design. b The viral RNA-dependent RNA polymerase (RdRp) gene copies were quantified by probe-based RT-qPCR (n = 5–7) or c subgenomic envelope (sgE) mRNA copies were quantified by probe-based RT-qPCR (n = 5–7) or d viral titer quantification with plaque assay (n = 5–7). All data were obtained from three times of independent experiments and shown as mean ± SD. Statistical differences were determined with one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test in b–d. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 7

Immunohistochemistry and H&E analysis of the infected mice lung tissue. a Representative images of immunohistochemistry staining visualizing the SARS-CoV-2 nucleocapsid protein (brown, indicated by black arrows) in the lung of the infected mice at 4 dpi. (n = 5–7). Scale bar, 200 µm. b Representative images of H&E staining visualizing the virus-induced pathology in the lung of the infected mice at 4 dpi. (n = 5–7). Representative images of alveoli, bronchioles, and blood vessels of the lung indicated by yellow dashed circle were enlarged with numbers 1, 2, and 3, respectively. Scale bar, 500 µm

Fig. 8

Effects of resistance mutations on the inhibition of SARS-CoV-2 M^pro by SY110 and Nirmatrelvir. a Inhibition of SY110 and Nirmatrelvir against SARS-CoV-2 M^pro polymorphisms. IC₅₀ fold increase is relative to the WT. b IC₅₀ determination of SY110 and Nirmatrelvir against M^pro mutants. Data are shown as mean ± SD based on three independent experiments