SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model

Abstract

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (M^pro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing M^pro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 M^pro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of M^pro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

Fig. 1. Schematic diagram of the design of novel SARS-CoV-2 M^pro inhibitors.

Fig. 2. Overall structure of SARS-CoV-2 M^pro–MI-23 complex.

(A) Cartoon view of the M^pro dimer (molecule A, cyan; molecule B, purple). Three domains (I, II, and III) of each monomer are marked. The catalytic dyad Cys¹⁴⁵-His⁴¹ is located in the cleft between domains I and II. MI-23 in both molecules is shown in purple or orange. The N and C termini of each M^pro are labeled. Labels for molecule B are in italics. (B) The chemical structure of MI-23. (C) The MI-23 binding pocket of M^pro. F_o – F_c density map (gray mesh, σ = 2.5) is shown for MI-23 (purple). Cys¹⁴⁵ and His⁴¹ are shown in yellow and blue, respectively. The covalent bond is formed by Cys¹⁴⁵ and the warhead aldehyde. F_o − F_c density map (σ = 2.5) is shown in gray. (D) Interactions between M^pro and MI-23; the hydrogen bonds between them are shown as black dashed lines. Ser¹ from molecule B interacts with Glu¹⁶⁶ and Phe¹⁴⁰ in molecule A (red dashed lines) to support S1 pocket formation. The warhead carbon is marked with a black asterisk in (B), (C), and (D). Images in (A), (C), and (D) were prepared using PyMOL (https://pymol.org).

Fig. 3. Antiviral activity of six compounds against SARS-CoV-2 in cell-based assays.

(A) Vero E6 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 and treated with different concentrations of test compounds (MI-09, MI-12, MI-14, MI-28, MI-30, and MI-31). At 3 dpi, the cytopathic effect caused by SARS-CoV-2 infection was quantitatively analyzed using CCK8 according to the manufacturer’s protocol. Data are means ± SD; n = 3 biological replicates. (B) HPAEpiC cells were infected with SARS-CoV-2 at an MOI of 0.01 and treated with different concentrations of test compounds (MI-09, MI-12, MI-14, MI-28, MI-30, and MI-31). At 2 dpi, viral RNA copies (per ml) were quantified from cell culture supernatants by RT-qPCR. Data are means ± SD; n = 2 biological replicates.

Fig. 4. MI-09 and MI-30 reduce lung viral loads and lung lesions in a SARS-CoV-2 infection transgenic mouse model.

(A and B) Chemical structures and summary of in vitro activity data and bioavailability of MI-09 and MI-30. (C) Overview of in vivo study design. (D) Viral loads in the lungs of SARS-CoV-2–infected hACE2 transgenic mice. Mice infected with the indicated dose of SARS-CoV-2 were treated with MI-09, MI-30, or vehicle solution, and then were killed at 1 or 3 dpi. Five lung lobes of each mouse were collected to determine viral loads. Data (means ± SD) represent the median of five lung lobes of individual mice. The horizontal dotted line shows the viral load limit of detection (LOD) of 1.0 log₁₀ RNA copies. Data below the LOD are shown at the LOD. *P < 0.05, **P < 0.01 (two-tailed unpaired Student’s t test). (E) Representative images of lung histopathological changes from SARS-CoV-2–infected hACE2 mice (5 × 10⁶ TCID₅₀) at 3 dpi. Magnified views of the boxed regions for each image are shown below. Black arrows indicate alveolar septal thickening; red arrows point to inflammatory cell infiltration. See fig. S4 for whole-lung tissue scan images of SARS-CoV-2–infected hACE2 mice at 3 dpi. (F) Representative chemokine and cytokine assessment of the lung tissues (n = 3) of the indicated groups, as detected in lung tissue homogenate at 3 dpi. Data are means ± SD. *P < 0.05, **P < 0.01 versus the vehicle group (one-way analysis of variance). (G and H) Infiltration analysis for neutrophils and macrophages in the lungs of SARS-CoV-2–infected hACE2 mice (5 × 10⁶ TCID₅₀) at 3 dpi. (G) Percentages of macrophages and neutrophils in the lungs. *P < 0.05, **P < 0.01 (unpaired Student’s t test). (H) Representative images of fluorescence staining. White triangle and arrow indicate macrophage and neutrophil, respectively.