Proof-of-concept studies with a computationally designed Mpro inhibitor as a synergistic combination regimen alternative to Paxlovid

Abstract

As the SARS-CoV-2 virus continues to spread and mutate, it remains important to focus not only on preventing spread through vaccination but also on treating infection with direct-acting antivirals (DAA). The approval of Paxlovid, a SARS-CoV-2 main protease (M^pro) DAA, has been significant for treatment of patients. A limitation of this DAA, however, is that the antiviral component, nirmatrelvir, is rapidly metabolized and requires inclusion of a CYP450 3A4 metabolic inhibitor, ritonavir, to boost levels of the active drug. Serious drug-drug interactions can occur with Paxlovid for patients who are also taking other medications metabolized by CYP4503A4, particularly transplant or otherwise immunocompromised patients who are most at risk for SARS-CoV-2 infection and the development of severe symptoms. Developing an alternative antiviral with improved pharmacological properties is critical for treatment of these patients. By using a computational and structure-guided approach, we were able to optimize a 100 to 250 μM screening hit to a potent nanomolar inhibitor and lead compound, Mpro61. In this study, we further evaluate Mpro61 as a lead compound, starting with examination of its mode of binding to SARS-CoV-2 M^pro. In vitro pharmacological profiling established a lack of off-target effects, particularly CYP450 3A4 inhibition, as well as potential for synergy with the currently approved alternate antiviral, molnupiravir. Development and subsequent testing of a capsule formulation for oral dosing of Mpro61 in B6-K18-hACE2 mice demonstrated favorable pharmacological properties, efficacy, and synergy with molnupiravir, and complete recovery from subsequent challenge by SARS-CoV-2, establishing Mpro61 as a promising potential preclinical candidate.

Keywords: Mpro61; SARS-CoV-2; drug synergy; molnupiravir; protease inhibitor.

Fig. 1.

Computational and structure-guided optimization of the docking hit compound, perampanel. The representative optimization steps shown here highlight reorganization of the scaffold rings (Mpro6), modification to a uracil group (Mpro8), oxyalkyl extension into additional subsites to achieve submicromolar inhibition (Mpro24 and Mpro43), and methylation of the uracil group to improve potency in cells (Mpro61). IC₅₀ and EC₅₀ values were previously published (–14).

Fig. 2.

Crystal structure of compound Mpro61 bound by the SARS-CoV-2 main protease (2.3 Å, PDB ID: 8UR9). Carbon atoms of Mpro61 are shown in cyan, carbon atoms of the catalytic dyad (C145 and H41) are shown in purple, and carbon atoms of residues involved in hydrogen bonding with Mpro61 are shown in orange. Hydrogen bonds are shown as dashed lines. The surface view (Right) shows the fit of Mpro61 within the active site.

Fig. 3.

Panel of 33 targets including CYP450 isoforms, ions channels, and receptors examined for off-target effects. Color-coding shows the percentage inhibition of each target in the presence of 10 μM inhibitor. Mpro61 showed no significant off-target effects except slight inhibition of HERG ion channel at high concentrations.

Fig. 4.

Synergistic inhibition of SARS-CoV-2 replicon replication by combination of Mpro61 with molnupiravir, shown using MacSynergy II 3D plots. Peaks above the horizontal plane indicate synergistic inhibition. The results are from three experiments involving triplicate determination.

Fig. 5.

Concentrations of Mpro61-HCl in the serum (Left) and various tissues (Right) of C57BL/6 mice. Mice were dosed with two 150 mg/kg capsules 10 to 12 h apart, and tissues were analyzed at 24 h by Q-TOF-EST-MS. Mpro61-HCl concentrations were determined as described in the Materials and Methods.

Fig. 6.

Efficacy and synergy studies in K18-hACE2 mice infected with SARS-CoV-2 WA-1 nLuc virus. Mice were split into four groups for dosing: vehicle control (n = 4), twice daily 150 mg/kg Mpro61 (n = 6), twice daily 100 mg/kg molnupiravir (n = 4), and twice daily Mpro61 150 mg/kg in combination with molnupiravir 100 mg/kg (n = 4). Molnupiravir was given for 3 d, while Mpro61 was given for 6 d (A). Bioluminescent imaging of virus in mice, with images taken in the ventral (v) and dorsal (d) positions (B). Quantification of nLuc signal as flux (photons/sec) in the whole body (C) and brain (D). Changes in mouse body weight, with the initial body weight set as 100% (E). Kaplan–Meier survival curves of the mice in each dosing group (n = 4 to 6 per group) (F). Fold changes in viral mRNA expression (G) and viral loads (nLuc activity/mg tissue) (H) in the brain, lung, and nose of mice following death or 14 dpi (G). Grouped data in (C–E) and (G and H) were analyzed by two-way ANOVA followed by Tukey’s multiple comparison tests. Statistical significance for group comparisons to vehicle is shown in black. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001; ns not significant; mean values ± SD are depicted.

Fig. 7.

Efficacy and synergy studies in B6-K18-hACE2 mice infected with SARS-CoV-2 WA-1 nLuc virus, as described in Fig. 6. Bioluminescent imaging of virus (A) and quantification of nLuc signal as flux (photons/sec) (B) in isolated tissues as indicated following necropsy at 14 dpi. Fold changes in cytokine mRNA expression in the brain (C) and lung (D) as well as mRNA expression of Krt8 in the lung (E) of mice following death or 14 dpi. Grouped data in (B–D) were analyzed by two-way ANOVA followed by Tukey’s multiple comparison tests. The data in (E) were analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. Statistical significance for group comparisons to vehicle is shown in black. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001; ns not significant; mean values ± SD are depicted.