Exploring diverse reactive warheads for the design of SARS-CoV-2 main protease inhibitors

Abstract

SARS-CoV-2 main protease (M^pro) is a validated antiviral drug target of nirmatrelvir, the active ingredient in Pfizer's oral drug Paxlovid. Drug-drug interactions limit the use of Paxlovid. In addition, drug-resistant M^pro mutants against nirmatrelvir have been identified from cell culture viral passage and naturally occurring variants. As such, there is a need for a second generation of M^pro inhibitors. In this study, we explored several reactive warheads in the design of M^pro inhibitors. We identified Jun11119R (vinyl sulfonamide warhead), Jun10221R (propiolamide warhead), Jun1112R (4-chlorobut-2-ynamide warhead), Jun10541R (nitrile warhead), and Jun10963R (dually activated nitrile warhead) as potent M^pro inhibitors. Jun10541R and Jun10963R also had potent antiviral activity against SARS-CoV-2 in Calu-3 cells with EC₅₀ values of 2.92 and 6.47 μM, respectively. X-ray crystal structures of M^pro with Jun10541R and Jun10221 revealed covalent modification of Cys145. These M^pro inhibitors with diverse reactive warheads collectively represent promising candidates for further development.

Keywords: 3CL protease; Antiviral; COVID-19; Main protease; SARS-CoV-2.

Figure 1.

SARS-CoV-2 ${\text{M}}^{\text{pro}}$ inhibitors with diverse reactive warheads.

Figure 2.

Synthesis of covalent SARS-CoV-2 ${\text{M}}^{\text{pro}}$ inhibitors through Ugi-4CR. The R and S chirality refers to the chiral center at the pyridine substitution. w=warhead.

Figure 3.

SARS-CoV-2 ${\text{M}}^{\text{pro}}$ inhibitors with substituted acrylamide warheads or vinyl sulfonamides. Results of Jun9513 and Jun10382 were reported.[22]

Figure 4.

SARS-CoV-2 ${\text{M}}^{\text{pro}}$ inhibitors with substituted propiolamide warheads.

Figure 5.

SARS-CoV-2 ${\text{M}}^{\text{pro}}$ inhibitors with substituted nitrile warheads.

Figure 6.

SARS-CoV-2 ${\text{M}}^{\text{pro}}$ inhibitors with miscellaneous warheads.

Figure 7.

${\text{K}}_{\text{i}}$ plots for four potent ${\text{M}}^{\text{pro}}$ inhibitors Jun10221R, Jun1112R, Jun10541R, and Jun10963R.

Figure 8.

Antiviral activities of Mpro inhibitors Jun10541R and Jun10963R against SARS-CoV-2 in Calu-3 cells. The antiviral ${\text{EC}}_{50}$ curve and the cytotoxicity ${\text{CC}}_{50}$ curve were shown in blue and green, respectively.

Figure 9.

Complex structures of SARS-CoV-2 ${\text{M}}^{\text{pro}}$ with inhibitors Jun10541R (2.50 Å resolution) and 10221 (2.55 Å). (A) 2Fo-Fc map, shown in grey and contoured at 1 $\text{σ}$, illustrates the continuous electron density between the catalytic cysteine C145 and Jun10541R, which forms a covalent adduct through its nitrile warhead. (B) Jun10221 also forms a covalent adduct via its alkyne warhead, but has two conformations corresponding to both diastereomers. Here, the 2Fo-Fc map, shown in grey, is contoured at 0.5 $\text{σ}$ to highlight the positioning of the $\text{α}$-methylbenzene sidechain more clearly. (C) The conformations for both diastereomers of Jun10221 are shown from a different angle, offering an unobstructed view of the pyridyl stereocarbon, indicated in the figure, which causes the $\text{α}$-methylbenzene sidechain to occupy either the S2 pocket for the (R, S) diastereomer (turquoise) or flip into the solvent exposed S3 site for the (S, S) diastereomer (pale blue). (D) 2F_o-F_c electron density map for inhibitor Jun10221. Sidechain density for the two diastereomers of Jun10221 is less defined at 1 σ (left panel), than 0.5 σ (right panel).