Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies

Abstract

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (M^pro) to cleave viral proteins. Consequently, M^pro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of M^pro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-M^pro complexes reveal that an alternative binding pocket in M^pro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na⁺ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for M^pro inhibitor design.

Keywords: COVID-19; Crystallography; GC376 analogs; Main protease; Protease inhibitor; Structure-guided design.

Graphical abstract

Fig. 1

Developmental workflow of SARS-CoV-2 M^proinhibitors. The process of inhibitor design and modification carried out in this publication is depicted. Evaluation of inhibitors was carried out at each step by FRET studies, crystallography, and viral assays.

Fig. 2

Binding sites of SARS-CoV-2 M^procatalytic cleft. The active cysteine-145 residue (C145, yellow) as well as side-pockets S1’, S1, S2, S4 and surface depression S3 are highlighted.

Fig. 3

Crystal structures and schematic depictions of inhibitors bound to SARS-CoV-2 M^pro. (A) 1a bound to the active site of SARS-CoV-2 M^pro. The benzyl ring at the P3 position of the inhibitor can be seen interacting with the S3 surface depression, similarly to the binding mode of GC373 (PDB 7LCS). (B) 1i bound to the active site of SARS-CoV-2 M^pro. The presence of a fluorine atom at the 3-position on the benzyl ring directs the aromatic moiety into the S4 binding pocket (PDB 7LCO). (C) 1f bound to the active site of SARS-CoV-2 M^pro. Substitution of the benzyloxy group with a 4-methoxyindole prompts binding of the P3 portion of the inhibitor to the S4 pocket of the enzyme. This is a result of favorable interactions between the indole nitrogen and the carbonyl of Glu166 as well as that of the methoxy group with the binding pocket itself (PDB 7LDL). (D) 1g bound to the active site of SARS-CoV-2 M^pro. The phenyl ring of (S)-methyl benzyl group can be seen being directed towards the S4 site of the enzyme via the presence of the additional methyl group (PDB 7LCT).

Fig. 4

Key lead compounds identified as improved SARS-CoV-2 M^proinhibitors. Both structures contain a cyclopropyl group at the P2 position. Differentiation occurs at the P3 position, where 2c contains a 3-fluorobenzyl group and 2d contains a 3-chlorophenylethyl group. Both inhibitors show substantially improved potency as demonstrated by IC₅₀ and EC₅₀ values, both with and without CP-100356, as compared to the parent compound GC376.