Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease

Abstract

Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease (M^pro) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the M^pro/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor's keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the M^pro active site adapt to the inhibitor, which appears to be an intrinsic property of M^pro. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with M^pro which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.

Fig. 1. Active site architecture of SARS-CoV-2 M^pro and chemical structures of protease inhibitors.

a Substrate binding subsites are indicated as S5-S1′, including the oxyanion hole. b Reversible covalent inhibitors showing chemical groups P5-P1′ that bind in the subsites S5-S1′, respectively.

Fig. 2. Joint X-ray/neutron crystal structure of deuterated SARS-CoV-2 M^pro with BBH-1.

a Electron and b nuclear density for BBH-1 c covalently bound in the M^pro active site. d Direct and water-mediated hydrogen bond interactions between BBH-1 and M^pro. Protein homodimer represented with one protomer as white surface and the other as salmon cartoon. Electron (pink mesh) and nuclear (purple mesh) density of BBH-1 (cyan ball and sticks) contoured to 1 σ. Hydrogen bonds are represented as black dashes while C-H···O interaction with Gln189 is represented as dash-dots, with distances in angstroms (Å). Deuterium atoms are colored in light orange.

Fig. 3. Direct observation of BBH-1 electrostatic interactions with M^pro determined by neutron crystallography.

a Cys145 reacts with the keto-benzothiazole warhead of BBH-1 to form a covalent hemithioketal group with an alkoxy anion directed towards the oxyanion hole and hydrogen bonding to an accompanied D₂O molecule. b Hydrogen bond network involved with the P1 γ-lactam of BBH-1 in the M^pro S1 pocket. c The catalytic His41 in M^pro/BBH-1 is singly protonated on Nδ1 and forms a C-D· · ·N interaction (orange dashes) with His164. d The P1′ benzothiazole group of BBH-1 sterically swings His41 from its position in the ligand-free XN structure of M^pro (7JUN) and evicts the catalytic water molecule. Protonation states of His41 and His164 change from doubly protonated to singly protonated. BBH-1 as cyan ball and sticks. Ligand-free M^pro colored in salmon. Neutron 2Fo-Fc map (purple mesh) contoured at 1.0 σ. Omit nuclear Fo-Fc for D atoms on His41 and His164 are shown as green mesh contoured at 4 and 2.8 σ respectively. Hydrogen bonds are represented as black dashes. Distance is given in angstroms (Å). Deuterium atoms are colored in light orange.

Fig. 4. Room-temperature X-ray crystal structures of M^pro in complex with nitrile warhead inhibitors BBH-2, NBH-2, and PF-037321332.

a–c Inhibitors BBH-2, NBH-2, and PF-037321332 bind to the active site of M^pro and form a covalent thioimidate with Cys145. Polder omits maps of inhibitor atoms (blue mesh) contoured to 3σ. d–f Hydrogen bonds between inhibitors and M^pro are shown as black dashes with distances in angstroms (Å).

Fig. 5. Active site structural plasticity of M^pro bound to nitrile warhead inhibitors.

Comparison of ligand-free M^pro (7JUN, white cartoon) with the BBH-2, NBH-2, and PF-037321332 complexes shows significant structural shifts in protein subsites S2, S4, and S5 responsible for binding to inhibitor P2, P4, and P5 moieties respectively. Distances are given in angstroms (Å). Superposition is calculated by the least-square-fitting of Cα atoms.