Progress in Developing Inhibitors of SARS-CoV-2 3C-Like Protease

Abstract

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral outbreak started in late 2019 and rapidly became a serious health threat to the global population. COVID-19 was declared a pandemic by the World Health Organization in March 2020. Several therapeutic options have been adopted to prevent the spread of the virus. Although vaccines have been developed, antivirals are still needed to combat the infection of this virus. SARS-CoV-2 is an enveloped virus, and its genome encodes polyproteins that can be processed into structural and nonstructural proteins. Maturation of viral proteins requires cleavages by proteases. Therefore, the main protease (3 chymotrypsin-like protease (3CL^pro) or M^pro) encoded by the viral genome is an attractive drug target because it plays an important role in cleaving viral polyproteins into functional proteins. Inhibiting this enzyme is an efficient strategy to block viral replication. Structural studies provide valuable insight into the function of this protease and structural basis for rational inhibitor design. In this review, we describe structural studies on the main protease of SARS-CoV-2. The strategies applied in developing inhibitors of the main protease of SARS-CoV-2 and currently available protein inhibitors are summarized. Due to the availability of high-resolution structures, structure-guided drug design will play an important role in developing antivirals. The availability of high-resolution structures, potent peptidic inhibitors, and diverse compound scaffolds indicate the feasibility of developing potent protease inhibitors as antivirals for COVID-19.

Keywords: COVID-19; SARS-CoV-2; antivirals; drug discovery; protease inhibitor; protein structures.

Figure 1

Genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Viral proteins encoded by the viral genome. The nonstructural proteins (nsps), structural proteins, and accessory proteins (Orf3a to Orf9b) are shown. (b) Membrane topology of several nonstructural proteins. The transmembrane domains of proteins are shown as cylinders. Arrows indicate cleavage sites of: papain-like cysteine protease (PL^pro; red) and 3 chymotrypsin-like protease (3CL^pro; blue). Other nonstructural proteins are shown as spheres. The sphere has not been drawn to actual scale of individual proteins. More information can be obtained from https://viralzone.expasy.org/764.

Figure 2

Structure of SARS-CoV-2 3CL^pro. The N-terminal seven residues (N-finger), domains I, II, III, and the linker of domains II and III of both protomers are shown in red, light blue, wheat, green, and purple, respectively. The linker in the two protomers is shown in ribbon mode. Other domains in one protomer are shown in surface mode except the linker region, and corresponding domains in the other protomer are shown in ribbon mode. The structure (PDB ID 6Y2G) is used in this figure.

Figure 3

De novo drug discovery versus drug repurposing. The time required from hit identification to lead optimization (upper panel) is saved in drug repurposing (lower panel). In the case of SARS-CoV-2 3CL^pro, virtual screening, biochemical, and cell-based assays were applied to identify protease inhibitors from FDA-approved drugs. The duration required in individual processes is based on [59], which gives detailed information for drug repurposing. It is worth mentioning that the timeline for COVID-19 might be different from other diseases due to its pandemic status. HTS, high throughput screening. FDA, the Food and Drug Administration.

Figure 4

The substrate binding site of SARS-CoV-2 3CL^pro. (a) A cocrystal structure of SARS-CoV-2 3CL^pro with an inhibitor (N3) is shown. (b) Surface charge analysis of the active site of the protease. The structure (PDB ID 6LU7) is shown using PyMOL (https://pymol.org/2/). The protease in the absence (a) and presence (b) is shown in the same orientation. The inhibitor is shown as green sticks. Only domains I and II of one protomer of the protease is shown for clarity.

Figure 5

Some peptidic inhibitors of SARS-CoV-2. Structures and their half maximal inhibitory concentration values/ half-maximal effective concentration (IC₅₀s/EC₅₀s) against SARS-CoV-2 3CL^pro are shown. The binding site 11a with SARS-CoV-2 3CL^pro is shown. The inhibitor 11a is shown as sticks, and the protease is shown as a surface. S1 and S2 indicate the binding sites for P1 and P2 residues of the inhibitor.

Figure 6

Compounds identified through high-throughput screening. Structures, half maximal inhibitory concentration values/ half-maximal effective concentration (IC₅₀s/EC₅₀s) (when applicable) of compounds are shown. Please refer to [54] for more details.