SARS-CoV-2 Mpro: A Potential Target for Peptidomimetics and Small-Molecule Inhibitors

Abstract

The uncontrolled spread of the COVID-19 pandemic caused by the new coronavirus SARS-CoV-2 during 2020-2021 is one of the most devastating events in the history, with remarkable impacts on the health, economic systems, and habits of the entire world population. While some effective vaccines are nowadays approved and extensively administered, the long-term efficacy and safety of this line of intervention is constantly under debate as coronaviruses rapidly mutate and several SARS-CoV-2 variants have been already identified worldwide. Then, the WHO's main recommendations to prevent severe clinical complications by COVID-19 are still essentially based on social distancing and limitation of human interactions, therefore the identification of new target-based drugs became a priority. Several strategies have been proposed to counteract such viral infection, including the repurposing of FDA already approved for the treatment of HIV, HCV, and EBOLA, inter alia. Among the evaluated compounds, inhibitors of the main protease of the coronavirus (M^pro) are becoming more and more promising candidates. M^pro holds a pivotal role during the onset of the infection and its function is intimately related with the beginning of viral replication. The interruption of its catalytic activity could represent a relevant strategy for the development of anti-coronavirus drugs. SARS-CoV-2 M^pro is a peculiar cysteine protease of the coronavirus family, responsible for the replication and infectivity of the parasite. This review offers a detailed analysis of the repurposed drugs and the newly synthesized molecules developed to date for the treatment of COVID-19 which share the common feature of targeting SARS-CoV-2 M^pro, as well as a brief overview of the main enzymatic and cell-based assays to efficaciously screen such compounds.

Keywords: COVID-19; SARS-CoV-2 Mpro; coronavirus; peptidomimetics; protease inhibitors; remdesivir.

Figure 1

Sketched view of the SARS-CoV-2 replication cycle.

Figure 2

3D structure of SARS-CoV-2 M^pro in two different views. One protomer of the dimer is shown in light blue, the other one in orange [12].

Figure 3

The differences between SARS-CoV M^pro and SARS-CoV-2 M^pro structures. The overall structure of both M^pro with the different amino acids are marked in black (SARS-CoV M^pro) and blue (SARS-CoV-2 M^pro). Close-up of the active site cavity and bound N3 inhibitor into SARS-CoV (black sticks) and SARS-CoV-2 (blue sticks) M^pro. The catalytic water molecule that resembles the position of the third member of the catalytic triad adopted from the cysteine proteases is shown (red sticks) and the proteins’ structures are shown in surface representation. Position 46 located, near the entrance of the active site, is labelled with an asterisk (*) [22].

Figure 4

Hydrolysis mechanism of SARS-CoV-2 M^pro. Amino acids of the catalytic dyad and the substrate are depicted in blue and orange, respectively.

Figure 5

(a) Structure of compound 1 bearing at P2 an aliphatic group as side chain. (b) X-ray structure of the complex 1/M^pro, with the most important interactions. (c) Structure of compound 2 bearing at P2 an aromatic group as side chain. (d) X-ray structure of the complex 2/M^pro, with the most important interactions [37].

Figure 6

(a) 3 is a prodrug used to treat the FIP (feline infectious peritonitis). In vivo, the bisulfite compound converts into the aldehyde inhibitor of M^pro 4. (b) 4 covalently linked to SARS-CoV-2 M^pro [89].

Figure 7

(a) Structure of 5, the best peptidomimetic inhibitor of M^pro discovered so far in terms of IC₅₀ values. (b) X-ray of 5 in complex with SARS-CoV-2 M^pro [91].

Figure 8

Structure of 6 and 7, two derivatives of 5 with strong inhibition potency towards Vero E6 cells infected with SARS-CoV-2.

Figure 9

Structure and biological activity of 8.

Scheme 1

Design of M^pro inhibitors bearing a α-ketoamide as warhead proposed by Zhang et al. [13]. 9, originally developed as inhibitor of other coronaviral proteases, underwent chemical modifications towards peptidic backbone. 11 was demonstrated to effectively block M^pro activity and reduce proliferation of SARS-CoV-2 in Calu-3 lung cells. 12 showed no antiviral activity.

Figure 10

Main interactions of 11 with M^pro binding cavity [12].

Figure 11

Chemical structure and biological activity of the calpain inhibitor tripeptidyl α-ketoamide 13.

Figure 12

(a) Chemical structure and biological activity of the tetrapeptidyl vinyl ester 14; (b) X-ray structure of 14 in complex with SARS-CoV-2 M^pro [20].

Figure 13

(a) 15 is an irreversible inhibitor of SARS-CoV-2 M^pro bearing the MFMK moiety as warhead. (b) Predicted binding model of 7 covalently linked inside the pocket of SARS-CoV-2 M^pro [98].

Figure 14

Chemical structure of 16 and 17. 16 is a reversible inhibitor of SARS-CoV M^pro bearing the TFMK moiety as a warhead. The peptidyl DFMK ketone 17 manifested antiviral effect in Vero E6 cells infected with hCoV-229E.

Figure 15

(a) Hydroxymethyl ketone 19 and the phosphate prodrug are depicted. 18 is currently under clinical trials by Pfizer Inc. to treat COVID-19. (b) Cocrystal structure of 19 covalently bound to the active site of the M^pro [102].

Figure 16

Chemical structure and biological activity of 20.

Figure 17

Chemical structure and biological activity of the first-in-class cyclic peptide UCI-1.

Figure 18

(a) Chemical structure of 21, a suicide inhibitor of M^pro. Inside the binding pocket of M^pro, 21 is converted into 22 and covalently binds Cys145, leading to an irreversible inhibition of the protease. (b) X-ray complex of 22 and M^pro [110].

Figure 19

Biochemical activation of 21 within the M^pro binding pocket involved a E1cB-like reaction mechanism.

Figure 20

Chemical structure of the pyrimidotriazine-dione walrycin B (19).

Figure 21

Chemical structure of 24, an irreversible inhibitor of M^pro. Differential scanning fluorimetry using 1:1 molar ratio of M^pro and the inhibitor, shifted the melting temperature of the protein to lower temperatures (ΔT_m M^pro = 5.59 °C).

Figure 22

(a) Structure of the reversible inhibitor 25, the major component extracted from the roots of a Chinese herb. (b) The main interactions between 25 and M^pro [36].

Figure 23

Chemical structure of the three flavonoids active as M^pro inhibitors, baicalin (26), baicalein (27), and scutellarein (28).

Figure 24

Representative description of biosensor that lights up when M^pro is active in the cell.