Medicinal chemistry strategies towards the development of non-covalent SARS-CoV-2 Mpro inhibitors

Abstract

The main protease (M^pro) of SARS-CoV-2 is an attractive target in anti-COVID-19 therapy for its high conservation and major role in the virus life cycle. The covalent M^pro inhibitor nirmatrelvir (in combination with ritonavir, a pharmacokinetic enhancer) and the non-covalent inhibitor ensitrelvir have shown efficacy in clinical trials and have been approved for therapeutic use. Effective antiviral drugs are needed to fight the pandemic, while non-covalent M^pro inhibitors could be promising alternatives due to their high selectivity and favorable druggability. Numerous non-covalent M^pro inhibitors with desirable properties have been developed based on available crystal structures of M^pro. In this article, we describe medicinal chemistry strategies applied for the discovery and optimization of non-covalent M^pro inhibitors, followed by a general overview and critical analysis of the available information. Prospective viewpoints and insights into current strategies for the development of non-covalent M^pro inhibitors are also discussed.

Graphical abstract

Figure 1

SARS-CoV-2 genomic organization and viral proteins. (A) Sequence of SARS-CoV-2 polyprotein; M^pro cleavage sites are indicated by red arrows, and PL^pro cleavage sites are indicated by gray arrows. (B) Diagram highlighting the role of M^pro in the replicative cycle of SARS-CoV-2.

Figure 2

Structure and catalytic mechanism of SARS-CoV-2 M^pro. (A) Crystal structure of the M^pro homodimer and close-up view of its catalytic site (PDB ID: 7JUN). Domains I, II and III are represented with green, red and orange cartoons, respectively. All structures hereinafter are visualized by Pymol (pymol.org). (B) Proposed catalytic mechanism of M^pro.

Figure 3

Residues and sub-pockets at the dimerized SARS-CoV-2 M^pro catalytic site (PDB ID: 7JUN). The second monomer is not shown in the figure.

Figure 4

Verified non-covalent hit compounds from virtual screening targeting M^pro.

Figure 5

Screening-based modification of hydantoin-based M^pro inhibitors. (A) Structures and activities of hits/compounds (7–10) from two rounds of screening. (B) Structures and activities of compounds 11–13. (C) Co-crystal structures overlay and residue interactions of 7 (green, PDB ID: 7B2U), 11 (blue, PDB ID: 7O46) and 12 (magenta, PDB ID: 7QBB). Hydrogen bonds are shown as magenta dashed lines, π–π stacking are shown in green dashed lines.

Figure 6

The discovery, optimization, and co-crystal study of dihydroquinolinone derivatives. (A) Starting hits and their screening-based optimization. (B) Preliminary SAR of the screened compounds. (C) Co-crystal structure of compound 21 and M^pro demonstrating space occupancy and residue interactions (PDB ID: 7P2G). Hydrogen bonds are shown as magenta dashed lines. π–π stacking are shown in green dashed lines. Halogen bonds are shown in orange dashed lines.

Figure 7

Structures, activities and co-crystal analysis of compounds 23 (A, PDB ID: 7LTJ) and 24 (B, PDB ID: 7RLS). Hydrogen bonds are shown as magenta dashed lines. π–π stacking are shown in green dashed lines.

Figure 8

Hit compounds discovered with the assistance of artificial intelligence and machine learning.

Figure 9

Non-covalent SARS-CoV-2 M^pro inhibitors identified by HTS. (A) Chemical structures and activities of compounds 28 and 29. (B) Chemical structure, activity and crystal structure of 30 in complex with SARS-CoV-2 M^pro (PDB ID: 7TVX).

Figure 10

Hit compounds from DEL-based M^pro inhibitor screening. (A) Chemical structures and IC₅₀ values of 31–34. Common structures are highlighted in purple/blue. Chiralities of 31 and 34 are unspecified. (B) The antiviral activity towards various strains, in vivo half-life and oral bioavailability of 33. (C) Residue interactions of 33 revealed by its co-crystal structure with SARS-CoV-2 M^pro (PDB ID: 7EN8). Hydrogen bonds are shown as magenta dashed lines. π–π stacking are shown in green dashed lines. Halogen bonds are shown in orange dashed lines. Amnio–π interactions are shown in red dashed lines.

Figure 11

(A) Structures and activities of 35 and 36. (B) M^pro binding mode revealed by X-ray crystallography. 36 (magenta)/35 (cyan). Hydrogen bonds are shown as magenta dashed lines. π–π stacking are shown in green dashed lines (PDB ID: 7KX5).

Figure 12

Discovery of the trisubstituted piperidine analogs. (A) Rational design based on Ugi-generated scaffold; (B) Structure and activity of representative compound 37.

Figure 13

Discovery and co-crystal studies of perampanel derivatives. (A) Optimization process starting from compound 38. (B) Co-crystal structure of compounds 39 (left) and 40 (right) with M^pro illustrating H-bond interactions (magenta) and spatial occupancy (PDB ID: 7L10).

Figure 14

Modifications of benzotriazole derivatives. All the IC₅₀ values presented are for SARS-CoV-2. (A) Prior modifications led to compounds 43 and 44. (B) Rational design based on 44. (C, D) The co-crystal structure of 43 (C, PDB ID: 7LME) and 47 (D, PDB ID: 7LMD) in complex with M^pro. Hydrogen bonds are shown as magenta dashed lines.

Figure 15

Optimization of fragment 48. (A) Structures and activities of compounds 48, 49; (B) (C) Co-crystal structures of 48/49 with SARS-CoV-2 M^pro (PDB ID: 5RF7 and 7NBT).

Figure 16

Rational design and co-crystal study of trisubstituted piperazine M^pro inhibitors. (A) Structure and activity of 50 and 51. (B) Co-crystal structure of 50 with SARS-CoV-2 M^pro (PDB ID: 8ACD). (C) Co-crystal structure of 51 with SARS-CoV-2 M^pro (PDB ID: 8ACL). Hydrogen bonds are shown as magenta dashed lines. π–π stacking are shown in green dashed lines. Halogen bonds are shown in orange dashed lines.

Figure 17

Design and development of ensitrelvir (54, S-217622). (A) Structural optimization and representative compounds in the development of ensitrelvir. (B) Co-crystal structure of compound 52 (hit) with M^pro. (C) Co-crystal structure of ensitrelvir with M^pro and close-up view showing the conformational change of His41. (D) Binding pose comparison of compounds 52 and 54. Hydrogen bonds are shown as magenta dashed lines. π–π stacking are shown in green dashed lines.

Figure 18

Fragment-based screening and optimization of SARS-CoV-2 M^pro inhibitors. (A) Chemical structures of 55 (x0967), 56 (x0434) and 57 (SX013) and biological activities of 57. Superpositions of fragments occupying subsites S1, S2 and S4 subsites are shown in panels (B), (C) and (D), respectively.

Figure 19

Chemical structure, biological activity and crystal structure of compound 58 in complex with SARS-CoV-2 M^pro (PDB ID: 7P51). Hydrogen bonds are shown as magenta dashed lines.

Figure 20

Derivatives (60–63) identified through lead discovery based on an aminoisoquinoline fragment 59.

Figure 21

Schematic diagram and outcome of the ranking model driven by COVID-19 moonshot fragments. (A) Activity ranking based on pharmacophore fingerprints of two compounds; (B) Structures and activities of top training model compound 64 and predicted compound 65.

Figure 22

(A) Chemical structures and biological activities of the discussed natural products (66–69) targeting M^pro; (B) Co-crystal structure of shikonin and M^pro (PDB ID: 7CA8); (C) Co-crystal structure of baicalein and M^pro (PDB ID: 6M2N). H-bonds are shown in magenta dashed lines. π–π stacking interactions are shown in green dashed lines.

Figure 23

Metal complexes targeting M^pro. (A) Chemical structures and biological activities of zinc salts/coordinates inhibiting SARS-CoV-2 M^pro. (B) Crystal structure of zinc ion bound to the M^pro catalytic site (PDB ID: 7DK1). (C) Chemical structures and activity of zinc–hinokitol complexes.

Figure 24

Peptides as non-covalent inhibitors of SARS-CoV-2 M^pro. (A) Amino acid sequences and biological activities of peptides 76–79. (B) Co-crystal structure of 76 in complex with M^pro (PDB ID: 7RNW). The gray dashed line represents undetermined atoms.

Figure 25

Small molecules targeting non-catalytic sites. (A, B) Chemical structures, activities, and binding sites of 80 (pelitinib), 81 (AT7519) and 82 (x1187). (C) Co-crystal structures of pelitinib bound at the M^pro dimer surface (PDB ID: 7AXM). (D) Ligand interactions of 81 with M^pro (PDB ID: 7AGA). (E) Conformational changes of Arg298 and Tyr154 in the 81-bound M^pro structure (black) compared with apo-M^pro (light gray). (F) Co-crystal structure of 82 bound at the M^pro dimer surface (PDB ID: 5RFA)^,.

Figure 26

Overview of key pharmacophores and privileged groups in non-covalent M^pro inhibitors.