Targeting SARS-CoV-2 Main Protease for Treatment of COVID-19: Covalent Inhibitors Structure-Activity Relationship Insights and Evolution Perspectives

Abstract

The viral main protease is one of the most attractive targets among all key enzymes involved in the SARS-CoV-2 life cycle. Covalent inhibition of the cysteine¹⁴⁵ of SARS-CoV-2 M^PRO with selective antiviral drugs will arrest the replication process of the virus without affecting human catalytic pathways. In this Perspective, we analyzed the in silico, in vitro, and in vivo data of the most representative examples of covalent SARS-CoV-2 M^PRO inhibitors reported in the literature to date. In particular, the studied molecules were classified into eight different categories according to their reactive electrophilic warheads, highlighting the differences between their reversible/irreversible mechanism of inhibition. Furthermore, the analyses of the most recurrent pharmacophoric moieties and stereochemistry of chiral carbons were reported. The analyses of noncovalent and covalent in silico protocols, provided in this Perspective, would be useful for the scientific community to discover new and more efficient covalent SARS-CoV-2 M^PRO inhibitors.

Figure 1

(a) Sequence of events necessary to activate the digestion process of M^PRO: (1) virus entry into lung epithelial cell by interaction with ACE2 receptor, (2) release of the genomic material of SARS-CoV-2, (3 and 4) virus protein synthesis in human ribosomes, and (5) M^PRO digestion. (b) SARS-CoV-2 M^PRO binding site with viral polyproteins. (c) X-ray structure of dimeric SARS-CoV-2 M^PRO (PDB code 6Y2F). (d) Focus on the catalytic site, with the four regions S1, S1′, S2, and S3/S4 highlighted.

Figure 2

(a) Covalent inhibition of SARS-CoV-2 M^PRO. P1′, P1, P2, and P3 labels reflect the chemical analogies with the viral substrate. Warheads P1′ are in green, while fragments P1, P2, and P3 are shown in red, purple, and blue, respectively. Subregions of the binding pocket are labeled with S numbering complementary to the fragments of the inhibitor. (b) Kinetic scheme of covalent inhibition. E, I, EI, and E–I stand for enzyme, inhibitor, noncovalent enzyme–inhibitor complex, and covalent enzyme–inhibitor complex, respectively.

Figure 3

Electrophilic warheads characterizing covalent SARS-CoV-2 M^PRO inhibitors.

Figure 4

General mechanism of action of covalent carbonyl inhibitors.

Figure 5

(a) Chemical structures of peptidomimetic aldehyde derivatives 1–3; for derivative 1, as an example for the whole series, the four key moieties P1′, P1, P2, and P3 for the interaction with SARS-CoV-2 M^PRO are highlighted. (b) X-ray structure of SARS-CoV-2 M^PRO in complex with compound 1 (PDB code 6LZE). (c) X-ray structure of SARS-CoV-2 M^PRO in complex with compound 2 (PDB code 6M0K). In both crystal structures, the amino acids involved in the interaction with the ligand and the four cavities of the binding pocket (S1′, S1, S2, and S3/S4) are shown.⁻

Figure 6

(a) Chemical structures of GC-373 (electrophilic P1′ warheads in green, P1 in red, P2 in purple, and P3 in blue), GC-376, analogues 4 and 5, UAWJ247, and NK01-63 (coronastat).⁻ (b) X-ray structure of SARS-CoV-2 M^PRO in complex with GC-373 (PDB code 6WTJ(31)). (c) X-ray structure of SARS-CoV-2 M^PRO in complex with 4 (PDB code 7LCO(29)).

Figure 7

(a) Chemical structures of boceprevir, telaprevir, derivatives MI-09, MI-23, and MI-30, and analogues UAWJ9-36-1 and UAWJ9-36-3. (b) X-ray crystal structure of MI-23 in complex with SARS-CoV-2 M^PRO (PDB code 7D3I).^,

Figure 8

Structures of tripeptidyl inhibitors of SARS-CoV-2 M^PRO: MPI3, MPI8, and MGI-132.^,,

Figure 9

Chemical structures of the repurposed calpain inhibitors I and II as new SARS-CoV-2 M^PRO modulators.^,,

Figure 10

Chemical structure of peptidomimetic compound 6.

Figure 11

(a) Chemical structures of a series of acyloxymethyl ketone derivatives with SARS-CoV-2 M^PRO inhibitory activity. (b) PF-00835231 in complex with SARS-CoV-2 M^PRO (PDB code 6XHM).^,−

Figure 12

Structure of α,α-difluoromethyl ketone 9.

Figure 13

Bispidine compound 10, a representative example of a nonpeptidomimetic ketone as a SARS-CoV-2 M^PRO inhibitor.

Figure 14

General mechanism of action of SARS-CoV-2 M^PRO α-ketoamide inhibitors.

Figure 15

(a) Chemical structures of α-ketoamide derivatives 11 and 12. (b) X-ray structure of SARS-CoV-2 M^PRO in complex with compound 12 (PDB code 6Y2F).

Figure 16

Chemical structures of hydantoin derivatives 13–15.(60)

Figure 17

Chemical structures of GC-376 α-ketoamide analogues UAWJ246 and UAWJ248.

Figure 18

Chemical structure of calpain inhibitor XII.^,,

Figure 19

FDA-approved α-ketoamide drugs: boceprevir and telaprevir.

Figure 20

Chemical structures of potential SARS-CoV-2 M^PRO inhibitors 16–18.(59)

Figure 21

In silico designed SARS-CoV-2 M^PRO inhibitors 19–21.(64)

Figure 22

Chemical structure of the anticancer compound 22 with activity on SARS-CoV-2.

Figure 23

General mechanism of inhibition conjugate systems (α,β-unsaturated carbonyl warhead is reported as an example).

Figure 24

(a) Chemical structures of N3 and analogues 23–26.^,− (b) X-ray crystal structure of 26 in complex with SARS-CoV-2 M^PRO.

Figure 25

Chemical structures of peptidomimetic irreversible M^PRO inhibitors 27 and 28.

Figure 26

SIMR-2418, a promising nonpeptidomimetic SARS-CoV-2 M^PRO inhibitor.

Figure 27

Examples of nonpeptidomimetic SARS-CoV-2 M^PRO inhibitors with a covalent mechanism of action.^,

Figure 28

Chemical structures of ZINC database inhibitors of SARS CoV-2 M^PRO.

Figure 29

Chemical structures of natural products and analogues as potential SARS-CoV-2 M^PRO inhibitors.⁻

Figure 30

(a) Chemical structure of the flavonoid compound myricetin. (b) X-ray crystal structure of the covalent complex between myricetin and SARS-CoV-2 M^PRO (PDB code 7B3E). (c) Hypothetical mechanism of action of myricetin in inhibiting SARS-CoV-2 M^PRO.^,

Figure 31

Chemical structures of promising myricetin analogues as covalent SARS-CoV-2 M^PRO inhibitors.

Figure 32

General mechanism of action of an α-haloacetamide warhead.

Figure 33

(a) Chemical structure of the α-chloroacetamide derivative 36, developed as a SARS-CoV-2 M^PRO covalent inhibitor. (b) X-ray crystal structure of the covalent complex between derivative 36 and SARS-CoV-2 M^PRO (PDB code 7MLF).

Figure 34

Chemical structures of the dichloroacetamide, tribromoacetamide, and chloro-fluoro-acetamide derivatives 38 and 40 developed as SARS-CoV-2 M^PRO covalent inhibitors.

Figure 35

Chemical structure of α-chloroacetamide SARS-CoV-2 M^PRO inhibitor 41.

Figure 36

Chemical structures of pyrazoline-based compounds (R)-EN82, HW-2-010B, and QUB-00006-Int-07 as SARS-CoV-2 M^PRO inhibitors.^,

Figure 37

General mechanism of SARS-CoV-2 M^PRO inhibition with the nitrile warhead.

Figure 38

(a) Chemical structure of the orally bioavailable compound PF-07321332. (b) X-ray crystal structure of PF-07321332 in complex with SARS-CoV-2 M^PRO (PDB code 7VH8).

Figure 39

Chemical structure of the nitrile derivative 42.

Figure 40

Mechanism of acylation of SARS-CoV-2 M^PRO mediated by ester derivatives.

Figure 41

Chemical structures of chloropyridinyl esters GRL-0820, GRL-0920, and GRL-1720 repurposed as SARS-CoV-2 M^PRO inhibitors.^,,

Figure 42

Chemical structures of indole derivatives 43 and 44.

Figure 43

(a) Chemical structure of MAC-5576, a chloropyridinyl ester SARS-CoV-2 M^PRO inhibitor. (b) Crystal structure of the covalent complex SARS-CoV-2 M^PRO with MAC-5576 (PDB code 7JT0).

Figure 44

Chemical structures of 5-chloropyridinyl ester derivatives of nonsteroidal anti-inflammatory agents 45 and 46.

Figure 45

(a) Chemical structure of ebselen. (b) Proposed mechanism of action of ebselen with SARS-CoV-2 M^PRO. (c) Proposed mechanism for selenylation of the catalytic site of SARS-CoV-2 M^PRO. (d) Crystal structure of the selenylated cysteine¹⁴⁵ in the SARS-CoV-2 M^PRO binding site (PDB code 7BAK).⁻

Figure 46

Ebselen derivatives 50 and 51.

Figure 47

Chemical structures of phenyl-substituted ebselen derivatives 56–60.

Figure 48

Chemical structures of ebsulfur and ebselen/ebsulfur analogues 61 and 62.

Figure 49

Chemical structure of ebsulfur analogue 63.

Figure 50

Chemical structures of the promising ebsulfur derivatives 64–69.