Targeting Sars-Cov-2 main protease in the treatment of COVID-19: Focus on covalent inhibition

The Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic b-coronavirus, that emerged in late 2019, leading to the global pandemic known as COronaVIrus Disease 2019 (COVID-19). This disease poses a significant threat to both human health and public safety. Despite the remarkable contribution of various vaccines in containing the COVID-19 pandemic, the application and ongoing research of new pharmacological antiviral therapies can help to manage the progression of viral infections, particularly in severe cases in "fragile" patients. Antiviral strategies for the treatment of SARS-CoV-2 infection continue to be a topic of great interest and importance to the scientific community. Among the druggable targets against SARSCoV-2, the main protease (Mpro) plays a crucial role in the processing of the viral polyproteins into functional units necessary for viral replication.5 The ability of this nonstructural protein to recognize the glutamine (Gln) cleavage site is unique and not found in any other human protease. This distinct feature reduces the likelihood of off-target effects, making Mpro an attractive drug target. Structurally, Mpro is a homodimeric cysteine protease. Each protomer (A and B) consists of 306 amino acids, divided into 3 domains. The catalytic domains for each homodimer consist of β-sheets forming a substrate binding cleft, the C-terminal domain involves α-helices that function as dimerization platforms by interacting with N-terminal residues of the second protomer. The binding pocket is organized into four subsites, S1’, S1, S2, and S3/S4, which are occupied by the P1’, P1, P2, and P3 portions of the native polyproteins, respectively.2,3 In region S1’, between domains I and II, is located the catalytic site, characterized by the catalytic dyad (Cys145 and His41). During the hydrolysis of the peptide bond, His41 activates the thiol side chain of Cys145 by deprotonation, with subsequent stabilization of the adduct by the so-called “oxyanion hole”. The S1 subregion is highly specific for the glutamine residue. The S2 subregion is highly plastic and consists of hydrophobic amino acids. The S3/S4 are particularly exposed to the solvent.3 Covalent inhibition of the Cys145 residue of SARS-CoV-2 Mpro with selective antiviral drugs can disrupt the replication process and effectively stop SARS-CoV-2 infection, without affecting human catalytic pathways. Compared to conventional non-covalent agents, the covalent inhibitors provide better efficacy, higher potency, longer residence time in the receptor binding site, sustained pharmacological effect, and the ability to overcome resistance.4-6 The overall efficiency of covalent inhibition is described by the second-order rate constant Kinact/Ki: this value combines the affinity of the initial reversible interaction and the maximum potential rate of covalent bond formation. In the development of covalent inhibitors, it is crucial to achieve an exergonic process for the formation of the enzymeinhibitor complex (E-I), along with minimizing the activation energy barriers. Additionally, the energy barriers at the transition from E-I back to the reactants (E:I) control whether the inhibition is reversible or irreversible. The right balance between irreversibility and reversibility is essential to optimize both the efficacy and the safety profiles of the inhibitors.3,5,7 From the perspective of covalent drugs, this presentation aims to provide an overview of the most representative covalent inhibitors of SARS-CoV-2 Mpro. The Protein Data Bank (PDB) currently lists more than two hundred structures of covalently bound competitive Mpro inhibitors, providing a rich data set for structural and mechanistic analyses. Among the molecules studied, the dipeptide and tripeptide mimetic compounds represent the largest class described so far in the literature due to their intrinsic ability to mimic the native polyprotein of SARS-CoV-2. Following the Schechter-Berger notation, the main structural moieties P1’, P1, P2, P3 of the peptide mimetic covalent inhibitors are correlated with the SARS- CoV-2 Mpro sub-regions (S1’, S1, S2, S3/S4). A successful covalent SARSCoV-2 Mpro binder must meet specific structural requirements: a reactive electrophilic warhead at P1’ to covalently bind the catalytic Cys145; as P1 moiety, a recurring fivemembered g-lactam ring analogous to glutamine, to recognize the Gln cleavage site; a hydrophobic P2 (as examples: leucine isopropyl, cyclopropyl, benzyl and bicyclic proline) and a generally bulky P3 moieties (such as benzyl, carboxy benzyl and indole groups), which are projected into the S2 and S3/S4 pockets, establishing hydrophobic interactions and conserved H-bounds with Glu166 backbone, His163 side chain, and/or Cys145 and Gly143 backbones (the oxyanion hole). The studied SARS-CoV-2 Mpro covalent inhibitors will be classified into different categories according to the type of their electrophilic warhead (aldehyde, ketone, a-ketoamide, Michael acceptor, nitrile) and the biological data, such as antiviral effect in vitro and in vivo results, will be examined. Most of the SARS-CoV-2 Mpro inhibitors provides IC50 and EC50 values in the low micromolar/nanomolar range, showing a good correlation with the available values of the equilibrium-binding constants Ki and Kinact/Ki. Particular attention will be paid to GC-373 (aldehyde warhead) and its pro-drug GC376. Originally studied as veterinary drugs,9 these two compounds were later repurposed as new anti-COVID-19 agents, showing potent inhibitory activity on SARS-CoV-2 Mpro. Coronastat (NK01-63), a GC- 376 analogue, with a CF3 substituent on the aromatic ring at the P3 moiety, demonstrates improved inhibitory and drug-like properties, with no significant toxicity, high metabolic stability and high concentration in plasma and lung after both oral and intraperitoneal administration.10 PF- 00835231, with a hydroxymethyl-ketone warhead, exhibits interesting values for IC50 and Ki (6.9 nM and 0.27 nM, respectively) against the target protein. Compared to GC-376 or other anti-SARS CoV-2 agents such as the adenosine analogue remdesivir, PF-00835231 shows similar or higher potency, low cytotoxicity, and reasonable tolerability in in vitro model of human airway epithelium (HAEC).11,12 However, the clinical use of PF-00835231 is limited due to its poor solubility and bioavailability. To overcome these limitations, the phosphate pro-drug PF-07304814 (lufotrelvir) was developed through a lead optimization process. Lufotrelvir exhibits comparable antiviral and Mpro inhibitory activity to PF-00835231 but better solubility and pharmacokinetics in in vivo animal models. The obtained Ki value (174 nM) of lufotrelvir is 644-fold higher than that of PF- 00835231; this long-lasting inhibition makes lufotrelvir an irreversible covalent competitive inhibitor of SARS-CoV-2 Mpro.13 PF-07321332 (nirmatrelvir), a peptidomimetic derivative with a nitrile warhead, is the first orally bioavailable covalent SARS-CoV-2 Mpro inhibitor and is currently approved in combination with ritonavir (PAXLOVID) in the treatment of COVID-19. Its less peptidomimetic structure (fewer H-bond donors and lower polarity compared to the parent compound lufotrelvir) and the inclusion of a trifluoroacetamide moiety ensure excellent intestinal barrier permeation and oral bioavailability.14,15 In addition, we will analyze a few examples of non-peptidomimetic SARS-CoV-2 Mpro covalent inhibitors: natural compounds with Michael acceptor warhead and molecules containing an electrophilic selenium atom. In particular, the selenium-heterocyclic compound ebselen, previously studied as an antioxidant and anti-inflammatory agent,16 is one of the most interesting examples of repurposing of investigational drugs against SARSCoV- 2. Ebselen shows remarkable IC50 values against Sars Cov 2 Mpro, forming a covalent adduct with Cys145 via a two-step mechanism of action: a selenyl sulfide bond followed by the irreversible selenylation of the cysteine.16 Based on the data presented, GC-376, nirmatrelvir, lufotrelvir, ebselen and the newgeneration nitrile warhead compounds represent promising lead candidates for the future development of more effective covalent inhibitors against Sars Cov 2 Mpro.