Self-Masked Aldehyde Inhibitors of Human Cathepsin L Are Potent Anti-CoV-2 Agents

Abstract

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, 1), for which the P1 Phe group contains a 1′-hydroxy group, effectively, an o-tyrosinyl aldehyde (Cbz-Phe-o-Tyr-CHO, 2; (Li et al. (2021) J. Med. Chem. 64, 11,267–11,287)). Compound 2 and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of Trypanosoma cruzi, results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L (Ki = 11–60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott et al. (2021) ACS Chem. Biol. 16, 642–650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC50 = 2–8 μM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC50 = 0.3–0.6 μM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound (9) inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line.

Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: An Update

“…287 Additionally, some of the selfmasked aldehyde inhibitors of cruzain, the main protease of Trypanosoma cruzi, were also tested against SARS-CoV-2 (vide infra). 288,289 Beyond aldehydes, many other well-known types of carbonyl-derived warheads have been employed to target the SARS-CoV-2 M pro , including α-ketoamides, 290 ketobenzothiazoles 291,292 and α-acyloxymethyl ketones 293 (see also section 2.6.3) but also nitriles. 294,295 However, an extensive discussion of them is out of the scope of this Perspective, and the reader is referred to the cited publications.…”

“…Further aldehyde derivatives based on the bicyclic core scaffold of the approved antiviral drugs boceprevir or telaprevir, such as 148 and 149 (Figure ), were also developed using a similar covalent design strategy to obtain a good PK profile in rats . Additionally, some of the self-masked aldehyde inhibitors of cruzain, the main protease of Trypanosoma cruzi , were also tested against SARS-CoV-2 ( vide infra ). , Beyond aldehydes, many other well-known types of carbonyl-derived warheads have been employed to target the SARS-CoV-2 M pro , including α-keto­amides, keto­benzo­thiazoles , and α-acyloxy­methyl ketones (see also section ) but also nitriles. , However, an extensive discussion of them is out of the scope of this Perspective, and the reader is referred to the cited publications.…”

Recent Advances on Targeting Proteases for Antiviral Development

“…The authors have found that 29 is a selective inhibitor of CatL, with higher inactivation rates of the latter than cathepsins B and K, and it inhibits viral entry in four host cells models (Vero E6, HeLa/ACE2, A549/ACE2 and Calu-3/2B4) with nanomolar potency. Following these results, Zhu et al aimed to develop a series of SMAIs that could inhibit both CatL and SARS-CoV-2 M pro [ 126 ] by finding a unique peptide scaffold that would be recognized by both proteases. Although none of the compounds were able to inhibit M pro directly, they found that some of the SMAIs were able to produce an inhibition of the cytopathic effect on Vero E6 and A549/ACE2 cells infected with SARS-CoV-2, indicating that a bi-functional strategy might be feasible with proper optimization, while also indicating that targeting the host’s proteases is also a possible strategy to prevent viral infection.…”

Overview of Antiviral Drug Therapy for COVID-19: Where Do We Stand?

“…Zhu J. et al investigated a potential anti-SARS-CoV-2 activity of a novel class of self-masked aldehyde inhibitors (SMAIs). They characterized the human cathepsin L inhibition of these compounds and found that propargyl analogue of K777 significantly blocked the SARS-CoV-2-induced cytopathic effect in Vero E6 and A549/ACE2 cells [ 79 ]. A therapeutic approach leading to a decreased infection rate and improved efficacy could be a synergistic block of crucial factors in the viral replication cycle.…”