3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials

Abstract

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pK_a of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases.

Keywords: 3CLpro; SARS-CoV-2; novel mechanism of catalysis; preclinical and clinical trials; triad.

Figure 1

3CLpro structure and function: (a) The three-dimensional structure of 3CLpro homodimer colored based on chain (PDB ID 7WOF) [31]; (b) 3Clpro monomer has three distinct domains: I, II, and III. Domains I (residues 10–99) and II (residues 100–182) have six antiparallel β-strands, forming a stage for the catalytic site [46,47]. Meanwhile, Domain III (residues 198–303) unveils a captivating structure featuring a globular cluster of five α-helices, steering 3CLpro dimerization regulation through salt bridge interactions. (c) The substrate binding site of 3CLpro is a composition of the subsites S1, S1’, S2, and S3/S4, (d) following the Schecter–Berger nomenclature for proteases. 3CLpro’s active site features S1’, S1, S2, and S3/S4 subsites, with Pis and Pi’s denoting substrate positions. The S1–S1’ interface marks the cleavage site, initiating numbering. X-ray crystal structure studies confirm the subsites S1’, S1, S2, and S3/S4 [44,45]. The S1 subsite (F140, C145, H163, E166) ensures structural stability and substrate recognition, featuring key π–π interactions. S1’ (T25, T26, H41, L27, N142, G143) is exposed to the aqueous environment, accommodating smaller side chains of the substrate. S2 (M49, Y54, P52, H164, M165, D187, R188, Q189) is highly hydrophobic, and S3/S4 (M165, E166, L167, P168, F185, T190, A191) completes the hydrophobic subsite composition [44,45].

Figure 2

Proposal of a novel mechanism of catalysis for 3CLpro: (a) Domains I, II, and III are denoted as DI, DII, and DIII. (b) Between domains I and II lies the active site, where the proposed catalytic triad H41, C145, and D187 is located. (c) The proposed mechanism outlined in this review delineates two distinct phases: acylation (step I) followed by deacylation (step II), wherein the roles of specific residues mirror those of the catalytic triad observed in 3CLpro. In the acylation step, the active site of the main protease, Cys145 functions as a nucleophile. Initially, His41 serves as a base, taking a Cys145 proton, thereby activating it as a nucleophile. Next, the nucleophilic Cys145 initiates an attack on the carbon atom of the carbonyl group of the glutamine of the substrate, forming a tetrahedral intermediate. This enzymatic action leads to the cleavage of the peptide bond, resulting in the release of a substrate fragment with an alcohol group and the formation of an acyl bond between Cys145 and the residual fragment of the substrate. The deacylation step (step II) is the subsequent phase, where a water molecule assumes the role of a nucleophile and His41 functions as a base activating water as a nucleophile. Water makes a nucleophilic attack on the carbon atom of the acyl group bound to Cys145. Consequently, the acyl bond is cleaved, and the active form of the main protease is regenerated. Simultaneously, the second fragment with a carbonyl group of the substrate is released, thereby completing the enzymatic reaction. Asp187 may participate in modulating the pK_a of His41, allowing His41 to act as a base in the acylation and deacylation phases.

Figure 3

Pharmacodynamics optimization for PF-00835231. Biological activities (IC₅₀ and/or K_i and EC₅₀) are shown for PF-00835231 and PF–7304814.

Figure 4

Pharmacokinetic optimization for α-ketoamide derivatives. Biological activities (IC₅₀ and EC₅₀) are shown for compounds 11r, 13a, 13b, and 14b. ND = not determined.