Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19

Abstract

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins which are cleaved at specific sites by a cysteine 3C-like protease (3CL pro) in a post-translational processing step that is critical for coronavirus replication. The 3CL pro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CL pro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CL pro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19. (~450 kDa) and pp1ab (~750 kDa) that contain overlapping sequences and include a 3C-like cysteine protease (3CL pro). The function of this internally encoded 3CL pro is integral to the processing of these proteins and critical for viral replication. 7 The SARS CoV-1 3CL pro shares a high degree of structural homology and similar substrate specificity with the coronavirus 3C-like cysteine proteases of hCoV-229E and TGEV 8 , but is most similar to the SARS CoV-2 3CL pro. Specifically, the SARS CoV-1 and SARS CoV-2 share 96% identity between their respective 3CL pro sequences and 100% identity in the active site. 8 There are numerous reports of reversible cysteine protease inhibitors which include aldehydes 9-12 , thio-or oxymethylketones 13 , cyclic ketones 14 , amidomethylketones 15 , nitriles 16,17 or various 12-283,039 ± 22,586 13 Me 220 ± 0.5 14 cyc-propyl 182 ± 6 15 tert-butyl 230 ± 5 16 Ph 86 ± 3 17 4-OMe-Ph 79 ± 3 18 4-Me-Ph 87 ± 2 19 4-CN-Ph 53 ± 1 20 4-F-Ph 82 ± 3 21 4-Cl-Ph 97 ± 3 22 2,6-(Cl)2-Ph 62,993 ± 2,501 23 2,6-(F)2-Ph 12,776 ± 594 24 2-OH-4-Cl-Ph 11,525 ± 40 25 2-F, 4-CN-Ph 13,321 ± 2,309 26 2,6-(Me)2-Ph 74 ± 4 27 2,6-(MeO)2-Ph 205 ± 2 28 2-CN-Ph 17 ± 2 a See Experimental Section for details on assay methods, values were calculated from at least eight data points with at least two independent determinations.

Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development

“…Calpain inhibitor XII was from Cayman Chemicals (Ann Arbor, MI). PF-00835231 was prepared as previously outlined ( Hoffman, Kania et al 2020 ). The identity of each compound in DMSO solution was verified by LC-MS (data not shown).…”

“…PF-07304814 is a phosphate prodrug with enhanced solubility that is metabolized to the active form PF-00835231 ( Boras, Jones et al 2020 ). In vitro, the inhibitory activity of PF-00835231 is comparable to that of remdesivir against a broad range of coronaviruses ( de Vries, Mohamed et al 2020 ), with a marked preference for coronavirus proteases over human enzymes ( Hoffman, Kania et al 2020 ). GC376, originally licensed for veterinary use in feline coronavirus infections, is now considered for the treatment of COVID-19 due to its high level of potency against SARS-CoV-2 ( Ma, Sacco et al 2020 ).…”

Drug targets, mechanisms of drug action, and therapeutics against SARS-CoV-2

“…However, in vivo studies of these ligands can only yield their true efficacy against M pro . Similarly, several covalent inhibitors, such as ketone-based covalent inhibitors [69] , cysteine focussed covalent inhibitors [70] , etc. were proposed for the inhibition of M pro .…”

Multiscale Simulations of SARS-CoV-2 3CL Protease Inhibition with Aldehyde Derivatives. Role of Protein and Inhibitor Conformational Dynamics in the Reaction Mechanism

“…The plausibility of the proposed water-mediated mechanism can be confirmed comparing the structure obtained for the reaction TS with the x-ray structure of the protein with the PF-00835231 inhibitor (PDB code 6XHM). 12 This recently proposed inhibitor of the SARS-CoV-2 main protease presents a ketone group as a warhead and the aldehyde hydrogen atom has been substituted by a hydroxymethyl group. As observed in Figure 6e the hydroxyl moiety of the PF-00835231 inhibitor in the 6XHM structure occupies exactly the same position that the bridging water molecule in our TS.…”

“…Up to know, several families of inhibitors have been proposed and tested in vitro against the 3CL protease of SARS-CoV-2, including Michael acceptors, 8 a-ketoamides, 9 aldehyde derivatives 10,11 and ketones. 12 These compounds first bind into the active site of the protease forming a noncovalent complex (EI) and then react with the thiol group of the catalytic cysteine to form a stable covalent acyl-enzyme complex (E-I), see Figure 1a. The design and improvement of these compounds is usually guided by the information provided by the x-ray structure of the covalent complex.…”