Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity

Abstract

The main protease (M pro ) is a validated antiviral drug target of SARS-CoV-2. A number of M pro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 M pro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376 , these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent M pro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 M pro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.

Selective covalent targeting of SARS-CoV-2 main protease by enantiopure chlorofluoroacetamide

“…S8 † ). 38 On the other hand, the most stable pose of ( R , S )-18 was S N 2-non-reactive due to the unpreferable orientation of the C–Cl bond for the nucleophilic attack by Cys145. The rotation of the CFA unit of ( R , S )-18 could give an S N 2-reactive pose, but the resulting rotamer lacks any stabilizing interaction with the M pro active site.…”

Invalidation of dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) as SARS-CoV-2 main protease inhibitors and the discovery of PGG as a papain-like protease inhibitor

“…The Flip-GFP M pro and PL pro assays were performed as previous reported [ 15 , 23 , 24 , 37 ]. Briefly, the assay started with seeding 293T-ACE2 in 96-well, black, clear bottomed plate (Greiner, catalog No.…”

Ginkgolic acid and anacardic acid are reversible inhibitors of SARS-CoV-2 3-chymotrypsin-like protease

“…Covalent inhibitors usually use electrophilic moieties, prominently nitrile, disulfide or cyanoacrylate to react with a corresponding site in its target [ 8 ]. The majority of current reported SARS-CoV-2 3CL pro inhibitors are peptidomimetic covalent inhibitors with a reactive warhead such as ketone, aldehyde or ketoamide [ 9 ]. A highlighting milestone is Nirmatrelvir (PF-07321332), a covalent inhibitor carrying a nitrile warhead that targets SARS-CoV-2 3CL pro , which plus Ritonavir has received its first conditional authorization on 31 December 2021 for the treatment of COVID-19 in the United Kingdom [ 10 ].…”