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A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals

Journal Article · · European Journal of Medicinal Chemistry
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  1. Texas A & M Univ., College Station, TX (United States)
  2. Sorrento Therapeutics, Inc. San Diego, CA (United States)
  3. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
  4. Texas A & M Univ., College Station, TX (United States); Texas A & M Univ., Houston, TX (United States)
+ Show Author Affiliations
Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and 293T cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and cytotoxicity and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a β-(S-2-oxopyrrolidin-3-yl)-alanyl (Opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. Here, the P1 Opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency to inhibit ectopically expressed MPro in human 293T cells. In general, inhibitors with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to cytotoxicity tests on 293T cells and antiviral potency tests on three SARS-CoV-2 variants. They all have relatively low cytotoxicity and high antiviral potency with EC50 values around 1 μM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.
Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS)
Sponsoring Organization:
National Institutes of Health (NIH); Robert A. Welch Foundation; USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
2422634
Alternate ID(s):
OSTI ID: 1875748
Journal Information:
European Journal of Medicinal Chemistry, Journal Name: European Journal of Medicinal Chemistry Journal Issue: C Vol. 240; ISSN 0223-5234
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
English

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Figures / Tables (6)

Figure 1(p. 5)figure Figure 1
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Figure 5(p. 17)figure Figure 5
Table 1(p. 23)table Table 1

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Related Subjects

60 APPLIED LIFE SCIENCES
COVID-19
Carbamate
Covalent inhibitor
Main protease
Pharmacology & Pharmacy
SARS-CoV-2