Dual Inhibitors of SARS-CoV-2 3CL Protease and Human Cathepsin L Containing Glutamine Isosteres Are Anti-CoV-2 Agents

Journal Article · Wed Jan 01 23:00:00 EST 2025 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/jacs.4c11620· OSTI ID:2575241

Kumar, Vivek ^[1]; Zhu, Jiyun ^[1]; Chenna, Bala C. ^[1]; Hoffpauir, Zoe A. ^[2]; Rademacher, Andrew ^[1]; Rogers, Ashley M. ^[1]; Tseng, Chien-Te ^[3]; Drelich, Aleksandra ^[3]; Farzandh, Sharfa ^[2]; Lamb, Audrey L. ^[2]; Meek, Thomas D. ^[1]

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1. Texas A & M Univ., College Station, TX (United States)
2. Univ. of Texas at San Antonio, TX (United States)
3. Univ. of Texas Medical Branch at Galveston, TX (United States)

SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P₂ position of a peptidomimetic inhibitor. At the P₁ position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P₁ position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from K_i = 0.6–18 nM (cathepsin L) and K_i = 2.6–124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC₅₀ = 0.47–15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys₁₄₅, and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P₃ position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

• Research Organization: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
• Grant/Contract Number: AC02-76SF00515
• OSTI ID: 2575241
• Journal Information: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 2 Vol. 147; ISSN 0002-7863; ISSN 1520-5126
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English