Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Apr 7;13(1):1891.
doi: 10.1038/s41467-022-29413-2.

Development of optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for treatment of COVID-19

Hengrui Liu  1 , Sho Iketani  2   3 , Arie Zask  4 , Nisha Khanizeman  1 , Eva Bednarova  1 , Farhad Forouhar  5 , Brandon Fowler  1 , Seo Jung Hong  6 , Hiroshi Mohri  2 , Manoj S Nair  2 , Yaoxing Huang  2 , Nicholas E S Tay  1 , Sumin Lee  1 , Charles Karan  7 , Samuel J Resnick  6   8 , Colette Quinn  9 , Wenjing Li  9 , Henry Shion  9 , Xin Xia  4 , Jacob D Daniels  10 , Michelle Bartolo-Cruz  4 , Marcelo Farina  4   11 , Presha Rajbhandari  4 , Christopher Jurtschenko  9 , Matthew A Lauber  9 , Thomas McDonald  9 , Michael E Stokes  4 , Brett L Hurst  12 , Tomislav Rovis  13 , Alejandro Chavez  14 , David D Ho  15 , Brent R Stockwell  16   17
Affiliations

Development of optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for treatment of COVID-19

Hengrui Liu et al. Nat Commun. .

Abstract

The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses.

PubMed Disclaimer

Conflict of interest statement

S.I., H.L., A.Z., B.R.S., A.C., T.R., N.K., E.B., F.F., N.E.S.T., S.L., and D.D.H. are inventors on invention disclosures and patent applications submitted based on this work. B.R.S. is an inventor on additional patents and patent applications related to small molecule therapeutics, and co-founded and serves as a consultant to Inzen Therapeutics, Nevrox Limited, Exarta Therapeutics, and ProJenX Inc., and serves as a consultant to Weatherwax Biotechnologies and Akin Gump Strauss Hauer & Feld LLP. A.Z. is an inventor on additional patents and patent applications related to small molecule therapeutics, and co-founded and serves as a consultant to ProJenX Inc. C.Q., W.L., H.S., C.J., M.A.L., and T.M. are employed by Waters Corporation. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Compound 4, GC376, MAC-5576, and ebselen inhibited SARS-CoV-2 3CL protease.
a Structures of compound 4, GC376, MAC-5576, and ebselen. b The dose-dependent effects of compound 4, GC376, MAC-5576, and ebselen on the activity of 200 nM SARS-CoV-2 3CL protease were tested. The fluorogenic peptide MCA-AVLQSGFR-Lys(DNP)-Lys-NH2, corresponding to the nsp4/nsp5 cleavage site in the virus was applied as substrate. Data are plotted as the mean ± s.d., n = 3 biological replicates. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Evaluation of lead compounds for cellular antiviral potency, metabolic stability, and solubility.
a Ability of compound 4, GC376, MAC-5576, and ebselen to inhibit SARS-CoV-2 viral infection in cell culture. Stocks of SARS-CoV-2 strain 2019-nCoV/USA_WA1/2020 were propagated and titered in Vero-E6 cells. Serial dilutions of the test compound were prepared in cell media (EMEM + 10% FCS + penicillin/streptomycin), overlaid onto cells, and then virus was added to each well at an MOI (multiplicity of infection) of 0.2. Cells were incubated at 37 °C under 5% CO2 for 72 h before viral RNA extraction from the supernatant and quantification against a RNA standard by quantitative reverse-transcriptase PCR (qRT-PCR). Data are plotted as the mean ± s.d., n = 3 biological replicates. b Half-life of compound 4 and GC376 in human and mouse (CD-1) plasma and liver microsomes (with NADPH). c Solubility test (light scattering) of compound 4 and GC376 in PBS with 1% DMSO. Data are plotted as the mean ± s.d., n = 3 biological replicates. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Analogs of GC376 developed as SARS-CoV-2 3CL protease inhibitors.
Structures of GC376 and its analogs were shown, with modifications highlighted in red.
Fig. 4
Fig. 4. Co-crystal structures of GC376 analogs with SARS-CoV-2 3CL protease.
a The co-crystal structure of 3CL-EB46 complex. b The co-crystal structure of 3CL-EB48 complex. c The co-crystal structure of 3CL-SL-4-241 complex. d The co-crystal structure of 3CL-NK01-14 complex. e The co-crystal structure of 3CL-NK01-63 complex. f The co-crystal structure of 3CL-EB54 complex. g The co-crystal structure of 3CL-NK01-48 complex. h The co-crystal structure of 3CL-EB56 complex. i Alignment of the co-crystal structure of 3CL-EB46, 3CL-EB48, 3CL-EB54, 3CL-EB56, 3CL-SL4-241, 3CL-NK01-14, 3CL-NK01-48, 3CL-NK01-63, and 3CL-GC376 complex.
Fig. 5
Fig. 5. Pan-coronavirus inhibition potency of optimized GC376 analogs.
a Evaluations of GC376 analogs in a cell-based SARS-CoV, SARS-CoV-2, and MERS-CoV 3CL protease inhibitor assay. Expression of viral 3CL protease suppressed the viability of the 293T cells. Inhibitors of the protease rescue cell growth and boost the amount of crystal violet cell staining which is read on an absorbance plate reader. Cells expressing EYFP were included as control. Experiments were performed in quartet. Data are plotted as the mean ± s.d., n = 4 biological replicates. Source data are provided as a Source Data file. b Ability of EB46, EB54, and NK01-63 to inhibit viral infection in cell culture.
Fig. 6
Fig. 6. In vivo toxicity and pharmacokinetic study of NK01-63.
a Body weight change of C57BL/6 mouse treated with 20 mg/kg NK01-63 or water vehicle via intraperitoneal (IP) or oral (PO) dose for 14 consecutive days on an in vivo toxicity study. Data are plotted as the mean ± s.d., n = 10 biological replicates. Two-tailed t test were performed on weight of 14 days post initial treatment. Pns of IP = 0.07, Pns of PO = 0.68. b, c Molar concentration of NK01-63 in plasma or lung of C57BL/6 mouse 0 h, 2 h, 4 h, 8 h, or 24 h after treatment with 20 mg/kg NK01-63 via intraperitoneal (IP) or oral (PO) dose. Data are plotted as the mean ± s.d., n = 8 biological replicates. EC90 value of NK01-63 is from Caco-2 cell-based SARS-CoV-2 antiviral assay. Source data are provided as a Source Data file.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hu, B., Guo, H., Zhou, P. & Shi, Z. L. Characteristics of SARS-CoV-2 and COVID-19. Nat. Rev. Microbiol., 10.1038/s41579-020-00459-7 (2020). - PMC - PubMed
    1. Wu F, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579:265–269. - PMC - PubMed
    1. Zhou P, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–273. - PMC - PubMed
    1. Musuamba F, et al. Advanced methods for dose and regimen finding during drug development: summary of the EMA/EFPIA Workshop on Dose Finding (London 4–5 December 2014) CPT: Pharmacomet. Syst. Pharmacol. 2017;6:418–429. - PMC - PubMed
    1. Yu J, et al. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science. 2020;369:806. - PMC - PubMed

MeSH terms