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A Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2

J. Trae Hampton, Tyler J. Lalonde, Jeffery M. Tharp, Yadagiri Kurra, Yugendar R. Alugubelli, Christopher M. Roundy, Gabriel L. Hamer, Shiqing Xu, Wenshe Ray Liu
doi: https://doi.org/10.1101/2022.07.06.498864
J. Trae Hampton
aTexas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
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Tyler J. Lalonde
aTexas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
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Jeffery M. Tharp
aTexas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
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Yadagiri Kurra
aTexas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
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Yugendar R. Alugubelli
aTexas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
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Christopher M. Roundy
bDepartment of Entomology, Texas A&M University, College Station, TX 77843, USA
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Gabriel L. Hamer
bDepartment of Entomology, Texas A&M University, College Station, TX 77843, USA
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Shiqing Xu
aTexas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
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Wenshe Ray Liu
aTexas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
cInstitute of Biosciences and Technology and Department of Translational Medical Sciences, College of Medicine, Texas A&M University, Houston, TX 77030, USA
dDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA
eDepartment of Molecular and Cellular Medicine, College of Medicine, Texas A&M University, College Station, TX 77843, USA
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  • For correspondence: wsliu2007@tamu.edu
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ABSTRACT

Using the regioselective cyanobenzothiazole condensation reaction with the N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and ACE2, the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2 showed that one macrocyclic peptide is highly potent against viral reproduction in Vero E6 cells with an EC50 value of 3.1 μM. The AlphaLISA-detected IC50 value for this macrocyclic peptide was 0.3 μM. The current study demonstrates that two kinetically-controlled reactions toward N-terminal and internal cysteines, respectively, are highly effective in the construction of phage-displayed macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike epitopes is a promising methodology in the identification of peptidyl antivirals.

  • Phage display
  • macrocyclic peptides
  • Asymmetric cyclization
  • SARS-CoV-2
  • antiviral

Competing Interest Statement

The authors have declared no competing interest.

  • ABBREVIATION

    S
    Spike
    RBD
    receptor binding domain
    ACE2
    angiotensin converting enzyme 2
    ORF
    open reading frame
    CBT
    cyanobenzothiazole
    CAmCBT
    N-chloroacetyl 2-chloro-N-(2-cyanobenzo[d]thiazol-6-yl)acetamide
    eGFP sfGFP
    superfolder green fluorescent protein
    TEV
    tobacco etch virus
    TCEP
    triscarboxyethylphosphine
  • Copyright 
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    Posted July 06, 2022.
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    A Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2
    J. Trae Hampton, Tyler J. Lalonde, Jeffery M. Tharp, Yadagiri Kurra, Yugendar R. Alugubelli, Christopher M. Roundy, Gabriel L. Hamer, Shiqing Xu, Wenshe Ray Liu
    bioRxiv 2022.07.06.498864; doi: https://doi.org/10.1101/2022.07.06.498864
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    A Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2
    J. Trae Hampton, Tyler J. Lalonde, Jeffery M. Tharp, Yadagiri Kurra, Yugendar R. Alugubelli, Christopher M. Roundy, Gabriel L. Hamer, Shiqing Xu, Wenshe Ray Liu
    bioRxiv 2022.07.06.498864; doi: https://doi.org/10.1101/2022.07.06.498864

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