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De novo design of picomolar SARS-CoV-2 miniprotein inhibitors

Longxing Cao, Inna Goreshnik, Brian Coventry, James Brett Case, Lauren Miller, Lisa Kozodoy, Rita E. Chen, Lauren Carter, Lexi Walls, Young-Jun Park, Lance Stewart, Michael Diamond, David Veesler, David Baker
doi: https://doi.org/10.1101/2020.08.03.234914
Longxing Cao
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
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Inna Goreshnik
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
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Brian Coventry
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
3Molecular Engineering Graduate Program, University of Washington, Seattle, WA 98195
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James Brett Case
4Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
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Lauren Miller
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
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Lisa Kozodoy
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
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Rita E. Chen
4Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
5Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
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Lauren Carter
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
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Lexi Walls
1Department of Biochemistry, University of Washington, Seattle, WA 98195
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Young-Jun Park
1Department of Biochemistry, University of Washington, Seattle, WA 98195
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Lance Stewart
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
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Michael Diamond
4Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
6The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO 63110, USA
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David Veesler
1Department of Biochemistry, University of Washington, Seattle, WA 98195
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David Baker
1Department of Biochemistry, University of Washington, Seattle, WA 98195
2Institute for Protein Design, University of Washington, Seattle, WA 98195
7Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195
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  • For correspondence: dabaker@uw.edu
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Abstract

We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC50 values ranging from 35 pM to 35 nM; the most potent of these — 56 and 64 residue hyperstable proteins made using the second approach — are roughly six times more potent on a per mass basis (IC50 ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted August 03, 2020.
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De novo design of picomolar SARS-CoV-2 miniprotein inhibitors
Longxing Cao, Inna Goreshnik, Brian Coventry, James Brett Case, Lauren Miller, Lisa Kozodoy, Rita E. Chen, Lauren Carter, Lexi Walls, Young-Jun Park, Lance Stewart, Michael Diamond, David Veesler, David Baker
bioRxiv 2020.08.03.234914; doi: https://doi.org/10.1101/2020.08.03.234914
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De novo design of picomolar SARS-CoV-2 miniprotein inhibitors
Longxing Cao, Inna Goreshnik, Brian Coventry, James Brett Case, Lauren Miller, Lisa Kozodoy, Rita E. Chen, Lauren Carter, Lexi Walls, Young-Jun Park, Lance Stewart, Michael Diamond, David Veesler, David Baker
bioRxiv 2020.08.03.234914; doi: https://doi.org/10.1101/2020.08.03.234914

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