De novo design of ACE2 protein decoys to neutralize SARS-CoV-2

AbstractThere is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (∼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

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Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

Science Translational Medicine ◽

10.1126/scitranslmed.abd6990 ◽

2021 ◽

pp. eabd6990

Author(s):

Sang Il Kim ◽

Jinsung Noh ◽

Sujeong Kim ◽

Younggeun Choi ◽

Duck Kyun Yoo ◽

...

Keyword(s):

B Cells ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

De Novo ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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De novo design of self-assembling helical protein filaments

Science ◽

10.1126/science.aau3775 ◽

2018 ◽

Vol 362 (6415) ◽

pp. 705-709 ◽

Cited By ~ 48

Author(s):

Hao Shen ◽

Jorge A. Fallas ◽

Eric Lynch ◽

William Sheffler ◽

Bradley Parry ◽

...

Keyword(s):

De Novo ◽

Computational Design ◽

Building Blocks ◽

Repeat Units ◽

Self Assembling ◽

Wide Range ◽

Helical Protein ◽

Monomeric Proteins

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

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De novo design of selective Sortase-A inhibitors: Synthesis, structural and in vitro characterization

Chemical Data Collections ◽

10.1016/j.cdc.2018.04.007 ◽

2018 ◽

Vol 15-16 ◽

pp. 126-133 ◽

Cited By ~ 2

Author(s):

Kranthi Raj K ◽

Pardhasaradhi Mathi ◽

Mutyala Veera Venkata Vara Prasad ◽

Mahendran Botlagunta ◽

Ravi M ◽

...

Keyword(s):

De Novo ◽

De Novo Design ◽

Sortase A ◽

In Vitro Characterization

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Peptide-Bound Dinitrosyliron Complexes (DNICs) and Neutral/Reduced-Form Roussin’s Red Esters (RREs/rRREs): Understanding Nitrosylation of [Fe–S] Clusters Leading to the Formation of DNICs and RREs Using a De Novo Design Strategy

Inorganic Chemistry ◽

10.1021/ic201529e ◽

2011 ◽

Vol 50 (20) ◽

pp. 10417-10431 ◽

Cited By ~ 26

Author(s):

Zong-Sian Lin ◽

Feng-Chun Lo ◽

Chih-Hsiang Li ◽

Chih-Hao Chen ◽

Wei-Ning Huang ◽

...

Keyword(s):

De Novo ◽

Design Strategy ◽

De Novo Design ◽

Reduced Form

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ChemInform Abstract: De Novo Design, Synthesis, and in vitro Evaluation of Inhibitors for Prokaryotic tRNA-Guanine Transglycosylase: A Dramatic Sulfur Effect on Binding Affinity.

ChemInform ◽

10.1002/chin.200222208 ◽

2010 ◽

Vol 33 (22) ◽

pp. no-no

Author(s):

Emmanuel A. Meyer ◽

Ruth Brenk ◽

Ronald K. Castellano ◽

Maya Furler ◽

Gerhard Klebe ◽

...

Keyword(s):

Binding Affinity ◽

De Novo ◽

De Novo Design ◽

Design Synthesis ◽

In Vitro Evaluation ◽

Sulfur Effect

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A defined structural unit enables de novo design of small-molecule–binding proteins

Science ◽

10.1126/science.abb8330 ◽

2020 ◽

Vol 369 (6508) ◽

pp. 1227-1233 ◽

Cited By ~ 3

Author(s):

Nicholas F. Polizzi ◽

William F. DeGrado

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Small Molecules ◽

Structural Unit ◽

De Novo ◽

Computational Design ◽

De Novo Design ◽

X Ray ◽

X Ray Crystallography ◽

Chemical Groups

The de novo design of proteins that bind highly functionalized small molecules represents a great challenge. To enable computational design of binders, we developed a unit of protein structure—a van der Mer (vdM)—that maps the backbone of each amino acid to statistically preferred positions of interacting chemical groups. Using vdMs, we designed six de novo proteins to bind the drug apixaban; two bound with low and submicromolar affinity. X-ray crystallography and mutagenesis confirmed a structure with a precisely designed cavity that forms favorable interactions in the drug–protein complex. vdMs may enable design of functional proteins for applications in sensing, medicine, and catalysis.

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De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

Science ◽

10.1126/science.abe0075 ◽

2020 ◽

Vol 370 (6521) ◽

pp. 1208-1214 ◽

Cited By ~ 4

Author(s):

Thomas W. Linsky ◽

Renan Vergara ◽

Nuria Codina ◽

Jorgen W. Nelson ◽

Matthew J. Walker ◽

...

Keyword(s):

Protein Design ◽

De Novo ◽

High Specificity ◽

Spike Protein ◽

Host Proteins ◽

Angiotensin Converting Enzyme 2 ◽

Cryo Electron Microscopy ◽

De Novo Protein Design ◽

Single Spike

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo–electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

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