- Hunt, Andrew C;
- Case, James Brett;
- Park, Young-Jun;
- Cao, Longxing;
- Wu, Kejia;
- Walls, Alexandra C;
- Liu, Zhuoming;
- Bowen, John E;
- Yeh, Hsien-Wei;
- Saini, Shally;
- Helms, Louisa;
- Zhao, Yan Ting;
- Hsiang, Tien-Ying;
- Starr, Tyler N;
- Goreshnik, Inna;
- Kozodoy, Lisa;
- Carter, Lauren;
- Ravichandran, Rashmi;
- Green, Lydia B;
- Matochko, Wadim L;
- Thomson, Christy A;
- Vögeli, Bastian;
- Krüger, Antje;
- VanBlargan, Laura A;
- Chen, Rita E;
- Ying, Baoling;
- Bailey, Adam L;
- Kafai, Natasha M;
- Boyken, Scott E;
- Ljubetič, Ajasja;
- Edman, Natasha;
- Ueda, George;
- Chow, Cameron M;
- Johnson, Max;
- Addetia, Amin;
- Navarro, Mary Jane;
- Panpradist, Nuttada;
- Gale, Michael;
- Freedman, Benjamin S;
- Bloom, Jesse D;
- Ruohola-Baker, Hannele;
- Whelan, Sean PJ;
- Stewart, Lance;
- Diamond, Michael S;
- Veesler, David;
- Jewett, Michael C;
- Baker, David
New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.