Fig. 1. Design and characterization of de novo ACE2 decoys.

(A) ACE2 (gray) and its binding motifs (H1 19-52, orange; H2 55-84, green; EE3 346-360, blue) in complex with SARS-CoV-2 RBD (pink). Three starting structures were simultaneously used as targets (see main text); 6VW1 is shown. (B) De novo secondary structure elements (magenta) were computationally generated to stabilize H1, H2, and EE3. Seven combinations of secondary structure elements were considered. Circles are α-helices, triangles are β-sheets, filled circles are helices oriented forward, and empty circles are helices oriented backward. We used Rosetta to generate fully connected backbones (using the “protein_mimic_designer” algorithm) and amino acid sequences predicted to fold into the target structure. In all cases, the binding interface of ACE2 with the SARS-CoV-2 RBD was preserved intact (see the materials and methods). (C) Automatic computational filtering based on eight metrics selected the best candidates. The RMSD of the binding motifs to ACE2 was also used as a quality check. The dots indicate the mean computational score for each design scored against the three target RBD structures. Designs selected for experimental testing are shown in black. Our best design, CTC-445, is shown in red. The blue boxes indicate the filtering thresholds (see the materials and methods). (D) Designs that passed filtering were subjected to biased forward folding simulations (see the materials and methods), here shown for CTC-445, including the unsalted biased simulation (brown), the native-salted simulation (orange), and relaxation (blue). (E) The top 196 designs were selected for yeast display screening using a combination of Rosetta score per residue, the ddG Rosetta filter, and the folding simulations (see the materials and methods). The designs were individually assessed for specific binding to SARS-CoV-2 spike RBD (Fc fusion, 200 nM). The plot for CTC-445 is shown. (F) CTC-445 was recombinantly expressed and purified by affinity chromatography (see the materials and methods). Analytical size exclusion chromatography (SEC) for CTC-445 revealed the presence of oligomeric species. (G and H) CTC-445 was optimized by directed evolution and rational combination of the observed favorable mutations (G), leading to CTC-445.2 (SEC), which is mainly monomeric in solution (H) and ~1000× more potent to compete with ACE2 than its parent [see (G)]. We further optimized the potency of our molecule by generating a bivalent version named CTC-445.2d. (I) Potency of designs to outcompete binding of SARS-CoV-2 RBD to ACE2, as measured by competition enzyme-linked immunosorbent assay (ELISA) using a constant concentration of 0.4 nM ACE2. (J) Timeline of the de novo protein design and optimization pipeline. Timewise, green indicates phases that we believe were performed optimally, red indicates those that can potentially be avoided in future efforts, and yellow indicates phases that can potentially be expedited by using more advanced and/or automated methods for gene synthesis, cloning, and high-throughput screening.