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

Abstract

Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer-generated scaffolds were either built around an ACE2 helix that interacts with the spike receptor binding domain (RBD) or docked against the RBD to identify new binding modes, and their amino acid sequences were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concentration (IC₅₀) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC₅₀ ~ 0.16 nanograms per milliliter). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.

Fig. 1. Overview of the computational design approaches.

(A) Design of helical proteins incorporating ACE2 helix. (B) Large-scale de novo design of small helical scaffolds (top) followed by RIF docking to identify shape and chemically complementary binding modes.

Fig. 2. High-resolution sequence mapping of AHB2, LCB1, and LCB3 before sequence optimization.

(A, C, and E) (Left) Designed binding proteins are colored by positional Shannon entropy from site saturation mutagenesis, with blue indicating positions of low entropy (conserved) and red those of high entropy (not conserved). (Right) Zoomed-in views of central regions of the design core and interface with the RBD. (B, D, and F) Heat maps representing RBD-binding enrichment values for single mutations in the design model core (left) and the designed interface (right). Substitutions that are heavily depleted are shown in blue, and beneficial mutations are shown in red. The depletion of most substitutions in both the binding site and the core suggest that the design models are largely correct, whereas the enriched substitutions suggest routes to improving affinity. Full SSM maps over all positions for AHB2 and all eight de novo designs are provided in figs. S6 and S7.

Fig. 3. The optimized designs bind with high affinity to the RBD, compete with ACE2, and are thermostable.

(A) ACE2 competes with the designs for binding to the RBD. Yeast cells displaying the indicated design were incubated with 200 pM RBD in the presence or absence of 1 μM ACE2, and RBD binding to cells (y axis) was monitored with flow cytometry. (B) Binding of purified miniproteins to the RBD monitored with BLI. For LCB1 and LCB3, dissociation constants (K_d) could not be accurately estimated because of a lack of instrument sensitivity and long equilibration times below 200 pM. (C) Circular dichroism spectra at different temperatures and (D) CD signal at 222-nm wavelength, as a function of temperature. The fully de novo designs LCB1 and LCB3 are more stable than the ACE2 scaffolded helix design AHB2.

Fig. 4. Cryo-EM characterization of the LCB1 and LCB3 minibinders in complex with SARS-CoV-2 spike protein.

(A) Molecular surface representation of LCB1 bound to the SARS-CoV-2 spike ectodomain trimer viewed along two orthogonal axes (left, side view; right, top view) (B) Superimposition of the computational design model (silver) and refined cryo-EM structure (magenta) of LCB1 (using the map obtained through local refinement) bound to the RBD (cyan). (C and D) Zoomed-in views of computational model (silver) of LCB1/RBD complex overlaid on the cryo-EM structure (cyan for RBD and pink for LCB1), showing selected interacting side chains. (E) Molecular surface representation of LCB3 bound to the SARS-CoV-2 spike ectodomain trimer viewed from the side and top of the spike trimer. (F) Superimposition of the computational design model (silver) and refined cryo-EM structure (pink) of LCB3 (using the map obtained through local refinement) bound to the RBD (cyan). (G and H) Zoomed-in view of the interactions between LCB3 (pink) and the SARS-CoV-2 RBD (cyan), showing selected interacting side chains. In (A) and (E), each spike protomer is colored distinctly (cyan, pink, and yellow). For (B) and (F), the RBDs were superimposed to evaluate the binding pose deviations between designed models and refined structure of each minibinder.

Fig. 5. Neutralization of live virus by designed miniprotein inhibitors.

(A and B) Neutralization activity of (A) AHB1 and AHB2 or (B) LCB1-5 were measured with a focus reduction neutralization test. Indicated concentrations of minibinders were incubated with 100 FFU of authentic SARS-CoV-2 and subsequently transferred onto Vero E6 monolayers. AHB1, AHB2, LCB1, and LCB3 potently neutralize SARS-CoV-2, with median effective concentration (EC₅₀) values <50 nM (AHB1 and AHB2) or <50 pM (LCB1 and LCB3). Data are representative of two independent experiments, each performed in technical duplicate.