Yeast Display Technology Enables Rapid Discovery of Low-Nanomolar Macrocyclic Peptide Inhibitors of Human Angiotensin-Converting Enzyme 2

Figures

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  1. Selection of yeast-encoded MPs toward hACE2 protein. (a) General flowchart applied to identify yeast-encoded MP binders against hACE2. Five different yeast-encoded MP libraries (1–5) were pooled together and incubated with hACE2 through two rounds of MBs-based separation followed by four rounds of FACS-based selection. The biotinylated hACE2 immobilized on MBs is represented as a black circle. In FACS-based selection, the soluble biotinylated hACE2 is represented as a gray circle and the fluorescently labeled anti-HA antibody (IF) as a white circle. (b) Density plots of the polyclonal population of yeast cells encoding different MPs against hACE2 that has been enriched from 14 to 53% through four cycles (I, II, III and IV) of FACS. Each dot represents two fluorescent signals of a single yeast cell. The fluorescence intensity on the y-axis is a measure of the amount of biotinylated hACE2 bound to the surface of a yeast cell (DyLight 650, “target binding”), whereas the fluorescence intensity on the x-axis is a measure of the number of MP molecules expressed on the surface of a yeast cell (DyLight 488, “MP display”). (c) Schematic representation depicting the increase of the binding…

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  2. Characterization of yeast-encoded MPs selected toward hACE2. (a) Schematic representation of the binding affinity determination using yeast surface titrations. Yeast cells expressing the desired MP on the cell surface are incubated with varying concentrations of soluble biotinylated hACE2. The binding is reported as mean fluorescence intensity (MFI), and it is proportional to the amount of hACE2 protein bound to the MP expressed on the surface of the yeast cell. (b) Binding isotherms of three “one-ring” (GR1.1, GR1.4, and GR2.1) and one “two-ring” (GR3.1) yeast-displayed MP clones to soluble biotinylated hACE2 are shown. The apparent equilibrium dissociation constants (K D app) of each individual selected clone were determined by normalizing the MFI from the binding signal to the MFI from the display signal (y-axis) as a function of hACE2 concentration (x-axis). The indicated K D app values are the results of three independent experiments and are presented as mean (dots) ± standard deviation (s.d.) (bars). (c) Heat map indicating the percentage of residual binding of the four selected yeast-encoded MPs against eight unrelated proteins (UP1–UP8): mouse serum albumin (UP1), human…

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  3. Determination of hACE2 inhibitory activity of yeast-encoded MPs. (a) Schematic representation of the amino acid sequence and topology of the two “one-ring” GR1.1 and GR1.4, and of the three possible isomers (GR3.1.1, GR3.1.2, and GR3.1.3) of the “two-ring” GR3.1 MP. Residual activity of hACE2 enzyme incubated with “one-ring” GR1.1 (red circles) and GR1.4 (pink circles) (b) and with the “two-ring” GR3.1.1 (light gray circles), GR3.1.2 (light blue circles), and GR3.1.3 (blue circles) isomers (c). Commercially available A0773 and DX600 inhibitors were used as positive controls (white and black circles, respectively). The inhibitory constant (K i) values are reported. (d) Residual activity of hACE1 enzyme incubated with “one-ring” GR1.1 (red circles) and GR1.4 (pink circles), the “two-ring” GR3.1.2 (light blue circles) and commercially available inhibitors A0773 (white circles) and DX600 (black circles). The K i values are reported. (e) Plot depicting pK i versus the calculated pK D of GR1.1 (red circle), GR1.4 (pink circle), and GR3.1.2 (light blue circle) identified by yeast display in the current work. The K i and K D values, specified in units of molar concentration (M), are…

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  4. Crystal structure of hACE2 in complex with “one-ring” GR1.4 MP. (a) Molecular surface representation of hACE2 (white) in complex with the “one-ring” GR1.4 (pale green) shown in two different orientations (90° rotation). (b) Zoomed-in view of the GR1.4 (pale green) bound to hACE2 (white surface). Amino acid side-chains are shown as sticks and colored by atom type (carbon: pale green, oxygen: firebrick, nitrogen: sky blue, sulfur: yellow orange). (c) Detailed view of hydrogen bond network (orange dashed lines) between GR1.4 and hACE2 residues. Only the side- and main-chains of the residues involved in key interactions are shown as sticks. The complex is shown in two different orientations (d; 90° rotation). (e) Schematic representation of molecular interactions between GR1.4 and hACE2. Intermolecular hydrogen bonds are shown as red dashed lines, while intramolecular ones are represented as blue dashed lines. Bent gray lines indicate residues of GR1.4 in close contact with hACE2 (distances shorter than 4.0 Å that are nonpolar intermolecular interactions). (f) Binding isotherms of four MP mutants (GR1.6, GR1.7, GR1.8, and GR1.9) of the “one-ring” yeast-displayed GR1.4 to soluble…

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  5. Crystal structure of hACE2 in complex with “two-ring” GR3.1.2 MP. (a) Molecular surface representation of hACE2 (white) in complex with the “two-ring” GR3.1.2 (light pink) shown in two different orientations (90° rotation). (b) Zoomed-in view of GR3.1.2 (light pink) bound to hACE2 (white surface). Amino acid side-chains are shown as sticks and colored by atom type (carbon: light pink, oxygen: firebrick, nitrogen: sky blue, sulfur: yellow orange). (c) Detailed view of hydrogen bond network (orange dashed lines) and π-π stacking interactions (green dashed lines) between GR3.1.2 and hACE2 residues. Only the side- and main-chains of the residues involved in key interactions are shown as sticks. The complex is shown in two different orientations (d; 180° rotation). (e) Schematic representation of molecular interactions between GR3.1.2 and hACE2. Intermolecular hydrogen bonds are shown as red dashed lines, while π-π stacking interactions are represented as dark green dashed lines. Bent gray lines indicate residues of GR3.1.2 in close contact with hACE2 (distances shorter than 4.0 Å that are nonpolar intermolecular interactions); (f) Zoomed-in view of “one-ring” GR1.4 (pale green) and…

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  6. Structural comparison of the binding mode of yeast-encoded GR1.4 and GR3.1.2 with those identified using phage and mRNA display technologies. (a) Detailed view of the hydrogen bond network (orange dashed line) of the superimposed BCY15291 (deep blue; PDB: 8BYJ), BCY15292 (light blue: PDB: PDB: 8B9P), and GR1.4 (pale green; PDB: 9RVT) molecules bound to hACE2. The selected amino acid chains are represented as sticks and colored by atom type (oxygen = firebrick, nitrogen = sky blue, sulfur = yellow orange, BCY15291-carbon: deep blue, BCY15292-carbon: light blue, GR1.4-carbon: pale green). The complexes are shown in two orientations (b). (c) Detailed view of the hydrogen bond network (orange dashed line) of the superimposed peptide1 (wheat; PDB: 8TOQ), peptide2 (ruby; PDB: 8TOR), peptide6 (light orange; PDB: 8TOS), and GR1.4 (pale green; PDB: 9RVT) molecules bound to hACE2. The selected amino acid chains are represented as sticks and colored by atom type (oxygen = firebrick, nitrogen = sky blue, sulfur = yellow orange, peptide1-carbon: wheat, peptide2-carbon: ruby, peptide6-carbon: light orange, and GR1.4-carbon: pale green). The complexes are shown in two orientations (d). (e)…