Journal: Journal of Medicinal Chemistry

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

doi: 10.1021/acs.jmedchem.5c02876

Figure Lengend Snippet: 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 serum albumin (UP2), neutravidin (UP3), streptavidin (UP4), carbonic anhydrase (UP5), α-chymotrypsin (UP6), aldolase (UP7), and ovalbumin (UP8). Binding was assessed by flow cytometry using 1 μM of soluble proteins against yeast-displayed MPs. Normalized binding/display signal intensities range from light to dark blue colors, indicating low and high titers, respectively. (d) Schematic representation of the recombinant production of GR1.1 (red circles), GR1.4 (pink circles), and GR3.1 (blue circles) as N-terminal fusions of HaloTag (gray oval) (GRF1.1, GRF1.4, and GRF3.1). (e) Schematic representation of the apparent binding affinity determination using titration on mammalian cells expressing the ectopic hACE2 receptor on the surface. Mammalian cells are incubated with varying concentrations of recombinantly produced soluble MP fusions. Binding isotherms of the three MP fusions GRF1.1 (red circles), GRF1.4 (pink circles), and GRF3.1 (blue circles) are shown. RBD protein was used as a positive control (white circles). The indicated K D app values are the results of three independent experiments and are presented as mean (dots) ± s.d. (bars). (f) Schematic representation of the binding affinity determination using surface plasmon resonance (SPR). hACE2 immobilized on the surface of a chip was incubated with varying concentrations of soluble MPs recombinantly produced as HaloTag fusions. (g) SPR sensorgram traces for the interaction of the immobilized hACE2 with “one-ring” (GRF1.1 and GRF1.4) and “two-ring” (GRF3.1) soluble MPs. Data were fitted using a 1:1 binding model. Raw data are shown as solid lines, while fit data are shown as dashed lines. (h) Plot of the K D values (nM) of RBD protein, “one-ring” (GR1.1 and GR1.4) and “two-ring” (GR3.1) MP ligands calculated using SPR (red circles), yeast surface titrations (YC, blue square), and mammalian cell titrations (MC, gray rhombus). (i) Schematic representation of the competitive binding assay of yeast-encoded MPs for binding to hACE2 in the presence (+) or absence (−) of well-known hACE2 ligands, DX600 inhibitor (left) and RBD protein (right). The determined residual fluorescence levels are reported in percentage (%). When the ligand recognizes the same hACE2 site of MP, a decrease in fluorescence is expected (red plots; “competitive binding”). In the absence of the ligand (gray plot; “no ligand”) or if the ligand binds a site of hACE2 other than the one recognized by MP (blue plot; “noncompetitive binding”), no decrease in fluorescence should be observed. ****Significant value ( P < 0.0001); *Not significant value.

Article Snippet: Yeast cells displaying the desired MP ligand were incubated for 1 h at room temperature with gentle shaking with a concentration 10-fold higher than their K D value of soluble biotinylated hACE2 alone or precomplexed with at least a 100-fold molar excess of well-known site-specific soluble ligand such as the cyclic peptide inhibitor DX600 (MedChemExpress, Monmouth Junction, NJ, USA) or the RBD protein.

Techniques: Binding Assay, Expressing, Incubation, Fluorescence, Clone Assay, Concentration Assay, Standard Deviation, Flow Cytometry, Recombinant, Titration, Produced, Positive Control, SPR Assay, Competitive Binding Assay

Journal: Journal of Medicinal Chemistry

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

doi: 10.1021/acs.jmedchem.5c02876

Figure Lengend Snippet: 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 converted to the pK i and pK D scale using pK i = −log 10 ( K i ) and pK D = −log 10 ( K D ), respectively. Higher values of pK i and pK D indicate exponentially greater potency. (f) ITC raw thermogram (differential power, μW) resulting from successive injections of GR1.4 and GR3.1.2 into the cell containing hACE2 (top). The corresponding integrated heat per injection (Δ H , kJ/mol) is plotted against the molar ratio of ligand to protein (bottom). (g) Stability of peptides GR1.1, GR1.4, GR3.1.2 and GR1.4L in human plasma at 37 °C. At each time point (0, 0.25, 0.5, 1, 2, 4, 8, 24, 48 h), the relative intensity of each peptide was estimated by LC-MS. The relative intensity at 0 h was defined as 100%. Half-lives ( t 1/2 ) were determined by nonlinear regression analysis of the mean of two technical replicates using GraphPad Prism. Data are presented as mean values ± s.d. ( n = 2).

Article Snippet: Yeast cells displaying the desired MP ligand were incubated for 1 h at room temperature with gentle shaking with a concentration 10-fold higher than their K D value of soluble biotinylated hACE2 alone or precomplexed with at least a 100-fold molar excess of well-known site-specific soluble ligand such as the cyclic peptide inhibitor DX600 (MedChemExpress, Monmouth Junction, NJ, USA) or the RBD protein.

Techniques: Activity Assay, Sequencing, Incubation, Concentration Assay, Injection, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy