Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand

Abstract

Fatty acid-induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor-receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.

Figure 1.

(A) Schematic of the affibody scaffold displayed on the surface of yeast. (B) Flow chart for the discovery and evolution of DR5 binders from the naïve ABY library. Yeast displaying the affibody population evolved for binding the extracellular domain of DR5 were labeled with mouse c-Myc antibody, followed by AF647-conjugated anti-mouse antibody as well as cellular lysate with DR5ΔCD-GFP (C) or LAG3-GFP (D) for two hours at room temperature. Affibody display and target binding were detected by flow cytometry. DR5-specific GFP signal indicates DR5-specific binding. Absence of GFP signal for AF647- cells indicates absence of non-specific DR5 binding to yeast as well as absence of truncated ABY binders.

Figure 2.

(A) Amino acid sequences of six clones from the evolved population against DR5 were aligned with the wild-type affibody to demonstrate amino acid sequence variances of the six clones. Horizontal bars: amino acids that are identical with the wild-type (B) Coomassie blue staining of soluble ABY protein. ABY variants were purified using affinity chromatography and analyzed by 4–20% SDS-PAGE gels. Open circle represents soluble affibody. (C) Binding of ABY_DR5 variants to DR5ΔCD-GFP expressing stable cells. HEK293 cells with stable expression of DR5ΔCD-GFP were incubated with 1 μM soluble ABY_DR5 variants. Binding was detected with AF647 conjugated anti-His₅ antibody by flow cytometry. Black: HEK293 stable cells; blue, cyan, green, magenta, purple and red: HEK293 stable cells treated with ABY_DR5 1–6, respectively. (D) Affinity titration of ABY_DR5–6. HEK293 cells with a stable expression of DR5ΔCD-GFP or TNFR1ΔCD-GFP or transient expression of LAG3-GFP were incubated with increasing concentrations of soluble ABY_DR5–6 Binding was detected by AF647-conjugated anti-His₅ antibody via flow cytometry. Data are presented as mean ± standard deviation of three independent experiments. The line represents the minimization of the sum of squared errors for a 1:1 binding model, which indicates K_d = 94 ± 5 nM.

Figure 3.

Lack of binding of ABY variants to DR4. (A) Affinity titration of ABY_DR5–6. HEK293 cells with a stable expression of DR5ΔCD-GFP or transient expression of DR4ΔCD-GFP were incubated with increasing concentrations of soluble ABY_DR5–6 Binding was detected by AF647-conjugated anti-His₅ antibody via flow cytometry. Data are presented as mean ± standard deviation of three independent experiments. (B) ABY_DR5–6-DR4 binding was determined by a pull-down assay with anti-His magnetic beads. Soluble ABY_DR5–6 (200 nM) was mixed with anti-His beads and incubated at 4 °C for 2 hours. The beads were then washed thrice to remove the unbound proteins. Soluble-DR4-Fc (100 nM) or Soluble-DR5-Fc (100 nM) was added to ABY_DR5–6 coated magnetic beads and rotated at 4 °C for 2–4 hours. Beads were washed thrice, and pulled-down proteins were resolved by SDS-PAGE and immunoblotted with anti-DR4 and anti-DR5 antibodies.

Figure 4:

Colocalization of membrane-bound DR5ΔCD-GFP and ABY_DR5–6 on HEK293 cell surface. The yellow color in the overlay of the red and green signals indicates colocalization of ABY_DR5–6 and DR5ΔCD-GFP. Scale bars correspond to 100 μm.

Figure 5:

ABY_DR5–6 inhibits TRAIL-induced apoptosis. (A) FACS data demonstrates surface expression of DR5 on Jurkat cells. Jurkat cells were incubated with anti-DR5 antibody, followed by AF647-conjugated mouse secondary antibody, and analyzed by flow cytometry. Red: fully labeled cells; green: cells lacking primary DR5 antibody; black: unlabeled cells. (B) ABY_DR5–6 inhibits TRAIL-induced cell death as determined by MTT assay. Jurkat cells were treated with increasing concentrations of soluble ABY_DR5–6 (1 pM - 10 μM) then stimulated with TRAIL (0.1 μg/mL; 3 nM) for 16 hours. The line represents the minimization of the sum of squared errors for a 1:1 inhibition model, which indicates IC₅₀ = 15 ± 1 nM. Data are presented as mean ± standard deviation of three independent experiments. (C) Effect of ABY_DR5–6 on TRAIL-induced FADD recruitment to DR5. Jurkat cells were incubated with 200 nM affibody then stimulated with FLAG-tagged TRAIL. TRAIL and associated molecules were immunoprecipitated on anti-FLAG-conjugated magnetic beads, resolved using SDS-PAGE gels, and subjected to western blotting using anti-DR5 and FADD antibodies. (D) Caspase-8 activity was measured in Jurkat cells treated with increasing concentrations of soluble ABY_DR5–6 (1 pM - 10 μM) and TRAIL (0.1 μg/mL). The line represents the minimization of the sum of squared errors for a 1:1 inhibition model, which indicates IC₅₀ = 160 ± 20 nM. Data are presented as mean ± standard deviation of three independent experiments.

Figure 6:

ABY_DR5–6 binds DR5 without competing TRAIL-DR5 complex formation. (A) HEK293 cells with stable expression of DR5ΔCD–GFP were incubated with TRAIL (50 nM) and TRAIL + soluble ABY_DR5–6 (200 nM). TRAIL binding was detected with rabbit anti-TRAIL antibody, followed by AF647-conjugated anti-rabbit antibody, as measured by flow cytometry. Black: HEK293 stable cells; blue: HEK293 stable cells treated with TRAIL only; and red: HEK293 stable cells treated with TRAIL and ABY_DR5–6. (B) TRAIL-DR5 binding was determined by a pull-down assay with anti-GFP magnetic beads. DR5ΔCD-GFP lysate was mixed with anti-GFP beads and incubated at 4 °C for 2 hours. The beads were then washed thrice to remove the unbound proteins. Soluble-TRAIL (50 nM) and TRAIL+ ABY_DR5–6 (200 nM) was added to DR5-GFP coated magnetic beads and rotated at 4 °C for 2–4 hours. Beads were washed thrice, and pulled-down proteins were resolved by SDS-PAGE and immunoblotted with anti-GFP, TRAIL and His₅ antibodies.

Figure 7:

Effect of ABY_DR5–6 on ligand-independent DR5-DR5 interactions was determined using live-cell TR-FRET measurements. For lifetime measurements, HEK293 cells with a stable expression of DR5ΔCD-GFP and DR5ΔCD-GFP-RFP were lifted with trypsin, washed thrice with PBS, and resuspended in PBS at a concentration of 1 million cells/mL. Then cells were treated with soluble ABY_DR5–6 (0.1 – 10 μM), BSA and non-binder, and incubated for 1 – 2 hours. After incubation cells were washed with PBS and dispensed (50 μL/well) into a 96 well glass-bottom plate. Donor lifetime was measured using a fluorescence lifetime plate reader. Note: *, **, and *** indicates statistically significant increase relative to no affibody control with p < 0.05, 0.01, and 0.001.

Figure 8:

Effect of ABY_DR5–6 on TRAIL-induced apoptosis in Huh-7 hepatocytes. Huh-7 cells grown in 96-well plates were treated with recombinant human TRAIL (0.1 μg/mL) for 16 h in the presence or absence of increasing concentrations of ABY_DR5–6 (0.001 nM - 10 μM). TRAIL-induced cell death was determined by MTT assay (A), Caspase-8 assay (B) and a mixture of cell-permeable Hoechst 33342 and impermeable Sytox Green DNA fluorescent dyes (C). Triangle and dotted lines represent TRAIL-induced cell death in the absence of affibody treatment and square represents cell death in untreated cells. Data are means ± SD of three independent experiments. All ABY_DR5–6 samples have lower death (A) and caspase-8 activity (B) than non-binder by two-tailed unpaired t test (P < 0.0001).