Inhibiting amyloid-β cytotoxicity through its interaction with the cell surface receptor LilrB2 by structure-based design

Abstract

Inhibiting the interaction between amyloid-β (Aβ) and a neuronal cell surface receptor, LilrB2, has been suggested as a potential route for treating Alzheimer's disease. Supporting this approach, Alzheimer's-like symptoms are reduced in mouse models following genetic depletion of the LilrB2 homologue. In its pathogenic, oligomeric state, Aβ binds to LilrB2, triggering a pathway to synaptic loss. Here we identify the LilrB2 binding moieties of Aβ (¹⁶KLVFFA²¹) and identify its binding site on LilrB2 from a crystal structure of LilrB2 immunoglobulin domains D1D2 complexed to small molecules that mimic phenylalanine residues. In this structure, we observed two pockets that can accommodate the phenylalanine side chains of KLVFFA. These pockets were confirmed to be ¹⁶KLVFFA²¹ binding sites by mutagenesis. Rosetta docking revealed a plausible geometry for the Aβ-LilrB2 complex and assisted with the structure-guided selection of small molecule inhibitors. These molecules inhibit Aβ-LilrB2 interactions in vitro and on the cell surface and reduce Aβ cytotoxicity, which suggests these inhibitors are potential therapeutic leads against Alzheimer's disease.

Figure 1. The ¹⁶KLVFFA segment of Aß binds to LilrB2 D1D2.

ELISA-based interaction assays of Aß42 and its constituent segments. LilrB2 D1D2 (black bars) or bovine serum albumin as a negative control (BSA, white bars) was immobilized on ELISA plates, and incubated with Aß segments at concentrations shown. The unbound segments were washed off and the amounts of bound Aß segments were measured by the Aß specific antibody 6E10 and quantified by absorbance at wavelength 450 nm (OD450, left panel), or measured by the fluorescence signal of fluorescein (FITC) conjugated to the amino-termini of the segments and quantified by fluorescence units (right panel). Notice that for Aß_1–42, Aß_1–21 and Aß_16–21-TR, the amounts of Aß segments bound to LilrB2 D1D2 were significantly higher than that to BSA, indicating interaction between these segments and LilrB2 D1D2. The absence of KLVFFA from the weak binder Aß_1–15, as well as its presence in the stronger binders Aß_1–21, and Aß_16–21-TR (sequence KLVFFAPDGKLVFFA), indicate ¹⁶KLVFFA is the key segment of Aß that binds to LilrB2. Segment sequences are shown in Supplementary Table 1. Data are means ± SD (n=3 independent experiments). Two-sided t tests were performed and detailed statistical analyses are reported in Supplementary Table 4. *, p<0.05; **, p<0.005; ***, p<0.0005, n.s., not significant; conc, concentration.

Figure 2. Crystal structure of LilrB2 D1D2 complexed with benzamidine.

a, Overview of the structure of LilrB2 D1D2 (shown in surface model, colored by hydrophobicity) complexed with benzamidine (Ben 1 to 4, shown in sticks). The chemical structure of benzamidine is shown at the top right corner. The black dashed line between Ben 3 and Ben 4 represents 7.5 Å. Hydrophobicity ranges from −1.7 (hydrophilic) to +3.7 (hydrophobic). Notice that the binding pockets of Ben 3 and Ben 4 are located at the groove between LilrB2 domains D1and D2, and the groove has an extended hydrophobic surface. b and c, detailed interaction of Ben 3 (b) and Ben 4 (c) with LilrB2. LilrB2 is shown as a cartoon and the side chains of the residues involved in benzamidine binding are shown as sticks. The black dashed lines represent distances between 2.4 Å and 4.8 Å.

Figure 3. Mutagenesis studies and Rosetta docking validate the Aß binding sites on LilrB2.

a, ELISA-based interaction assays using wild type LilrB2 D1D2 (WT) or designed mutants. The same amount of LilrB2 D1D2 WT (blue bars), D36G (red bars), V38W (green bars) and N168W (purple bars), as well as bovine serum albumin (BSA, white bars) was immobilized on an ELISA plate (loading control see Supplementary Fig. 4), and incubated with Aß_1–42 or Aß_16–21-TR at indicated concentrations. The amounts of bound Aß_1–42 were measured by antibody 6E10 and quantified by absorbance at wavelength 450 nm (OD450, left panel); the amounts of bound Aß_16–21-TR were measured by fluorescence signal of fluorescein and quantified by fluorescence units (right panel). Data are means ± SD (n=3 independent experiments, ***p<0.0005, ANOVA test); conc, concentration. For detailed statistical analysis see Supplementary Table 4. b-d, Model of two KLVFFA peptides binding to LilrB2 D1D2 calculated by Rosetta docking. In this model Phe from one KLVFFA chain and Phe^19’ from another chain bind to Ben 3 (d, left panel) and Ben 4 (d, right panel) pockets respectively. Three residues tested in mutagenesis studies (Asp³⁶, Val³⁸ and Asn¹⁶⁸) were used as restraints in Rosetta docking. Residue colors correspond to the key given in panel (a).

Figure 4. Selected small molecules inhibit the Aß-LilrB2 interaction in vitro.

a, immunoprecipitation assays of Aß42 with (black bars) or without (white bar) LilrB2 D1D2. 1 μM of Aß42 and 100 nM of LilrB2 D1D2 were mixed with 5 μM of Aß-LilrB2 inhibitors (ALI #1–12) or equal amounts of DMSO (vehicle) and the amount of bound Aß42 was quantified by ELISA. Data are presented as percentages relative to controls in which LilrB2 and vehicle was added. Data are means ± SD (n=3 independent experiments, ***p<0.0005, ANOVA test). For detailed statistical analysis see Supplementary Table 4. b, same immunoprecipitation assays using multiple concentrations of ALI6 (left) and ALI10 (right). ELISA absorbance values of samples without LilrB2 were subtracted as a background from those of samples with LilrB2. The data are presented as percentages relative to the samples with LilrB2 and vehicle. The percentage values of samples with inhibitors are plotted against the concentration of inhibitors. The name and chemical structure of inhibitors is shown on the top of each panel. c, Docking models of ALI6 (upper panel) and ALI10 (lower panel) binding to Ben 3 and 4 pockets. Residues involved in benzamidine binding are shown as stick models. d, K_i and IC₅₀ values calculated from the data are shown in (b) and Supplementary Fig. 6. In immunoprecipitation assays shown in (b) and (d), data are mean ± SD, n=3 independent experiments.

Figure 5. Selected inhibitors block LilrB2 induced cell attachment and inhibit toxicity of Aß.

a, Fluorescent images of HEK293T cells transfected with LilrB2-mRFP or mRFP (red), and treated with 500 nM fluorescein conjugated to Aß42 (FITC-Aß, green) and 10 μM selected Aß-LilrB2 inhibitors (or equal amounts of DMSO as vehicle control). DAPI, 4’, 6-diamidino-2-phenylindole. b, Quantification of FITC-Aß42 binding represented in (a). Aß42 binding was quantified as integrated intensity of green fluorescence in each well, normalized to LilrB2 expression level quantified as integrated intensity of red fluorescence in the same well (or normalized to cell confluency for cells transfect with mRFP), and then presented as a percentage relative to the controls, which are LilrB2-mRFP transfected HEK293T cells treated with vehicle. Data are means ± SD (n=4 independent experiments, **p<0.005, ***p<0.0005, ANOVA test). c, Cell viability (MTT) assays show that ALI6 reduces the toxicity of Aß42. HEK293T cells transfected with LilrB2-mRFP were treated with indicated concentrations of ALI6 or vehicle control, and then 500 nM of oligomeric Aß42 or PBS control was added. Cell viability is shown as a percentage relative to controls in which only PBS and vehicle are added. Data are means ± SD (n=3 independent experiments, **p<0.005, ***p<0.0005, ANOVA test). For detailed statistical analysis see Supplementary Table 4.

Figure 6. Validation of ALI6 using primary neurons.

a, Bright field and fluorescence images of primary neurons treated with 500 nM FITC-Aß (green) and 10 μM ALI6 (or equal amounts of DMSO). b, Quantification of FITC-Aß42 binding represented in (a). Aß42 binding was quantified as integrated intensity of green fluorescence in each well, normalized to cell confluency in the same well, and then presented as a percentage relative to cells treated with FITC-Aß42 and DMSO (**p<0.005, ANOVA test). c, Bright field and fluorescence images of primary neuron cells treated with 300 nM Aß42 and 5 μM ALI6 or equal amounts of DMSO, or treated with PBS and DMSO as vehicle control. Cell viability was measured by TUNEL assays and dead cells are shown as red puncta. d, Quantification of TUNEL cell viability assays. Cell viability is shown as a percentage of cell death calculated as the number of red puncta divided by the number of blue puncta (Hoechst stain) (***p<0.0005, two-sided t test). e, Primary neuron cells were treated with 150 nM Aß42 with 3 μM ALI6 or equal amounts of DMSO, and cofilin signaling levels were analyzed by Western blotting (left). Anti-Tubulin β−3 antibody detects neuronal tubulin and was used as a loading control. Quantification of cofilin phosphorylation (right) was calculated as the intensity of phosphorylated cofilin band divided by the intensity of cofilin band, and was normalized to the cells treated with PBS and DMSO (vehicle control) (**p<0.005, two-sided t test). All Data are means ± SD (n=4 independent experiments). For detailed statistical analysis see Supplementary Table 4.