A small-molecule inhibitor of lectin-like oxidized LDL receptor-1 acts by stabilizing an inactive receptor tetramer state

Abstract

The C-type lectin family member lectin-like oxidized LDL receptor-1 (LOX-1) has been object of intensive research. Its modulation may offer a broad spectrum of therapeutic interventions ranging from cardiovascular diseases to cancer. LOX-1 mediates uptake of oxLDL by vascular cells and plays an important role in the initiation of endothelial dysfunction and its progression to atherosclerosis. So far only a few compounds targeting oxLDL-LOX-1 interaction are reported with a limited level of characterization. Here we describe the identification and characterization of BI-0115, a selective small molecule inhibitor of LOX-1 that blocks cellular uptake of oxLDL. Identified by a high throughput screening campaign, biophysical analysis shows that BI-0115 binding triggers receptor inhibition by formation of dimers of the homodimeric ligand binding domain. The structure of LOX-1 bound to BI-0115 shows that inter-ligand interactions at the receptor interfaces are key to the formation of the receptor tetramer thereby blocking oxLDL binding.

Fig. 1. Cell based high throughput screening assay on LOX-1.

a Representative fluorescence microscopic pictures (n = 16) of AF647 labeled human oxLDL uptake into a CHO-K1 cell line with doxycycline inducible expression of the human LOX-1 receptor (-/+ DOX). The length of the scale bar corresponds to 20 µM. blue: Hoechst 33342, red: AF647-oxLDL b The HTS assay principle is based on the cellular internalization of AF594-labeled human oxLDL. AF594-oxLDL uptake in CHO-TREx-hLOX-1 is blocked by 30 nM mAb-LOX-1 (P value < 0.0001). The assay shows a window of factor 10 between background (-Tet+Alexa oxLDL) and full induction (+Tet+Alexa oxLDL). The error bars indicate the standard deviation of the measurements. c Representative example of a concentration response curve of BI-0115 with an IC₅₀ of 7.2 µM in the LOX-1 cellular uptake assay (n = 2 for each concentration, R² = 0.98). The chemical structure of BI-0115 is indented.

Fig. 2. Biophysical characterization of BI-0115-LOX-1 interaction.

a Characterization of small molecule binding to LOX129 by STD NMR. Aromatic region of the off-resonance spectrum showing all expected resonances of BI-0115 (labeled Off-resonance). STD spectrum (subtraction of on- and off-resonance spectrum) displays signals at the respective resonance frequencies of the small molecule indicative for binding to LOX-1 (BI-0115 STD +LOX-1). STD spectrum of BI-0115 in the absence of LOX-1 (BI-0115 STD -LOX-1) shows no signals supporting specific binding of the compound to LOX-1 in the middle row. b Surface plasmon resonance binding analysis of BI-0115 to immobilized LOX129. Binding of BI-0115 was assessed by measuring dose responsive changes (from 0.391 µM to 25 µM) in the refractive index. The Kd of 4.3 µM was determined by a fit to the change of the refractive index at equilibrium (n = 4). The figure shows a representative example of a sensorgramm and affinity fit. c ITC-Titration of 300 µM LOX129 dimer into 40 µM BI-0115 compound to determine the binding affinity (mean K_d = 6.99 µM, n = 3). The left graph presents the raw data. The integrated peak areas with the corresponding fit are shown at the right.

Fig. 3. Crystal structure of the BI-0115-LOX-1 complex.

a Two molecules of BI-0115 (yellow surface, LIG X1/LIG X2) bind to two dimers of LOX-1 CTLD. One dimer is formed by molecule A (Mol A in red) and molecule B (Mol B in salmon) and the second dimer by molecule C (Mol C in blue) and molecule D (Mol D in light blue). The amino and carboxyl-termini are indicated by N and C. b Detailed representation of the BI-0115 - LOX-1 interaction. Amino acids surrounding the binding site are shown as lines. C atoms are colored according to the LOX-1 molecule; O and N are in red and blue, respectively. c Close-up view of the asymmetric non-liganded binding site. Blue colored loop A233 to P239 of molecule C binds to the hydrophobic pocket of molecule A in red. d Superposition of multiple LOX-1 dimers. The right LOX129 C molecule has been used as reference for the superposition. The grey cylinder indicates the twofold rotation axis. The arrow at the left hand side indicates the large shift observed in the LOX129 AB dimer and the LOX143-BI-0115 AB dimer structures. e Close-up view of multiple unliganded LOX-1 monomers superposed on molecule B of the LOX143-BI-0115 complex. Residues near the ligand are highlighted.

Fig. 4. BI-0115 binding induces tetramerization in solution.

a LOX-1 tetrameric state detected at trace level without ligand. Increasing ligand concentration (a) 0 eq, (b) 2.5 eq, (c) 5 eq, (d) 10 eq induces LOX-1 tetramerization (each with n = 1). (i) (LOX-1)₂ dimer (MW: 33776 ± 1Da), (ii) (LOX-1)₄: 67565 ± 4Da, (iii) [(LOX-1)₄-BI-0115₂+ acetate]: 68202 ± 4 Da.

Fig. 5. Homology model of the tetrameric complex.

The CTLD part is based on the co-crystal structure of the BI-0115-LOX-1 complex, whereas the neck domain is modeled based on the Homer coiled-coil domain PDB ID 3CVE. The grey bar at the bottom represents the membrane. One dimer is built up by one molecule colored in red and the second molecule in salmon, the second dimer by one blue and one light blue molecule. CTLD: C-type lectin-like domain.