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

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.

Supplementary Figure 18: Sequence alignment of human C-type lectin like domains of OLR1, CLC7A, CL12B and CLC9A with residues in 5-Angstrom distance around the BI-0115 binding site highlighted as red triangles.
Supplementary Figure 19: ITC-Titration of 300 µM LOX129 dimer into buffer. The graph presents the raw data. No change of heat generation is indicated.

Experimental Part
The Examples that follow are intended to illustrate. The terms "ambient temperature" and "room temperature" are used interchangeably and designate a temperature of about 20 °C.
NMR experiments were recorded on Bruker Avance 400 MHz spectrometer at 303K. Sample BI-0115 was dissolved in 200μl DMSO-d6 and TMS was added as an internal standard and the sample BI-1580 was dissolved in 600μl DMSO-d6 and TMS was added as an internal standard. 1D-1H spectra were acquired with 30° pulse angle and a recycle delay of 2 sec with 64k number of points and a spectral width of 20.7 ppm. 1D-13C spectra were acquired with 90° pulse angle, proton broadband decoupling and a recycle delay of 4 sec, with 64k number of points and a spectral width of 276 ppm. Processing and analysis of spectra were performed with Bruker Topspin 3.2 software. The spectra were calibrated on TMS. Chemical shifts were reported in ppm on the δ scale.
HRMS data were recorded using a Waters Xevo G2 XS system with a Waters Acquity-UPLC.
Then, 20% aq. HCl is added slowly, a solid appeared. 118g product is obtained by filtration.