Abstract 9761: Novel Modulators of Low Density Lipoprotein Receptor Metabolism

Intervention with drugs to reduce Low Density Lipoprotein-cholesterol (LDL-C) has proven to decrease the risk of subsequent cardiovascular events, including mortality. Our goal is to develop novel, small molecule, LDL-C lowering drugs by targeting the Low Density Lipoprotein Receptor (LDLR) degradation pathway, which is modulated by the protease proprotein convertase subtilisin-like kexin type 9 (PCSK9). PCSK9 has been shown to bind to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR; it enhances the degradation of the LDLR by an unknown. We successfully integrated virtual screening methods and cell based assays into a simple, efficient procedure and identified compounds that interfere with the PCSK9-LDLR interaction. Using the atomic coordinates of the crystal structure of the PCSK9/LDLR-EGF-A complex, about a million drug-like compounds were docked in silico into the region of PCSK9 where LDLR-EGF-A binds. We selected 179 top scoring compounds that showed the best fit in the binding site as well as the greatest number of interactions with PCSK9. These compounds were tested in (a) our PCSK9/LDLR dependent HEK293 cell-based recombinant assay and (b) the endogenously expressed LDLR in HepG2/PCSK9 dependent cell based assay for their ability to upregulate the LDLR. From these screening campaigns, eleven compounds consistently exhibited concentration dependent increase in the LDLR as comparable to control, with IC 50 s in the low micromolar range. Three of the top picks were shown to exhibit a 5 to 10 fold up-regulation at 1.6 μM in two cell lines. In addition, these compounds also exhibited an increase in the fluorescently labeled DiI-LDL uptake in the nanomolar range in situ . Furthermore, in a two week animal study, subcutaneous injection of 4 mg/kg of one of these compounds resulted in a 32% reduction of the total cholesterol level in mice fed a high fat/high cholesterol diet as compared to 27% cholesterol reduction obtained with 40 mg/kg Atorvastatin. To our knowledge, no small molecule antagonist against PCSK9 has been reported, only mAbs. Thus, identifying such an oral small molecule PCSK9 inhibitor represents a tremendous advance and opportunity for drug development.