Structure-activity relationship studies of Niemann-Pick type C1-like 1 (NPC1L1) ligands identified by screening assay monitoring pharmacological chaperone effect

A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as pharmacological chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this pharmacological chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure-activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.