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.     

The Physical and Genetic Organization of A Viral Genom

Membrane proteins that bind and transport lipids face special challenges. Since lipids typically have low water solubility, both accessibility of the substrate to the protein and delivery to the desired destination are problematical. The amphipathic nature of membrane lipids, and their relatively large molecular size, also means that these proteins must possess substrate-binding sites of a different nature than those designed to handle small polar molecules. This review considers two integral proteins whose function is to bind and transfer membrane lipids within or across a membrane. The first protein, MsbA, is a putative lipid flippase that is a member of the ATP-binding cassette (ABC) superfamily. The protein is found in the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria such as E. coli, where it is proposed to move lipid A from the inner to the outer membrane (OM) leaflet, an important step in the lipopolysaccharide biosynthetic pathway. Cholesterol is a major component of the plasma membrane in eukaryotic cells, where it regulates bilayer fluidity. The other lipid-binding protein discussed here, mammalian NPC1 (Niemann-Pick disease, Type C1), binds cholesterol inside late endosomes/lysosomes (LE/LY) and is involved in its transfer to the cytosol as part of a key intracellular sterol-trafficking pathway. Mutations in NPC1 lead to a devastating neurodegenerative condition, Niemann-Pick Type C disease, which is characterized by massive cholesterol accumulation in LE/LY. The accelerating pace of membrane protein structure determination over the past decade has allowed us a glimpse of how lipid binding and transfer by membrane proteins such as MsbA and NPC1 might be achieved.     

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.     

Lipid-mediated motor-adaptor sequestration impairs axonal lysosome delivery leading to autophagic stress and dystrophy in Niemann-Pick type C

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Structure–activity relationship study of non-steroidal NPC1L1 ligands identified through cell-based assay using pharmacological chaperone effect as a readout

Niemann-Pick type C1-like 1 (NPC1L1) is an intestinal cholesterol transporter that is known to be the target of the cholesterol absorption inhibitor ezetimibe. We previously discovered steroidal NPC1L1 ligands by using a novel cell-based assay that employs pharmacological chaperone effect as a readout. Those steroid derivatives bound to a site different from both the sterol-binding domain and the ezetimibe-binding site, implying that they may be a novel class of NPC1L1 inhibitors with a distinct mode of action. As an extension of that work, we aimed here to find non-steroidal NPC1L1 ligands, which may be better candidates for clinical application than steroidal ligands, by using the same assay to screen our focused library of ligands for liver X receptor (LXR), a nuclear receptor that recognizes oxysterols as endogenous ligands. Here we describe identification of a novel class of NPC1L1 ligands with a ring-fused quinolinone scaffold, and an analysis of the structure–activity relationships of their derivatives as NPC1L1 ligands.