| Niemann-Pick type C (NPC) disease results from mutations in either of two different genes: npc1 or npc2. Most cases of NPC disease are linked to mutations in NPC1, a large membrane protein found in late endosomes and lysosomes. A small fraction of NPC cases present defects in NPC2, a soluble, cholesterol-binding, lysosomal protein.;No effective treatment is currently available for NPC disease, but recent studies involving administration of hydroxypropyl-beta-cyclodextrin in npc1-/- mice showed improvements in viability, hepatopathology and neuropathology, including clearance of cholesterol and glycosphingolipids. These animal studies were not designed to address the molecular and cellular mechanisms by which cyclodextrins (CD) could reduce the cholesterol accumulation in LSOs. Cyclodextrins are cyclical sugars that can solubilize many hydrophobic molecules such as cholesterol and various water-insoluble drugs. Research detailed herein focused on deciphering the molecular mechanism for cyclodextrin action using cultured cells from NPC patients. We were able to demonstrate that cyclodextrins are being endocytosed by the NPC-deficient cells and act from within the lumen of the lysosomal compartment to efflux cholesterol to the endoplasmic reticulum and eventually out of the cell and not by extracting cholesterol from the plasma membrane of the cells. Currently, hydroxypropyl-beta-cyclodextrin is being evaluated for therapy in two NPC patients. One major obstacle for successful cyclodextrin therapy is its very rapid excretion. Current efforts are underway to modify cyclodextrins to enhance their retention in cells.;In an effort to identify potential therapeutics for NPC disease we conducted a microscopy-based, high content screen to identify compounds that reduce cholesterol accumulation in NPC mutant cells. Research detailed herein centers on the proof and validation of LAL as the target for some of these compounds. Subsequently, we designed a series of analogues for the most potent and selective compounds we discovered. We were able to provide a detailed molecular mechanism of the compounds' inhibition of LAL which involved carbamoylation of the catalytic serine of LAL, similarly to what has been described for the inhibition of acetylcholinesterase by rivastigmine and acylation of various lipases by orlistat.;These studies open new and exciting venues for therapeutic intervention in NPC disease. |
