Developing an NPC1 activity assay using the novel fluorescent substrate AQ2 for use in basic research and drug discovery

I aim to develop the first direct assay for NPC1 activity, based on a novel fluorescent NPC1 substrate, AQ2, that I developed with Simon Pope (Cardiff). NPC1 is a lysosomal transmembrane protein belonging to the resistance-nodulation-division permease multi-substrate transporter superfamily, thought to transport cholesterol, sphingosine and Zn2+ out of lysosomes. Currently, there is no direct way to measure NPC1 activity in mammalian cells, which has limited studies into its function. AQ2 is a red fluorescent anthraquinone derivative, structurally similar to resistance-nodulation-division permease substrates. AQ2 traffics to the nucleus, at a speed correlated with NPC1 levels, and is trapped inside lysosomes in NPC1 null cells. I will validate AQ2 as an NPC1 substrate by measuring the amount of nuclear AQ2 following treatment with known NPC1 modulators using high-content imaging, and use the assay to screen drug libraries for NPC1 activity modulators. Loss of NPC1 function causes Niemann-Pick type C disease (NPC), whose cellular phenotypes include intralysosomal lipid accumulation, endocytic mistrafficking and lysosomal Ca2+ dyshomeostasis. I will assess the ability of NPC1 activators to improve phenotypes in NPC cells and zebrafish, and in patient fibroblasts from multiple lysosomal storage disorders, where NPC1 is dysfunctional. NPC1 is inhibited by tuberculosis infection, which prevents clearance of mycobacteria, and is necessary for certain filoviruses, including Ebola, to infect cells. NPC1 activators and inhibitors are therefore potential therapies for these infectious diseases, in addition to lysosomal disorders. Hits from the drug screen will be the subject of future funding applications to the Wellcome Trust and MRC.