Discovery of Autophagy Modulators that Increase I1061T NPC1 Expression and Promote Cholesterol Efflux in Niemann-Pick Type C Patient-Derived Fibroblasts

Autophagy, an evolutionarily conserved catabolic process, has been implicated as a potential therapeutic target in Niemann-Pick Type C (NPC) disease, a fatal lysosomal storage disorder. Our goal was to identify autophagy modulators that positively impact NPC-relevant phenotypes to further evaluate the role of autophagy in this disease. Using a phenotypic high-throughput screen for autophagy modulation and a subsequent secondary assay in homozygous I1061T NPC1 patient-derived fibroblasts, two compounds, 1 and 2, were identified that induced autophagy and reduced unesterified cholesterol accumulation to a level comparable to the reduction achieved by 2-hydroxypropyl-β-cyclodextrin. Global protein expression changes were evaluated in I1061T NPC1 fibroblasts following treatment with compounds 1 and 2 to identify affected pathways, and we observed a significant reduction of lysosomal hydrolase levels induced by compound 2. Additional mechanistic studies revealed that the expression of NPC1 protein is required for compound-induced amelioration of cholesterol accumulation and that compound 2 increases expression levels of I1061T NPC1 without broadly inhibiting proteasomal activity or exacerbating ER stress. These results have led to the hypothesis that compound 2 may serve as a proteostasis modulator or small-molecule chaperone that upregulates autophagy to a level that is predominantly cytoprotective and increases the proportion of properly folded mutant NPC1, thus increasing NPC1 expression levels and alleviating cholesterol accumulation and associated phenotypes. This work, along with future mechanistic studies, contributes to the development of novel strategies to modulate autophagy and proteostasis with potentially broad therapeutic applications.