Pharmaceutical research · 2026
Small Molecules to Elevate Rab7-GTPase Activity and Lower Cholesterol Accumulation in Niemann-Pick Type C Disease.
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Nguyen Mai K L, Nikenich Maya R, Seifert Kim, Pinkenburg Céline, Lai Felcia, Walther Hanna-Loisa, Hartmann Martje, Szulc Aleksandra, Sparkes Eric, Chen Shihui, Wikramanayake Ravi, Bernaus-Esqué Marc, Liu Yangjing, Tebar Francesc, Serwetnyk Michael, Wenninger Anna, Patzke Christopher, Blagg Brian S J, Ashfeld Brandon L, Groundwater Paul W, Hibbs David E, Hoy Andrew J, Rentero Carles, Enrich Carlos, Cho Ann-Na, Du Jonathan J, Grewal Thomas
Аннотация
PURPOSE: Niemann-Pick type C (NPC) disease caused by mutations in cholesterol transporters NPC1 or NPC2 is characterized by cholesterol accumulation in late endosomes/lysosomes (LE/Lys). The activation of alternative cholesterol export routes that can bypass NPC1/2 deficiency could provide therapeutic opportunities. We previously demonstrated that gene depletion of the Rab7-GTPase activating protein (GAP) TBC1D15, which hydrolyses active GTP-bound Rab7, led to elevated Rab7-GTP levels. This enabled cholesterol export from LE/Lys to reduce cholesterol accumulation in NPC1 mutant cells. Here we aimed to pharmacologically interfere with TBC1D15-mediated Rab7 inactivation to upregulate Rab7 activity and reduce cholesterol accumulation in NPC1 mutant models.
METHODS: The protein structure of the GAP domain of human TBC1D15 in complex with human Rab7-GTP served to perform in silico drug screening and identify small molecules with potentially high TBC1D15 binding affinity. Rab-GTP pulldown assays and fluorescence microscopy analyzed the ability of drug candidates to elevate Rab7-GTP levels and reduce cholesterol accumulation.
RESULTS: Four drug candidates reduced cholesterol accumulation in NPC1 mutant Chinese Hamster Ovary (CHO) M12 cells, NPC1 patient fibroblasts as well as differentiated SH-SY5Y neuronal cells and three-dimensional brain organoids treated with U18666A, a pharmacological NPC1 inhibitor. This was associated with elevated Rab7-GTP levels in drug-treated M12 and NPC1 patient fibroblasts. Moreover, drug candidates augmented 2-hydroxypropyl-β-cyclodextrin (HPβCD)-induced cholesterol removal from U18666A-treated SH-SY5Y cells. Notably, drug candidates did not negatively impact on cell viability or cause membrane damage.
CONCLUSION: Advancing small molecules that can elevate Rab7-GTPase activity could provide opportunities to overcome cholesterol transport defects in NPC mutant cells and offer applications in other Rab7-related neurological diseases.
METHODS: The protein structure of the GAP domain of human TBC1D15 in complex with human Rab7-GTP served to perform in silico drug screening and identify small molecules with potentially high TBC1D15 binding affinity. Rab-GTP pulldown assays and fluorescence microscopy analyzed the ability of drug candidates to elevate Rab7-GTP levels and reduce cholesterol accumulation.
RESULTS: Four drug candidates reduced cholesterol accumulation in NPC1 mutant Chinese Hamster Ovary (CHO) M12 cells, NPC1 patient fibroblasts as well as differentiated SH-SY5Y neuronal cells and three-dimensional brain organoids treated with U18666A, a pharmacological NPC1 inhibitor. This was associated with elevated Rab7-GTP levels in drug-treated M12 and NPC1 patient fibroblasts. Moreover, drug candidates augmented 2-hydroxypropyl-β-cyclodextrin (HPβCD)-induced cholesterol removal from U18666A-treated SH-SY5Y cells. Notably, drug candidates did not negatively impact on cell viability or cause membrane damage.
CONCLUSION: Advancing small molecules that can elevate Rab7-GTPase activity could provide opportunities to overcome cholesterol transport defects in NPC mutant cells and offer applications in other Rab7-related neurological diseases.