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Small Molecules to Elevate Rab7-GTPase Activity and Lower Cholesterol Accumulation in Niemann-Pick Type C Disease
Mai K L Nguyen1, Maya R Nikenich1, Kim Seifert1
1School of Pharmacy, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, 2006, Australia.
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.
Insights
New drugs targeting TBC1D15 show promise for Niemann-Pick type C disease by increasing Rab7-GTP levels, reducing cellular cholesterol buildup, and offering potential treatments for related neurological disorders.
Area of Science:
- Cell Biology
- Molecular Medicine
- Drug Discovery
Background:
- Niemann-Pick type C (NPC) disease involves cholesterol accumulation due to NPC1/2 transporter mutations.
- Elevating Rab7-GTP levels can promote cholesterol export, bypassing NPC1/2 deficiency.
- TBC1D15 inactivates Rab7; inhibiting TBC1D15 may upregulate Rab7 activity.
Purpose of the Study:
- To pharmacologically inhibit TBC1D15 and increase Rab7 activity.
- To reduce cholesterol accumulation in NPC disease models.
Main Methods:
- In silico drug screening based on TBC1D15-Rab7 structure.
- Assessing drug candidates' ability to increase Rab7-GTP levels via pulldown assays.
- Evaluating cholesterol reduction and cell viability using fluorescence microscopy.
Main Results:
- Four drug candidates effectively reduced cholesterol accumulation in NPC1 mutant cells and organoids.
- Elevated Rab7-GTP levels were observed in cells treated with these drug candidates.
- Drug candidates enhanced cholesterol removal and did not compromise cell viability.
Conclusions:
- Small molecules elevating Rab7-GTPase activity offer a therapeutic strategy for NPC disease.
- This approach may address cholesterol transport defects and benefit other Rab7-related neurological conditions.
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