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Purpose <p>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.</p> Methods <p>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.</p> Results <p>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.</p> Conclusion <p>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.</p>

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Small Molecules to Elevate Rab7-GTPase Activity and Lower Cholesterol Accumulation in Niemann-Pick Type C Disease

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月3日

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  • Mai K. L. Nguyen,
  • Maya R. Nikenich,
  • Kim Seifert,
  • Céline Pinkenburg,
  • Felcia Lai,
  • Hanna-Loisa Walther,
  • Martje Hartmann,
  • Aleksandra Szulc,
  • Eric Sparkes,
  • Shihui Chen,
  • Ravi Wikramanayake,
  • Marc Bernaus-Esqué,
  • Yangjing Liu,
  • Francesc Tebar,
  • Michael Serwetnyk,
  • Anna Wenninger,
  • Christopher Patzke,
  • Brian S. J. Blagg,
  • Brandon L. Ashfeld,
  • Paul W. Groundwater,
  • David E. Hibbs,
  • Andrew J. Hoy,
  • Carles Rentero,
  • Carlos Enrich,
  • Ann-Na Cho,
  • Jonathan J. Du,
  • Thomas Grewal

摘要

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.