Simultaneous Targeting of NPC1 and VDAC1 by Itraconazole Leads to Synergistic Inhibition of mTOR Signaling and Angiogenesis

Abstract

The antifungal drug itraconazole was recently found to exhibit potent antiangiogenic activity and has since been repurposed as an investigational anticancer agent. Itraconazole has been shown to exert its antiangiogenic activity through inhibition of the mTOR signaling pathway, but the molecular mechanism of action was unknown. We recently identified the mitochondrial protein VDAC1 as a target of itraconazole and a mediator of its activation of AMPK, an upstream regulator of mTOR. However, VDAC1 could not account for the previously reported inhibition of cholesterol trafficking by itraconazole, which was also demonstrated to lead to mTOR inhibition. In this study, we demonstrate that cholesterol trafficking inhibition by itraconazole is due to direct inhibition of the lysosomal protein NPC1. We further map the binding site of itraconazole to the sterol-sensing domain of NPC1 using mutagenesis, competition with U18666A, and molecular docking. Finally, we demonstrate that simultaneous AMPK activation and cholesterol trafficking inhibition leads to synergistic inhibition of mTOR, endothelial cell proliferation, and angiogenesis.

3.3 Å structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

“…4A), which could explain why overexpression of NPC1-I1061T in NPC1 −/− cells still retains late endosome localization and rescues the NPC1-deficient phenotype (25). In addition, previous crosslinking studies showed that mutation P691S in human, or P692S in mouse, abolishes binding of different ligands to the SSD (12,(14)(15)(16). In our structure, P691 is located in the TM5 helix and faces the SSD pocket that accommodates cholesterol or other small molecules (Fig.…”

“…The TM3-7 segment, termed sterol-sensing domain (SSD), is highly conserved within a subclass of membrane proteins in the cholesterol metabolism pathway (10,11). Biochemical and cell biological studies showed that small molecules such as U18666A and posaconazole can block cholesterol export from lysosomes and that a point mutation (P691S) in the NPC1-SSD can prevent binding of these ligands (12)(13)(14)(15)(16). Additionally, binding of some of these ligands is retained even after deletion of the NTD (13,15), which contains a classical cholesterol-binding site (17,18), suggesting that NPC1-SSD itself is able to bind multiple ligands of distinct structures.…”

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth

“…Briefly, A549 (for endogenous NPC1) or 293T cells overexpressing Myc-NPC1 [18] were grown in 6-cm dishes until confluent. Cells were pretreated with competitors (CEP and other cholesterol trafficking inhibitors) or DMSO for 30 min prior to the addition of 200 nM itraconazole photo-affinity probe.…”

Repurposing itraconazole as an anticancer agent

“…Aberrant activation of AKT is correlated with an increase in lipid raft formation, while the disruption of lipid rafts inhibits AKT activation (25). In HUVECs, itraconazole inhibited intracellular cholesterol trafficking to the plasma membrane by binding to Niemann-Pick C1 protein, resulting in cholesterol depletion (26). In glioblastoma cells, the redistribution of cholesterol was induced by the downregulation of sterol carrier protein (SCP2) (12), which is located in numerous organelles including mitochondria (27).…”