Itraconazole and posaconazole, inhibitors of NPC1 sterol transport, act as pharmacological chaperones after washout

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disorder primarily caused by mutations in the NPC Cholesterol Transporter 1 (NPC1) gene, resulting in cholesterol and lipid accumulation in late endosomes and lysosomes. While several therapeutic drugs show promise in reducing cholesterol accumulation, none of the current treatments are highly effective. Itraconazole and posaconazole, widely used antifungal drugs, have been shown to stabilize misfolded NPC1 proteins, enabling their escape from endoplasmic reticulum-associated degradation. This chaperone-like property makes them attractive candidates for testing chaperones as possible treatments for NPC disease, but both drugs also inhibit NPC1 function. In this study, we employed a washout approach to reverse the inhibitory effects of these drugs, leveraging the fact that wild-type NPC1 proteins have a half-life of about 42 h. Treating NPC1^{I1061T/I1061T} human fibroblasts with itraconazole or posaconazole for 72 h, followed by 24 to 48 h of washout, we observed a significant reduction in lysosomal cholesterol accumulation. A modest rebound was observed 72 h after drug removal, likely due to protein turnover. We also tested a repeated pulsed exposure treatment, in which short drug treatments were followed by extended washout periods. This strategy preserved the functional benefit of NPC1 stabilization while minimizing inhibitory effects. These findings indicate that a washout strategy can enhance the functional benefits of pharmacological chaperones, offering a potential future therapeutic approach for NPC disease.