Image-based screen capturing misfolding status of Niemann-Pick type C1 identifies potential candidates for chaperone drugs

Abstract

Niemann-Pick disease type C is a rare, fatal neurodegenerative disorder characterized by massive intracellular accumulation of cholesterol. In most cases, loss-of-function mutations in the NPC1 gene that encodes lysosomal cholesterol transporter NPC1 are responsible for the disease, and more than half of the mutations are considered to interfere with the biogenesis or folding of the protein. We previously identified a series of oxysterol derivatives and phenanthridine-6-one derivatives as pharmacological chaperones, i.e., small molecules that can rescue folding-defective phenotypes of mutated NPC1, opening up an avenue to develop chaperone therapy for Niemann-Pick disease type C. Here, we present an improved image-based screen for NPC1 chaperones and we describe its application for drug-repurposing screening. We identified some azole antifungals, including itraconazole and posaconazole, and a kinase inhibitor, lapatinib, as probable pharmacological chaperones. A photo-crosslinking study confirmed direct binding of itraconazole to a representative folding-defective mutant protein, NPC1-I1061T. Competitive photo-crosslinking experiments suggested that oxysterol-based chaperones and itraconazole share the same or adjacent binding site(s), and the sensitivity of the crosslinking to P691S mutation in the sterol-sensing domain supports the hypothesis that their binding sites are located near this domain. Although the azoles were less effective in reducing cholesterol accumulation than the oxysterol-derived chaperones or an HDAC inhibitor, LBH-589, our findings should offer new starting points for medicinal chemistry efforts to develop better pharmacological chaperones for NPC1.

Assessment of FDA-Approved Drugs as a Therapeutic Approach for Niemann-Pick Disease Type C1 Using Patient-Specific iPSC-Based Model Systems

“…In this context, pharmacological chaperones that specifically bind to and stabilize the target protein can be used, thereby correcting its pathogenic misfolding, promoting its trafficking to the subcellular destination, and restoring its functional capability. The utility of pharmacological chaperones in the context of NP-C1 has been repeatedly explored using different approaches by the working group of Ohgane [20,[36][37][38]. Oxysterols and their derivatives, such as 25-HC, were first identified to act as pharmacological chaperones for NP-C1 disease due to their conformational similarity to the natural ligand cholesterol [20,36].…”

“…To ultimately prove that abiraterone acetate and quinestrol are acting as pharmacological chaperones and not through an unknown mechanism, the direct binding should be investigated in the future. To this end, photo-crosslinking studies have been proven to be effective in assessing the binding of a compound to the NPC1 protein [20,38]. Despite this, on the basis of the in silico results predicting strong interaction of abiraterone acetate and the NTD of the NPC1 protein, as well as the increased amount of mature, Endo H-resistant NPC1 protein and lysosomal localization, we assume abiraterone acetate to act as a pharmacological chaperone.…”

“…First, analysis of filipin-stained cells revealed a significant decrease in LSO compartment ratio upon treatment with abiraterone acetate, indicating a decrease of cholesterol accumulation in all three cell types of NPC1 I1061T/I1061T cells (Figures 4B, 6B, and 7B). A diminished cholesterol accumulation in NPC1 I1061T/I1061T fibroblasts has also been shown upon treatment with compounds identified through other, image-based strategies, such as alexidine dihydrochloride [40] and itraconazole [38], albeit comparison of efficiency is hardly feasible due to different treatment conditions. In our case, decreased cholesterol levels were shown to be a consequence of increased mature NPC1 protein levels, rather than an increase of total NPC1 protein, probably through an enhancement of folding efficiency realized through the binding of abiraterone acetate to the NTD.…”

“…In summary, we showed a beneficial effect of abiraterone acetate and quinestrol on cholesterol accumulation. While previously proposed pharmacological chaperones, such as oxysterol-derivatives [20] and itraconazole [38], are thought to exert their effect through a binding site different than the NTD, we assume that the effect of quinestrol and abiraterone acetate is based on the strong interaction to the NTD, as predicted by in silico screening, and its subsequent stabilization of the misfolded protein.…”

Treatment Options in Congenital Disorders of Glycosylation

“…Pharmacological chaperones are small molecules capable of binding to the altered structure of mutated proteins and facilitating correct folding and thus increasing enzyme activity (Figure 2C). This principle has been explored in lysosomal storage disease and there are currently approved medications for Fabry disease (Fan et al, 1999;Germain et al, 2016) and Niemann-Pick C disease (Pipalia et al, 2019(Pipalia et al, , 2021Shioi et al, 2020), while such treatments for others are under investigation (Tropak et al, 2004;Parenti et al, 2014;Han et al, 2020). The advent of in silico screening of large compound libraries has greatly facilitated the identification of novel candidate compounds and is actively being explored in CDG (Yuste-Checa et al, 2016).…”