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.

Fig 1. An image-based screen for chaperone drugs that correct mis-localization of NPC1-I1061T-GFP.

(A) Schematic representation of the topology of NPC1 and the positions of P691S and I1061T mutations in the protein. The following abbreviations are used to denote the domains of NPC1; NTD, N-terminal domain; MLD, mid luminal domain; SSD, sterol-sensing domain; CTD, C-terminal luminal domain. (B) Structures of oxysterol-based pharmacological chaperones for NPC1-I1061T mutant protein. (C) Illustration of the image processing procedure used to extract the NPC1-I1061T-GFP localization pattern and relevant image features. From the resulting images, the cell area and GFP distribution patterns are extracted by applying automated thresholding. From the GFP patterns, morphological features that reflect localization changes from ER to LE/L, including circularity and smallObjRatio, were calculated and used for quantifying the chaperone effect.

Fig 2. Screening of an FDA-approved drug library identified several classes of potential NPC1 chaperones.

(A) Three image features (circularity, smallObjRatio, and GFPInt) capture the chaperone-mediated localization change of NPC1-I1061T-GFP. Data points are from a series of mo56HC-treated wells on 11 screening plates (duplicate dose-response analysis per plate). The image features are presented in a set of 2D plots and shown as robust S/N ratio (effect size normalized with median absolute deviation) after plate-wise normalization with respect to plate medians. (B) Dose-dependency of mo56HC examined by employing each of the three image features. Circularity gave the best separation of negative and positive data. Data points and error bars represent mean ± SD (n = 11 experiments, each performed in duplicate). (C) Screening results represented in a set of 2D plots (circularity, smallObjRatio, and GFPInt). Screening was performed at 10 μM. The dashed lines denote thresholds for each of the image features, and compounds that exceeded all the thresholds were selected as hits (red circles). (D) Chemical structures of the hits. (E) Heatmap representation of the screening results for the selected hits and closely related compounds in the library. The heatmap colors encode the robust S/N ratio of image features.

Fig 3. Validation of the hits by dose-response analysis and flow-cytometric analysis.

(A) Dose-response analysis of the hits and related compounds. (B) Chemical structures of the compounds used in (A) and not shown in Fig 2. (C) Stabilizing effect of the hits on the NPC1 mutant, examined by flow-cytometric analysis. Cells stably expressing NPC1-I1061T-GFP were treated as indicated for 21 h, and processed for flow-cytometric analysis to quantify the steady-state expression level of the mutant protein. Consistent with the stabilizing effect of mo56HC (upper panel), lapatinib (upper panel) and itraconazole (middle panel) both stabilized the mutant protein. (D) Dose-dependent increase in the mean GFP fluorescence intensity from the flow-cytometric data shown in (C). Itraconazole (left panel) increased the NPC1-I1061T-GFP level approximately 270% with an EC₅₀ of 0.15 μM, and digoxin (right panel) reduced the expression level to 45% with an EC₅₀ of 0.31 μM.

Fig 4. Effect of itraconazole and lapatinib on the hydrodynamic status on native-PAGE gels and on the ubiquitination status.

(A) High-resolution clear native PAGE (fluorescence-detectable native PAGE) analysis of NPC1-GFP from cells treated as indicated. Cells stably expressing the indicated NPC1-GFP were treated with vehicle, mo56HC (3 μM), itraconazole (3 μM), or lapatinib (10 μM) for 21 h, and DDM-solubilized lysates were subjected to native-PAGE analysis after normalization with respect to total protein concentration. The native-PAGE gel was imaged for GFP fluorescence (upper panel), and total protein was also visualized by TCE staining (lower panel). (B) Ubiquitination status of NPC1-GFPs upon treatment with itraconazole or lapatinib. Cells stably expressing the indicated NPC1-GFP were treated as shown for 18 h followed by treatment with CB5083 (3 μM) for 6 h. The ubiquitination status was first probed with anti-Ub antibody after immunoprecipitation of the FLAG-NPC1-GFP, and then the amount of the FLAG-NPC1-GFP was re-probed with anti-FLAG antibody. The extent of ubiquitination was quantified and normalized to the amount of detected FLAG-NPC1-GFP. The raw, uncropped blot images are available from Mendeley Data repository (http://dx.doi.org/10.17632/jr23ccpp46.3). (C) Quantification of the high-resolution clear native PAGE analysis from three independent experiments. The filled circles, error bars, and open circles represent mean, SEM, and raw data points from three independent experiments. Statistical significance was assessed by ANOVA and Tukey-Kramer multiple comparison test (***, p<0.001). The raw, uncropped gel images are available from Mendeley Data repository (http://dx.doi.org/10.17632/jr23ccpp46.3). (D) Quantification of the ubiquitination assay data collected from three independent experiments. Data was normalized with the ubiquitination level of WT for each experiment. The filled circles, error bars, and open circles represent mean, SEM, and raw data points. Statistical significance of the treatment was assessed similarly as in (C). *, p<0.05; p<0.1; ns, not significant (p = 0.24).

Fig 5. Direct binding of itraconazole to NPC1-I1061T mutant protein.

(A) Structures of photoaffinity probes and pull-down probes for itraconazole and lapatinib. (B) Chaperone activity of the probes on NPC1-I1061T mutant protein, examined by image-based chaperone assay. (C) Photoaffinity labeling of FLAG-tagged NPC1-I1061T-GFP or NPC1-P691S/I1061T-GFP with itraAZY probe. Membrane fractions were prepared from the cells stably expressing the indicated NPC1-GFP, and photo-crosslinking was performed in the presence or absence of the indicated competitors. After conjugation of biotin to the alkyne via click chemistry, the NPC1 was immunoprecipitated and the presence of biotin was probed with streptavidin-HRP conjugate, followed by re-probing with anti-FLAG antibody. The raw, uncropped blot images are available from Mendeley Data repository (http://dx.doi.org/10.17632/jr23ccpp46.3). (D) Quantification of the labeling. The extent of labeling was quantified and normalized with respect to the amount of the FLAG-NPC1-GFPs. Data is from independent experiments (n = 4 for I1061T and n = 3 for P691S/I1061T), and expressed as relative labeling respect to I1061T labeled with itraAZY. The filled circles, open circles, and error bars represent mean, raw data, and SEM, respectively. Statistical significance was assessed by the exact Wilcoxon rank sum test with correction of multiple comparisons by Benjamini-Hochberg method (*, p = 0.05; ns, not significant).

Fig 6. Chaperone effect of mo56HC and itraconazole is not affected by deletion of NTD but is abrogated by P691S mutation.

(A) Colocalization of ΔNTD-NPC1-I1061T-GFP and LAMP1. Cells stably expressing ΔNTD-NPC1-I1061T-GFP were treated as indicated (1 μM mo56AZK or 3 μM itraAZY for 21 h). After LAMP1 immunostaining, images were acquired on a confocal microscope FV-3000 (Olympus) equipped with x60 objective lens. Scale bar, 50 μm. (B) Quantitative colocalization analysis of ΔNTD-NPC1-I1061T-GFP and LAMP1. The filled black circles and error bars denote mean ± SD from 10 images, and gray points represent raw correlation coefficients obtained for each image. Statistical significance was evaluated by applying the Kruskal-Wallis rank sum test along with Dunn’s multiple comparison with the Benjamini-Hochberg-adjusted p value presented as *p<0.05 and **p<0.01 (two-sided). (C) Colocalization of ΔNTD-NPC1-P691S/I1061T-GFP and LAMP1, performed as in (A). (D) Quantitative colocalization analysis of ΔNTD-NPC1-P691S/I1061T-GFP and LAMP1 performed as in (B).

Fig 7. Effect of itraconazole on cholesterol accumulation in NPC fibroblasts bearing I1061T mutation.

NPC fibroblasts (GM18453, NPC1^{I1061T/I1061T}) were treated with the indicated compounds for 48 h and cholesterol accumulation was evaluated by means of filipin staining. The extent of cholesterol accumulation was quantified as fractional filipin intensity within filipin-positive puncta over total filipin staining intensity within cells. Statistical significance was evaluated by applying Dunnett’s multiple comparison test (two-sided) with adjusted p value (single-step method) presented as *p<0.05, **p<0.01, and ***p<0.001 versus vehicle, after confirming normality and variance homogeneity by means of the Shapiro-Wilk test and Bartlett test. Error bars represent means ± SD from n = 3 (for LBH589) or n = 4 (vehicle, mo56HC, and itraconazole) independent experiments.