High content screening for non-classical peroxisome proliferators

Abstract

Peroxisomes are ubiquitous cellular organelles that perform vital functions including fatty acid beta-oxidation, plasmalogen synthesis, and detoxification of harmful oxidative species. In rodents numerous compounds that increase peroxisome biogenesis also alleviate metabolic syndrome (MetS)/type 2 diabetes (T2D) symptoms. However, compounds that increase peroxisome biogenesis in rodents largely do not increase peroxisome biogenesis in humans. We designed a novel genetically encoded high throughput screening (HTS) high content assay to identify small molecule compounds that function as peroxisome proliferators in human cells. From this assay we have confirmed that 4-phenylbutyrate (PBA), a PPAR independent peroxisome proliferator and chemical chaperone, increases peroxisome proliferation in human cells and serves as a positive control for our screen. We performed a small pilot and larger 15,000 compound production screen with an overall Z' factor of 0.74 for 384-well plate format, providing a valuable screening tool for identifying peroxisome modulator compounds. From this screen we have identified 4 existing drugs and 10 novel compounds, some with common scaffolds 1000X more potent than PBA. It is hoped that these novel compounds may serve as scaffolds for testing for efficacy in alleviating MetS/T2D symptoms both in mouse models and ultimately human disease.

Figure 1

A) Enhanced peroxisome targeting fluorescent reporter (EPTFR) in HepG2 human cells. B) Double labeling of peroxisomal antigen PMP-70 (red) and EPTFR (Green) demonstrating co-localization. Note the general number, distribution and shape of peroxisomes is unchanged in EPTFR expressing cells (C, yellow). (Blue = Hoescht (nuclei)) (scale bar = 5 μm).

Figure 2

A) Spectral multiplexing of Hoechst (blue), GFP, and CellTracker-Red fluorescent probes. B) Flow cytometry analysis of a stable EPTFR-HepG2 line (green) and a wild-type HepG2 line showing suitability of the cell line for high-content screening purposes. The filled histogram shows a monophasic population of cells with substantial GFP expression (approximately 300× greater than the negative population). This cell population is sampled from the low-passage master cell-bank that was used in this screening effort.

Figure 3

PBA validation as a positive control for high content assay development and screening. (A) Dose-response curve for GFP fluorescence (inset structure of PBA) exhibiting a 2.4 mM EC50. PBA causes significant increases in peroxisomes visualized with EPTFR-HepG2 cells (blue – nuclei, green – peroxisomes) showing normal peroxisome distribution (B) (DMSO vehicle) and (C) increased peroxisomal content (with PBA).

Figure 4

Image segmentation showing (A) merged color image with blue nuclei, red cytoplasm and green peroxisomes, (B) nuclear binary mask, and (C) cytoplasmic mask. Each colored object in the masks indicates a unique cell, showing effective segmentation. (D) Peroxisomes masked (red) with nuclei (blue), and (E) assignment of peroxisomes to individual cells inside the respective cell mask.

Figure 5

Min-Max assay validation in two 384 well plates showing incubation with positive control (Max – 9.0 mM), and assay negative control (Min – DMSO carrier). Inset table shows assay statistics, resulting in a calculated Z of 0.72.

Figure 6

Pilot screening results from the Prestwick FDA-approved drug library. (A) Pilot screen scatter for the 1120 compound Prestwick library. Response in this assay is calculated as a percent-effect as compared with the positive control. A cursory hit threshold is drawn at <32% effect, representing the μ + 3σ (B) Dose response confirmation for the active compound niclosamide (inset) showing a 2.4 μM EC₅₀ and correlating western blot (left lane = DMSO, right lane = positive control) normalized to tubulin. Increased GFP expression correlates with increased peroxisomal specific antigen PMP-70 (↑33%). Abbrevation: U, arbitrary fluorescent units.

Figure 7

Production HCA screening data for 15 k compounds for peroxisomal biogenesis. (A) Scatterplot for 15,000 compounds screened, (B) histogram for the screen showing a normal (Gaussian) statistical distribution and (C) box plot for the screen showing distribution statistics and outliers (points).

Figure 8

20× color micrographs of Oil Red O staining of lipid droplets in HepG2 cells for untreated and PBA treated conditions showing a significant decrease in the size and intensity of staining with PBA. The respective histograms (lower panels) show the distribution of lipid droplet size per cell. Untreated mean lipid droplet area of ∼300 pixels² (0.02 μm²) compared 50 pixels² (0.003 μm²) for PBA treated.