High-Throughput Screening to Identify Compounds That Increase Fragile X Mental Retardation Protein Expression in Neural Stem Cells Differentiated From Fragile X Syndrome Patient-Derived Induced Pluripotent Stem Cells

Abstract

: Fragile X syndrome (FXS), the most common form of inherited cognitive disability, is caused by a deficiency of the fragile X mental retardation protein (FMRP). In most patients, the absence of FMRP is due to an aberrant transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. FXS has no cure, and the available treatments only provide symptomatic relief. Given that FMR1 gene silencing in FXS patient cells can be partially reversed by treatment with compounds that target repressive epigenetic marks, restoring FMRP expression could be one approach for the treatment of FXS. We describe a homogeneous and highly sensitive time-resolved fluorescence resonance energy transfer assay for FMRP detection in a 1,536-well plate format. Using neural stem cells differentiated from an FXS patient-derived induced pluripotent stem cell (iPSC) line that does not express any FMRP, we screened a collection of approximately 5,000 known tool compounds and approved drugs using this FMRP assay and identified 6 compounds that modestly increase FMR1 gene expression in FXS patient cells. Although none of these compounds resulted in clinically relevant levels of FMR1 mRNA, our data provide proof of principle that this assay combined with FXS patient-derived neural stem cells can be used in a high-throughput format to identify better lead compounds for FXS drug development.

Significance: In this study, a specific and sensitive fluorescence resonance energy transfer-based assay for fragile X mental retardation protein detection was developed and optimized for high-throughput screening (HTS) of compound libraries using fragile X syndrome (FXS) patient-derived neural stem cells. The data suggest that this HTS format will be useful for the identification of better lead compounds for developing new therapeutics for FXS. This assay can also be adapted for FMRP detection in clinical and research settings.

Keywords: Fibroblasts; Fragile X mental retardation protein assay; Fragile X syndrome; High-throughput screening; Induced pluripotent stem cells; Neural stem cells.

Figure 1.

Evaluation of the best pair of anti-FMRP antibodies in the time-resolved fluorescence resonance energy transfer (TR-FRET) assay. (A): Confirmation of the best pair of anti-FMRP antibodies for TR-FRET-based FMRP detection in a cell-based assay with control and fragile X syndrome (FXS) fibroblasts. The indicated numbers of control and FXS fibroblast cells were plated in 384-well plates, and the FMRP assay was performed. The y-axis shows the FMRP levels in control fibroblasts normalized to the levels in FXS fibroblasts. The numbers on the x-axis refer to the antibodies used in the assay and correspond to the ID# as described in supplemental online Table 1. The inset shows a diagrammatic representation of the basic principle of TR-FRET-based FMRP assay. Two different anti-FMRP antibodies, one labeled with a donor fluorophore, europium cryptate (K), and the other labeled with an acceptor (d2), are added to the cell lysate after incubation with the compounds. During detection, FRET can only occur if the two FMRP antibodies are in close proximity. This significantly reduces any nonspecific background signal that might result from the two individual antibodies. (B): FMRP assay using recombinant FMRP in 384-well plates. Twofold dilutions of the recombinant FMRP protein were measured in the TR-FRET assay. The purified recombinant protein in the FMRP assay was detectable at 2 fmol/µl and was linear up to 270 fmol/µl. Abbreviations: C, control; Em, emission; Ex, excitation; FMRP, fragile X mental retardation protein; KC, cryptate control with only FMRP antibody-K; NC, negative control without any antibodies.

Figure 2.

Optimization of the FMRP assay for HTS in 384-well and 1,536-well plates. (A, B): Indicated number of control and FXS (C10700, C10259, and C10147) fibroblasts were seeded in 384-well and 1,536-well plates, and the FMRP assay was performed as described in Materials and Methods. (C): Different numbers of control (BC1) and FXS (SC128) NSCs were plated in 1,536-well plate, and the FMRP levels were measured using the time-resolved fluorescence resonance energy transfer (TR-FRET) assay. (D): Neurons derived from BC1 and SC128 NSCs were seeded at 2,500 cells per well in 1,536-well plates, and the FMRP levels were measured using the TR-FRET assay. The y-axis shows the FMRP levels as the 665/615 nm TR-FRET ratio obtained in FXS cells over that observed in the control cells. Abbreviations: FMRP, fragile X mental retardation protein; FXS, fragile X syndrome; NSCs, neural stem cells.

Figure 3.

Scatter plot for signal-to-basal ratio and Z score in the time-resolved fluorescence resonance energy transfer (TR-FRET) FMRP assay. The TR-FRET assay was conducted with dimethyl sulfoxide (DMSO) plates in three different cell types [fibroblast (A), NSCs (B), and neurons (C)] to measure the performance parameters of this assay. Columns 1 and 2 (A, C) and column 1 (B) contained control cells indicating a positive response, and the remainder of the columns contained FXS cells with 0% response. All wells had an equal amount of DMSO added (0.5% final). The S/B ratio, Z score, and CV for each cell type are shown. Abbreviations: CV, coefficient of variation; FMRP, fragile X mental retardation protein; FXS, fragile X syndrome; NSCs, neural stem cells; S/B, signal-to-basal.

Figure 4.

Validation of hit compounds identified from screens of LOPAC and approved drug libraries using the FMRP assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR) for FMR1 mRNA. (A): Structure of hit compounds evaluated in secondary assays. (B): Dose responses of hit compounds in the FMRP assay with FXS NSCs. The cells were treated with the indicated concentration of the compounds for 24 hours and analyzed using the FMRP assay. The FMRP levels are shown as the percentage of those observed in the control cells. (C): Validation of hit compounds using qRT-PCR for FMR1 mRNA levels in FXS NSCs. Cells were treated with PPIX and SB216763 for 72 hours or geliomycin and tibrofan for 48 hours with the indicated concentrations. (D): Validation of hit compounds with qRT-PCR for FMR1 mRNA levels in FXS neurons. The cells were treated with SB216763 for 72 hours and tibrofan for 24 hours with the indicated concentrations. (C, D): FMR1 mRNA levels are expressed as a percentage of GAPDH mRNA. Results shown are an average from three independent drug treatments; error bars represent SD. In control NSCs, the FMR1 mRNA was ∼10% of GAPDH. Additional qRT-PCR data are shown in supplemental online Table 3. Abbreviations: DMSO, dimethyl sulfoxide; FMR1, fragile X mental retardation 1; FMRP, fragile X mental retardation protein; FXS, fragile X syndrome; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NSCs, neural stem cells; PPIX, protoporphyrin IX; WT, wild-type.