A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds

Abstract

Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved drugs, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts. These findings establish a novel approach to anti-progeroid drug discovery.

Keywords: FDA-approved compounds; HGPS; High-content imaging; High-throughput screening; Progerin; Retinoids.

Fig. 1.

Characterization of an inducible GFP-progerin fibroblast cell system. (A) Schematic representation of stable human dermal fibroblasts cell lines containing doxycycline-inducible GFP-tagged lamin A, progerin or progerin C661S mutant for use in immunofluorescence (IF) detection of GFP, lamin B1, γH2AX, and DNA DAPI stain. (B) Representative IF images of DNA DAPI stain (inset bottom left) and GFP signal of all three GFP-lamin inducible fibroblasts cell lines in the absence and presence of doxycycline for 4 days. Scale bar: 10 μM. (C) Western blot for uninduced and 4 days induced GFP-lamin fibroblast cell lines and quantification of β-actin normalized lamin protein levels. Values represent averages ± SD from 3 experiments. (p > 0.05). (D) Quantification of IF signals (see Section 2) of induced GFP-lamin protein levels as well as lamin B1, LAP2, HP1γ, tri-methylated lysine 27 on histone 3 (H3-K27–3M), γH2AX and 53BP1 HGPS markers. Values represent averages ± SD from 3 experiments (N > 300, *p < 0.05 between GFP-lamin A and GFP-progerin cell lines).

Fig. 2.

High-content imaging quantification of HGPS defects. (A) Schematic representation of the pipeline used for automated detection and quantification of HGPS defects in GFP-progerin inducible fibroblasts. (B, C, E) Representative image of nuclear segmentation based on the DNA DAPI signal in GFP-progerin inducible fibroblasts (4 days, 1 μg/ml doxycycline), shown for DAPI stain (A), GFP-progerin (B) and lamin B1 IF staining (D); scale bar: 10 μM. (D) Example of contrast-based segmentation of γH2AX foci in GFP-progerin inducible fibroblasts (4 days, 1 μg/ml doxycycline). Scale bar: 10 μM.

Fig. 3.

High-throughput screening of FDA-approved compound library in GFP-progerin inducible fibroblasts. (A) Overview of the screening assay. GFP-progerin inducible fibroblasts are plated in the presence of doxycycline and treated at the same time with FDA approved compounds that either have no effect on the induction of GFP-progerin and formation of lamin B1 and γH2AX HGPS aging defects (no hits), or alternatively are considered hits based on improving any of these endpoints. Untreated, induced GFP-progerin cells served as negative controls and positive controls are cells in which GFP-progerin remains either uninduced or is induced in the presence of FTI (see Section 2). (B) Immunofluorescence images of positive and negative controls for all assay endpoints. Scale bar: 10 μM. (C) Quantification (see Section 2) of panel B. N > 300; *p < 0.05. Values represent averages ± SD from 3 experiments. (D) Hit selection of candidate compounds identified from the screen and the respective biological classes they fall in. (E) Dose–response curves from screen identified retinoid compounds (tretinoin, and isotretinoin) based on the effect at 1, 3, 10, and 29 μM concentrations on the induction of GFP-progerin and on lamin B1 and γH2AX markers. Positive and negative control levels for all aging markers are indicated by dotted lines in green and red, respectively. Values represent averages ± SD from 3 experiments.

Fig. 4.

Validation screen of FDA-approved candidate compounds in HGPS patient fibroblasts. Quantitative analysis of immunofluorescence staining (see Section 2) for progerin, lamin B1, LAP2, HP1γ, H3-K273M, γH2AX and 53BP1 HGPS markers in HGPS patient fibroblasts that were treated for 8 days with candidate compounds identified in the primary screen. A dose of 10 μM compounds was used for each compound with the exception of doxofylline (1 μM), based on primary screening results. Values represent averages ± SD from 3 experiments. N > 300; *p < 0.05, untreated HGPS vs. compound treated HGPS. Compounds: #1, tretinoin; #2, tazarotene; #3, milrinone; #4, trequinsin hydrochloride; #5, doxofylline; #6, isoproterenol hydrochloride; #7, procaterol hydrochloride hemihydrates; #8, salbutamol sulfate; #9, hydrocortisone succinate; #10, deflazacort; #11, triamcinolone acetonide; #12, loteprednoletabonate; #13, resocortol butyrate; #14, diflucortolone valerate; #15, betapar; #16, prednisolone acetate; #17, diflorasonediacetate; #18, rimexolone; #19, fludrocortisones acetate; #20, 3,4-dihydroxy-2-(methylamino)acetophenone hydrochloride; #21, sumatriptan succinate; #22, cisplatin; #23, mozavaptan; #24, 2-ethylhexyl salicylate; #25, sulfacetamide; #26, benzethonium chloride; #27, albendazole.

Fig. 5.

Retinoids reduce the expression of A-type lamins and reverse aging defects in HGPS patient fibroblasts. (A) Representative immunofluorescence images of indicated HGPS markers in HGPS patient and wildtype fibroblasts treated with 10 lM of isotretinoin, tamibarotene, or tazarotene. Scale bar: 10 μM. (B) Western blot and quantification of the effect of all three retinoids on the protein levels of A-type lamins in HGPS patient fibroblasts. Values represent averages ± SD from 3 experiments. *p < 0.05 treated vs. untreated HGPS patient fibroblasts. (C) Expression analysis of lamin A, progerin and lamin C mRNA levels normalized for GAPDH mRNA expression in HGPS patient fibroblasts upon treatment with 10 μM of isotretinoin or tazarotene. Values represent averages ± SD from 3 experiments. *p < 0.05 treated vs. untreated HGPS patient fibroblasts.