An aggregation sensing reporter identifies leflunomide and teriflunomide as polyglutamine aggregate inhibitors

Abstract

Intracellular protein aggregation is a common pathologic feature in neurodegenerative diseases such as Huntington' disease, amyotrophic lateral sclerosis and Parkinson' disease. Although progress towards understanding protein aggregation in vitro has been made, little of this knowledge has translated to patient therapy. Moreover, mechanisms controlling aggregate formation and catabolism in cellulo remain poorly understood. One limitation is the lack of tools to quantitatively monitor protein aggregation and disaggregation. Here, we developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). httQ72-Luc fails to aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein such as Q80-cfp. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity. Using httQ72-Luc with Q80(CFP/YFP) as seeds, we screened the Johns Hopkins Clinical Compound Library and identified leflunomide, a dihydroorotate dehydrogenase inhibitor with immunosuppressive and anti-psoriatic activities, as a novel drug that prevents polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis, since neither uridine treatment nor other pyrimidine biosynthesis inhibitors affected polyQ aggregation. Inducible cell line and cycloheximide-chase experiments indicate that these drugs prevent incorporation of expanded polyQ into an aggregate. This study demonstrates the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. As current trials are under-way for teriflunomide in the treatment of multiple sclerosis, we propose that this drug be considered a possible therapeutic agent for polyQ diseases.

Figure 1.

Aggregation of httQ72-Luc reporter requires seeding by expanded polyQ. (A) Schematic representation of the httQ72-Luc reporter. The first 17 amino acids of the huntingtin protein and the flexible linker essential for proper luciferase folding are indicated in red and gray, respectively. (B) CFP-tagged polyQ proteins of different sizes (Q19-, Q72-, Q80-cfp) were co-transfected with httQ72-Luc or FLuc in HEK-293 cells. Forty-eight hours after transfection, lysates were prepared and protein aggregation analyzed by filter trap. Right panel: similar levels of expression in the same lysates were confirmed by western blot. (C) U2OS cells were co-transfected with indicated luciferase- and CFP-tagged constructs, and 48 h after transfection, luciferase staining was performed. httQ72-Luc but not FLuc was coaxed to aggregate into a polyQ aggregate. Scale bar, 20 μm.

Figure 2.

httQ72-Luc aggregates are inactive. (A–C) HEK-293 cells were co-transfected with httQ72-Luc and CFP/YFP-tagged polyQ of different sizes (Q19-, Q35-, Q72- and Q80(CFP/YFP)). Thirty-six hours after transfection, cells were split into sister 96-well plates for luciferase activity (A) or FRET (B) determinations. Single-point mutations of huntingtin that either increase (Q72K → R) or abolish (Q72M → P) httQ72-CFP aggregation were included. (C) A strict inverse correlation between luciferase activity and protein aggregation assessed by FRET was detected with a correlation coefficient of 0.9834. (D) HEK-293 cells were co-transfected with httQ72-Luc along with Q19-cfp or Q80-cfp. Forty-eight hours after transfection, live-cell BLI was performed. Fluorescence (CFP) and bright-field microscopy for the same field were recorded right before BLI. The arrow indicates a representative cell containing an httQ72-Luc/Q80-cfp co-aggregate devoid of luciferase activity. (E and F) HEK-293 cells were co-transfected as in (A), and 60 h later, lysates were prepared by sonication in the absence of detergents, spun for 10 min at 16 000g and total and cleared lysates assayed for luciferase and FRET.

Figure 3.

Flow chart describing the screening steps. For the primary screen, 5 μm of each compound was tested in triplicate on separate plates. Z_i scores were calculated for luciferase/(FRET/donor), and 20 drugs above a cutoff of 3 were pulled out. Fluorescent drugs that did not change luciferase activity were discarded. Z_i scores were calculated for luciferase as before, and 10 primary hits identified. Dose–response studies with commercially available drugs were performed on hits showing >1.5-fold increase in luciferase activity in the primary screen. Of the 1560 compounds screened, 4 hits were obtained.

Figure 4.

Leflunomide and its active metabolite teriflunomide increase luciferase activity of aggregated httQ72-Luc in a dose–response manner. HEK-293 cells were transfected with httQ72-Luc along with Q80(CFP/YFP) seeds (A) or Q19(CFP/YFP) seeds (B) and split into 96-well plates 24 h after transfection. After 12 h recovery, cells were treated with 0.001, 0.01, 0.1, 1, 5 or 10 μm of leflunomide or teriflunomide and incubated for additional 24 h. Cells were washed, lysed and luciferase activity was determined. Luciferase activity is plotted as percentage to DMSO control for each drug set.

Figure 5.

Leflunomide and teriflunomide increase httQ72-Luc solubility independently of pyrimidine biosynthesis inhibition. (A) HEK-293 cells were transfected with httQ72-Luc and Q80-CFP/YFP seeds and were split into 96-well plates 24 h later. After 12 h recovery, cells were treated with 100 μm teriflunomide or DMSO along with 100 μm uridine or vehicle. Luciferase activity was determined 24 or 48 h later and plotted as percentage to corresponding DMSO controls. (B) HEK-293 cells were transfected with httQ72-Luc along with Q19-CFP/YFP seeds or Q80-CFP/YFP seeds and were split into 96-well plates 24 h later. After 12 h recovery, cells were treated with 10 or 100 μm of the anti-pyrimidine drugs acivicin, 6-azauridine or lapachol, corresponding vehicles or teriflunomide for additional 24 or 48 h. Cells were washed and lysed and luciferase activity was determined. Luciferase activity is plotted as percentage to vehicle controls for each time set, as indicated.

Figure 6.

Leflunomide and its active metabolite teriflunomide decrease polyQ aggregate size. (A) Representative CFP images of HEK-293 cells transiently transfected with httQ72-CFP or Q80-CFP and treated with 100 μm leflunomide, 100 μm teriflunomide or DMSO vehicle for 48 h. (B) Aggregates per cell in Q80-CFP transfected and treated cells as in (A) were counted in 10 independent fields from 2 independent experiments. (C) Images from experiments as in (A) using Q80-CFP were analyzed for particle size using ImageJ as described in Materials and Methods. Frequency distributions were plotted as histograms using 25pixel² bins. Smaller aggregates (<300 pixel²) are more frequent in teriflunomide (red bars)- and leflunomide (white bars)-treated cells compared with vehicle (blue bars). Conversely, a decrease in aggregate size by leflunomide/teriflunomide was detected after a 300 pixel² cutoff (horizontal bar). (D) Leflunomide and teriflunomide treatment decreases the size of both httQ72-CFP and Q80-CFP aggregates. **P < 0.01 and ***P < 0.005.

Figure 7.

Teriflunomide blocks pure polyQ and expanded huntingtin aggregation by impeding incorporation into a polyQ aggregate. (A) HEK-293 cells were transiently transfected with httQ72-cfp or polyQ80-cfp and treated with 100 μm teriflunomide for 48 h. Lysates were prepared by sonication in the absence of detergents, and 20 μg of proteins were analyzed by filter trap using anti-GFP antibodies. Right panel: a representative western blot from three independent experiments to confirm similar levels of expression. (B) Tet-ON U2OS[Q80-cfp] cells were initially grown in the presence (−Tet) or absence (+Tet) of tetracycline for 3 days to preform aggregates and then replated in Tet-free or Tet-containing media to de novo formation of aggregates, respectively. Twelve hours after replating, cells were treated with 100 μm teriflunomide for 48 h and filter trap assays were done as before. Note that teriflunomide does not disaggregate preformed polyQ aggregates. (C) HEK-293 cells were co-transfected with httQ72-Luc and the indicated CFP/YFP-tagged polyQ proteins to seed the aggregation. Twenty-four hours after transfection, cells were split into 96-well plates and 12 h later, cells were treated with 100 μm teriflunomide for 24 h. Staggered CHX chase experiments were then performed for 0, 2 or 8 h in the presence of the drugs. Luciferase activity is represented as percentage to vehicle-only treatment at each time point. *P < 0.05, t-test (n = 8, from two independent experiments).