Figures and data
A whole-animal drug screen identifies LipoGlo-reducing compounds.
(A) Schematic summarizing drug screening paradigm. Drug treatments were prepared in 96-well plates, each plate with a negative control (vehicle), positive control (5 µM lomitapide), and serial dilutions (four-fold dilution; 8 µM, 4 µM, 2 µM, and 1 µM) of two different drugs of interest. Each treatment was prepared with 8 replicates. When animals were 3 dpf, when B-lp levels are relatively high (black arrow), they were dispensed into their drug treatment for a 48-hour incubation when luminescence was measured (red dashed arrow). (B) Boxplot of average Relative Luminescence Units (RLU) measured from fixed 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) animals treated for 48 hours with either negative (vehicle) or positive (5 µM lomitapide) control. Each data point represents the average of 8 independent samples measured from a single 96-well plate from 1381 independent experiments across the entire screen. We measured a 55.6% reduction in RLU in 5 µM treated animals. (C) An ordered plot of each average fold change of luminescence (log2 scale) measured from 5 µM lomitapide treated animals from each 96-well plate (n = 1381) relative to respective vehicle treatment. The solid black line at y=0 represents the divide in increased and decreased luminescence levels, the solid blue line at y = -1.33 represents the curve’s inflection point, and the dashed black line at y = -1.5 represents the fold change cutoff used to define a hit. (D) An ordered plot of each SSMD score measured from positive control (5 µM lomitapide) treated animals from each 96-well plate (n = 1381) relative to respective vehicle treatment. The solid black line at y = 0 represents the divide in increased and decreased SSMD score, the solid blue line at y = -1.41 represents the curve’s inflection point, and the dashed black line at y = -1 represents the SSMD (open circles) cutoff used to define a hit. (E) A plot of SSMD scores measured from each drug at each dose tested, each open circle represents the SSMD score of an individual drug at an individual dose. A total of 2762 drugs were tested, each at 4 different doses (8, 4, 2, and 1 µM; n = 11048). Dashed lines at y = ±1, ±1.25, ±1.645, ±2, ±3, ±5 represent common defining cutoffs of SSMD scores. (F) A dual flashlight plot of each dose of each drug (open circles) SSMD score (y-axis) against fold change (log2-scale, x-axis). Dashed lines at y = -1, y = 1, x = -1.5, and x = 1.5 represent the cut-off to define hits that significantly affect luminescence levels; all significant luminescence-reducing compounds are highlighted in red (n = 50).
Fifty total compounds significantly reduce LipoGlo levels.
Boxplot of the fold change for each of the 50 hit compounds from the Johns Hopkins Drug Library (JHDL) at the respective dose they met hit criteria (fold change (log2) ≤ -1.5 and SSMD ≤ -1). The luminescence of each drug at each dose was measured (n = 8), and fold change was calculated against the mean of vehicle treatment.
Validation of hits reveals different phenotypes for different drugs.
(A-D, F, H) Average fold change of Relative Luminescence Units (RLU) measured from homogenates of 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mCherry-2A-FireflyLuciferase) animals treated for 48 hours with either negative (vehicle), positive (5 µM lomitapide) control, or a serial dilution of the indicated compound. Each data point represents a measurement from an independent animal collected from two (A) or three (B, D, F, H) or four (C) independent experiments and normalized to the average vehicle RLU from each individual experiment. Sample size (n) is listed below each treatment. Statistical significance was determined by one-way ANOVA followed by Dunnett’s test against vehicle, with Bonferroni correction. Significant treatments (red asterisks) and their adjusted p-values are reported below. (A) Pomiferin treatment significantly reduced RLU levels (one-way ANOVA, F(3,40) = 61.85, p = 4.4×10−15). Significant Dunnett’s comparisons: 5 µM lomitapide (p = 7.6×10−11) and 4 µM pomiferin (p = 8.6×10−4). Representative whole-mount images of vehicle, 5 µM lomitapide, 2 µM pomiferin, and 4 µM pomiferin-treated animals are shown. (B) Riboflavin tetrabutyrate: one-way ANOVA F(3,54) = 68.85, p < 2×10−16; significant comparisons: 5 µM lomitapide (p = 5.0×10−7) and 8 µM riboflavin tetrabutyrate (p = 5.2×10−3). (C) Calcipotriene: one-way ANOVA F(3,80) = 61.47, p < 2×10−16; significant comparisons: 5 µM lomitapide (p = 8.0×10−17), 4 µM calcipotriene (p = 2.9×10−8), and 8 µM calcipotriene (p = 1.6×10−6). (D) Doxycycline: one-way ANOVA F(3,58) = 126.5, p < 2×10−16; significant comparison: 5 µM lomitapide (p = 1.7×10−14). (F) Thiethylperazine: one-way ANOVA F(3,48) = 82.76, p < 2×10−16; significant comparisons: 5 µM lomitapide (p = 5.0×10−7), 4 µM thiethylperazine (p = 1.4×10−10), and 2 µM thiethylperazine (p = 4.3×10−3). (H) Prochlorperazine: one-way ANOVA F(3,60) = 66.42, p < 2×10−16; significant comparisons: 5 µM lomitapide (p = 1.6×10−11), 8 µM prochlorperazine (p = 1.2×10−6), and 4 µM prochlorperazine (p = 7.0×10−7). (E, G, I) Representative native-PAGE images of luminescent B-lps from homogenates of 5 dpf animals treated with vehicle, 5 µM lomitapide, or the indicated compound for 48 hours. Each image is a composite of chemiluminescence (B-lps, cyan hot) and fluorescence (DiI-LDL, yellow). B-lps were binned into one of four classes: zero mobility (ZM), very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), or LDL, and visualized via boxplot. Each gel image is representative of one of three independent experiments. Statistical significance was determined by one-way ANOVA and post-hoc Dunnett’s test within each lipoprotein class. Significant comparisons (red asterisks) for each panel: (E) doxycycline ZM class: one-way ANOVA F(3,14) = 5.38, p = 1.1×10−2; significant comparison: 5 µM lomitapide (p = 1.3×10−2). Doxycycline VLDL class: one-way ANOVA F(3,14) = 47.69, p = 1.3×10−7; significant comparisons: 5 µM lomitapide (p = 3.0×10−2), 8 µM doxycycline (p = 3.5×10−3). (G) Thiethylperazine VLDL class: one-way ANOVA F(3,14) = 19.0, p = 3.3×10−5; significant comparisons: 5 µM lomitapide (p = 1.0×10−2), 8 µM thiethylperazine (p = 8.4×10−4). Thiethylperazine IDL class: one-way ANOVA F(3,14) = 20.4, p = 2.2×10−5; significant comparison: 5 µM lomitapide (p = 2.6×10−4). Thiethylperazine LDL class: one-way ANOVA F(3,14) = 24.8, p = 7.2×10−6; significant comparison: 8 µM thiethylperazine (p = 1.2×10−3). (I) Prochlorperazine VLDL class: one-way ANOVA F(3,14) = 24.8, p = 7.4×10−6; significant comparisons: 5 µM lomitapide (p = 3.8×10−6), 8 µM prochlorperazine (p = 9.1×10−4). Prochlorperazine IDL class: one-way ANOVA F(3,14) = 7.5, p = 3.0×10−3; significant comparison: 5 µM lomitapide (p = 2.4×10−3). Prochlorperazine LDL class: one-way ANOVA F(3,14) = 33.8, p = 1.1×10−6; significant comparisons: 5 µM lomitapide (p = 1.2×10−3), 8 µM prochlorperazine (p = 2.3×10−3).
Enoxolone reduces B-lps in larval zebrafish.
(A) Boxplot of the average fold change of Relative Luminescence Units (RLU) measured from fixed 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) animals treated for 48 hours with either negative (vehicle), positive (5 µM lomitapide) control, or an 8-fold serial dilution of enoxolone. Each data point represents a measurement from an independent animal collected from three independent experiments and normalized to the average vehicle RLU from each individual experiment. Several treatments significantly altered RLU levels (one-way ANOVA, F(9,221) = 32.79, p < 2×10−16). Lomitapide treatment (n = 24) significantly reduced RLU levels compared to vehicle treatment (n = 24, Dunnett’s test p = 5.6×10−10). Treatment with 8 µM enoxolone (n = 21, Dunnett’s test p = 7.8×10−11), 4 µM enoxolone (n = 24, Dunnett’s test p = 1.2×10−3), 2 µM enoxolone (n = 23, Dunnett’s test p = 3.2×10−4), 1 µM enoxolone (n = 22, Dunnett’s test p = 5.2×10−3), and 0.5 µM enoxolone (n = 23, Dunnett’s test p = 7.2×10−3) also reduced total RLUs. (B) Boxplot of the average fold change of RLUs measured from homogenized 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) animals that were treated for 48 hours with either vehicle, 5 µM lomitapide, or an 8-fold serial dilution of enoxolone. Several treatments significantly altered RLU levels (one-way ANOVA, F(9,226) = 54.2, p < 2×10−16). Lomitapide treatment (n = 24) significantly reduced RLU levels compared to vehicle treatment (n = 24, Dunnett’s test p = 1.7×10−15), as did 8 µM enoxolone treatment (n = 24, Dunnett’s test p = 6.1×10−6). (C) Boxplot of the average fold change of RLUs measured from untreated homogenates of 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) animals that were briefly treated with either vehicle, 400 nM NanoLuciferase inhibitor, or an 8-fold serial dilution of enoxolone to determine if enoxolone is an inhibitor of NanoLuciferase enzymatic activity. Only one treatment altered RLU levels (one-way ANOVA, F(9,221) = 78.31, p < 2×10−16), which was the positive control of 400 nM NanoLuciferase inhibitor (n = 22) when compared to vehicle treatment (n = 23, Dunnett’s test p = 3×10−14). No enoxolone treatment significantly altered RLU levels when compared to vehicle treatment. (D) Representative whole-mount images of 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) larvae following treatment of vehicle, 5 µM lomitapide, or 8 µM enoxolone for 48 hours. Lomitapide treatment induced a dark yolk phenotype, while no notable phenotypes followed enoxolone treatment. Scale bar represents 1 mm. (E) Representative image of a native-PAGE gel of luminescent B-lps from homogenates of 5 dpf animals treated with vehicle, 5 µM lomitapide, or 8 µM enoxolone for 48 hours. The image is a composite of chemiluminescence (B-lps, cyan hot) and fluorescence (DiI-LDL, yellow). For quantifications, B-lps were binned into one of 4 classes (ZM (zero mobility), very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), or LDL), and these values were visualized via boxplot. The gel image is a representative image of representative samples from one of the three independent experiments performed. * <0.05 as compared to vehicle
Pharmacological inhibition of HNF4⍺ reduces lipoproteins in the larval zebrafish.
(A) Boxplot of the average fold change of Relative Luminescence Units (RLU) measured from fixed 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) animals treated for 48 hours with either negative (vehicle), positive (5 µM lomitapide) control, or an 8-fold serial dilution of BIM5078 or BI6015. Each data point represents a measurement from an independent animal collected from three independent experiments and normalized to the average vehicle RLU from each individual experiment. Several treatments significantly altered RLU levels in the BIM5078 experiment (one-way ANOVA, F(9,216) = 29.61, p < 2×10−16) and BI6015 experiment (one-way ANOVA, F(9,219) = 43.6, p < 2×10−16). In the BIM5078 experiment, only lomitapide treatment (n = 21) significantly reduced RLU levels compared to vehicle treatment (n = 23, Dunnett’s test p = 1.1×10−18). In the BI6015 experiment, lomitapide treatment (n = 21) significantly reduced RLU levels compared to vehicle treatment (n = 24, Dunnett’s test p = 8.3×10−16). Treatment with 8 µM BI6015 (n = 21, Dunnett’s test p = 6.9×10−12) also reduced total RLUs. (B) Boxplot of the average fold change of RLUs measured from homogenized 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) animals that were treated for 48 hours with either vehicle, 5 µM lomitapide, or an 8-fold serial dilution of BIM5078 or BI6015. Several treatments significantly altered RLU levels in the BIM5078 experiment (one-way ANOVA, F(9,221) = 39.61, p < 2×10−16) and BI6015 experiment (one-way ANOVA, F(9,223) = 42.7, p < 2×10−16). In the BIM5078 experiment, lomitapide treatment (n = 22) significantly reduced RLU levels compared to vehicle treatment (n = 24, Dunnett’s test p = 2×10−16). Treatment with 8 µM BIM5078 (n = 24, Dunnett’s test p = 9.1×10−8), 4 µM BIM5078 (n = 24, Dunnett’s test p = 1.2×10−3), and 0.125 µM BIM5078 (n = 23, Dunnett’s test p = 8.3×10−3) also reduce total RLUs. In the BI6015 experiment, lomitapide treatment (n = 22) significantly reduced RLU levels compared to vehicle treatment (n = 24, Dunnett’s test p = 2.3×10−18). Treatment with 8 µM BI6015 (n = 24, Dunnett’s test p = 8.8×10−8), 4 µM BI6015 (n = 23, Dunnett’s test p = 1.2×10−5), and 1 µM BI6015 (n = 24, Dunnett’s test p = 2.1×10−2) also reduce total RLUs. (C) Boxplot of the average fold change of RLUs measured from untreated homogenates of 5 dpf Fus(ApoBb.1-NanoLuciferase); Tg(ubi:mcherry-2A-FireflyLuciferase) animals that were briefly treated with either vehicle, 400 nM NanoLuciferase inhibitor, or an 8-fold serial dilution of BIM5078 or BI6015 to determine if HNF4⍺ inhibitors interfere with NanoLuciferase enzymatic activity. Several treatments significantly altered RLU levels in the BIM5078 experiment (one-way ANOVA, F(9,218) = 90.21, p < 2×10−16) and BI6015 experiment (one-way ANOVA, F(9,224) = 108.7, p < 2×10−16). In the BIM5078 experiment, only the NanoLuciferase inhibitor treatment (n = 22) significantly reduced RLU levels compared to vehicle treatment (n = 23, Dunnett’s test p = 1.4×10−16) and BIM5078 treatment did not alter RLU levels. In the BI6015 experiment, only the NanoLuciferase inhibitor treatment (n = 24) significantly reduced RLU levels compared to vehicle treatment (n = 23, Dunnett’s test p = 7.3×10−17) and BI6015 treatment did not alter RLU levels. (D) Representative image of a native-PAGE gel of luminescent B-lps from homogenates of 5 dpf animals treated with vehicle, 5 µM lomitapide, or 8 µM BIM5078 or 8 µM BI6015 for 48 hours. The image is a composite of chemiluminescence (B-lps, cyan hot) and fluorescence (DiI-LDL, yellow). For quantifications, B-lps were binned into one of 4 classes (ZM (zero mobility), very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), or LDL), and these values were visualized via boxplot. The gel image is a representative image of representative samples from one of the two independent experiments performed.
HNF4⍺ is required for lipoproteins throughout larval development and for the lipoprotein-reducing effect of enoxolone.
(A) Boxplot of normalized Relative Luminescence Units (RLU) measured from homogenized Fus(ApoBb.1-NanoLuciferase)/+ whole animals that were either HNF4⍺+/+, HNF4⍺rdu14/+, or HNF4⍺rdu14/rdu14, collected at 1, 2, 3, 4, and 5 dpf. Data were collected from at least three independent experiments and normalized to the mean of 3 dpf HNF4⍺+/+ animals. Lipoprotein levels change throughout development (two-way ANOVA, F(1) = 261.206, p < 2×10−16) and due to the loss of HNF4⍺ (F(2) = 12.13, p = 6.4×10−6). Compared to their wild-type siblings, HNF4⍺ homozygotes have reduced lipoproteins at 1 dpf (Dunnett’s test, HNF4⍺rdu14/rdu14 n = 29 versus HNF4⍺+/+ n = 30, p = 1.6×10−6), 2 dpf (Dunnett’s test, HNF4⍺rdu14/rdu14 n = 33 versus HNF4⍺+/+ n = 34, p = 6×10−3), 3 dpf (Dunnett’s test, HNF4⍺rdu14/rdu14 n = 69 versus HNF4⍺+/+ n = 78, p = 1.8×10−19), 4 dpf (Dunnett’s test, HNF4⍺rdu14/rdu14 n = 38 versus HNF4⍺+/+ n = 41, p = 1.7×10−5). HNF4⍺ mutants have unchanged lipoproteins at 5 dpf. (B) Boxplot of normalized RLUs measured from homogenized Fus(ApoBb.1-NanoLuciferase)/+ whole animals that were either wild-type, heterozygous, or homozygous (HNF4⍺+/+, HNF4⍺rdu14/+, or HNF4⍺rdu14/rdu14 respectively) and treated with either vehicle or 8 µM enoxolone for 48 hours. The data were collected from two independent experiments and normalized to the mean of vehicle-treated HNF4⍺+/+ animals. Lipoprotein levels were significantly altered by the HNF4⍺ genotype (Two-way ANOVA, F(2) = 3.385, p = 3.6×10−2), drug treatment (F(1) = 22.736, p = 3.9×10−6), and the interaction of the HNF4⍺ genotype and drug treatment (F(2) = 3.136, p = 4.6×10−2). Enoxolone treatment reduced lipoproteins in HNF4⍺+/+ (Dunnett’s test, 8 µM enoxolone n = 28 versus vehicle n = 22, p = 1.7×10−4) and HNF4⍺rdu14/+ (Dunnett’s test, 8 µM enoxolone n = 48 versus vehicle n = 35, p = 4.2×10−2). However, enoxolone treatment did not significantly alter lipoprotein levels in HNF4⍺rdu14/rdu14 animals (Dunnett’s test, 8 µM enoxolone n = 23 versus vehicle n = 24, p = 0.8).
Differential expression analysis throughout enoxolone treatment affects lipid regulatory genes and is similar to the genetic loss of HNF4⍺.
(A) Heat map of differentially expressed (DE) genes following 4, 8-, 12-, 16-, and 24-hours post-enoxolone treatment (hpt), respectively, 39, 34, 57, 118, and 402 genes were differentially expressed with red colors depicting increased and blue colors depicting decreased relative expression levels (log2 fold change). Each column of each heatmap represents a single replicate. (B) Venn diagram of overlapping differentially expressed genes from each treatment duration. Of the total 471 differentially expressed genes, 115 are shared between at least two treatment durations, and only one gene, insig1, is shared by all durations. (C) The early response to enoxolone treatment features 14 differentially expressed genes. Gene ontology analysis of these 14 genes reveals enrichment of lipid regulating pathways. (D) The late response to enoxolone treatment features 34 differentially expressed genes. Gene ontology analysis of these 34 genes reveals carbohydrate-regulating and cell signaling pathway enrichment. (E) Table comparing differentially expressed genes following 4, 8-, 12-, 16-, and 24-hours post-enoxolone treatment to HNF4⍺ knockout, HNF4Ɣ knockout, and HNF4⍺/HNF4Ɣ double knockout. There is considerable overlap between differentially expressed genes following enoxolone treatment and HNF4⍺ knockout, but little overlap with HNF4Ɣ knockout.
