A whole-animal phenotypic drug screen identifies suppressors of atherogenic lipoproteins
- Department of Biology, Johns Hopkins University, United States
- Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, United States
- Department of Pharmacology and Molecular Sciences, Johns Hopkins University, United States
- Department of Chemistry, Johns Hopkins University, United States
- The Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University, United States
- Department of Genetic Medicine, Johns Hopkins University, United States
- Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, United States
Figures
Figure 1 with 1 supplement
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 (fourfold dilution; 8, 4, 2, and 1 µM) of two different drugs of interest. Each treatment was prepared with eight 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-hr 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 hr with either negative (vehicle) or positive (5 µM lomitapide) control. Each data point represents the average of eight 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 four different doses (8, 4, 2, and 1 µM; n = 11,048). Dashed lines at y = ±1, ±1.25, ±1.645, ±2, ±3, and ±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 cutoff to define hits that significantly affect luminescence levels; all significant luminescence-reducing compounds are highlighted in red (n = 50).
Figure 1—figure supplement 1
Forty-nine unique compounds reduce B-lp levels in a high-throughput drug screen to identify modulators of B-lps in larval zebrafish.
Boxplots of each of the 50 (49 unique) B-lp-lowering compounds from the initial drug screen of 2762 compounds, hits are compounds defined as having at least one dose test result in a fold change (log2 scale) of ≤–1.5 and strictly standardized mean difference (SSMD) ≤–1.0. Open circles represent each sample; sample size (n) and SSMD scores are listed below each treatment. The B-lp-reducing hits are (A) unknown, (B) 3-methylcholanthrene, (C) acetaminophen, (D) bismuth (III) oxychloride, (E) cetrimonium bromide, (F) cyproterone, (G) cytochalasin N, (H) calcipotriene, (I) cinnamon oil, (J) cupric chloride, (K) cytidine 5′-monophosphate, (L) cytidine 5′-diphosphate trisodium salt, (M) danazol, (N) danthron, (O) demecarium bromide, (P) diphenylboric acid, (Q) disodium fluorophosphate, (R) doxycycline, (S) emodin, (T) enoxolone, (U) fenbendazole, (V) fendiline hydrochloride, (W) frequentine, (X) fendiline, (Y) ferron, (Z) gentian violet, (AA) hydroxyurea, (AB) ketoprofen, (AC) medroxyprogesterone acetate, (AD) maleic acid, (AE) myristic acid, (AF) NADIDE, (AG) nabumetone, (AH) onion oil, (AI) pergolide mesylate, (AJ) pimethixene maleate, (AK) piperacillin sodium, (AL) pomiferin, (AM) peanut oil, (AN) polysorbate 65, (AO) prochlorperazine dimaleate, (AP) reserpine, (AQ) riboflavin tetrabutyrate, (AR) ricobendazole, (AS) strophanthin K, (AT) sulconazole, (AU) triptonide, (AV) thiethylperazine malate, (AW) thonzonium bromide, and (AX) verteporfin.
Figure 2
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.
Figure 3 with 1 supplement
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 hr 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 hr. 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).
Figure 3—figure supplement 1
Validation of B-lp levels following treatment of 30 hits identified from a high-throughput drug screen of B-lp modulators.
Of the 49 identified B-lp-reducing compounds, we subjected 30 to further validation studies. We examined the effects of each compound with an eightfold serial dilution from 8 to 0.0625 µM. Open circles represent each sample. Sample size (n) is listed below each treatment. Results were analyzed using one-way ANOVA to determine if the means of any treatment were significantly different from each other. When the one-way ANOVA was significant (p ≤ 0.05), a Dunnett’s test was conducted to determine which treatments differed significantly from vehicle treatment. Each p-value was adjusted for multiple comparisons with a Bonferroni correction and is listed on each graph. The compounds examined are (A) 3-methylcholanthrene, (B) acetaminophen, (C) cetrimonium bromide, (D) cyproterone, (E) calcipotriene, (F) cytidine 5’-monophosphate, (G) danazol, (H) danthron, (I) demecarium bromide, (J) doxycycline, (K) emodin, (L) enoxolone, (M) fenbendazole, (N) fendiline, (O) ferron, (P) hydroxyurea, (Q) ketoprofen, (R) medroxyprogesterone acetate, (S) maleic acid, (T) NADIDE, (U) nabumetone, (V) pergolide mesylate, (W) pimethixene maleate, (X) piperacillin sodium, (Y) pomiferin, (Z) prochlorperazine dimaleate, (AA) reserpine, (AB) riboflavin tetrabutyrate, (AC) ricobendazole, (AD) strophanthin K, (AE) sulconazole, (AF) triptonide, (AG) thiethylperazine malate, (AH) thonzonium bromide, and (AI) verteporfin.
Figure 4 with 1 supplement
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 hr with either negative (vehicle), positive (5 µM lomitapide) control, or an eightfold 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 hr with either vehicle, 5 µM lomitapide, or an eightfold 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 eightfold 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 hr. 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 hr. 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 four 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.
Figure 4—figure supplement 1
High-dose enoxolone-treated animals are shorter than vehicle-treated animals.
Boxplots of standard-length measurements of 5 dpf animals treated for 48 hr with vehicle, 5 µM lomitapide, and 8 or 4 µM enoxolone. After treatment, animals were imaged, and standard lengths were measured from the images. Animals treated with 5 µM lomitapide or 8 µM enoxolone were significantly shorter than vehicle-treated animals (one-way ANOVA, F(3,111) = 12.68, p = 3.5 × 10–7; Dunnett’s test 5 µM lomitapide n = 24 versus vehicle n = 32, p = 4.9 × 10–2; Dunnett’s test 8 µM enoxolone n = 27 versus vehicle n = 32, p = 9.1 × 10–6). Animals treated with 4 µM enoxolone lengths were unchanged compared to vehicle treatment (Dunnett’s test 4 µM enoxolone n = 32 versus vehicle n = 32, p = 0.095).
Figure 5
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 hr with either negative (vehicle), positive (5 µM lomitapide) control, or an eightfold 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 hr with either vehicle, 5 µM lomitapide, or an eightfold 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 eightfold 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 hr. 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 four 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.
Figure 6
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 hr. 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).
Figure 7 with 1 supplement
Differential expression analysis throughout enoxolone treatment affects lipid regulatory genes and is similar to the genetic loss of HNF4⍺.
(A) Heatmap of differentially expressed (DE) genes following 4, 8-, 12-, 16-, and 24-hr 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-hr 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.
Figure 7—figure supplement 1
Summary analysis of differentially expressed genes following enoxolone treatment.
(A) Principal component analysis (PCA) of RNAseq samples for treatment and the duration of treatment. (B) Boxplot summarizing the expression pattern of insig1 following the vehicle and 8 µM enoxolone treatment. Insig1 is highly expressed in enoxolone-treated animals at every duration measured. Each data point is the transcripts per million (TPM) of an individual sample. (C) Gene ontology (GO) analysis for the biological process of differentially expressed genes at 4-hr post-treatment (hpt). (D) GO analysis for biological process of differentially expressed genes at 8 hpt. (E) GO analysis for biological process of differentially expressed genes at 12 hpt. (F) GO analysis for biological process of differentially expressed genes at 16 hpt. (G) GO analysis for biological process of differentially expressed genes at 24 hpt.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Danio rerio) | Fus(ApoBb.1-NanoLuciferase); ‘LipoGlo’ | Thierer et al., 2019; https://doi.org/10.1038/s41467-019-11259-w | ZFIN: ZDB-ALT-191218-4 | NanoLuciferase knocked in at the endogenous apoBb.1 locus; reporter of ApoB-containing lipoproteins. Maintained in the S.A. Farber laboratory. |
| Genetic reagent (D. rerio) | hnf4a rdu14; HNF4⍺rdu14 | Davison et al., 2017; https://doi.org/10.1101/gr.220111.116 | ZFIN: ZDB-FISH-171107-24 | Loss-of-function allele provided by the J.F. Rawls laboratory; crossed into the Fus(ApoBb.1-NanoLuciferase) background for this study. |
| Sequence-based reagent | rdu14_F | This paper | PCR primer | 5′-TGATTCACACTACTTACTTGTCTAG-3′; forward primer for genotyping the rdu14 allele |
| Sequence-based reagent | rdu14_R | This paper | PCR primer | 5′-GATTAAAAGTAGTTATCTCATCCTCAG-3′; reverse primer for genotyping the rdu14 allele |
| Chemical compound, drug | Johns Hopkins Drug Library (JHDL) | Shim and Liu, 2014; https://doi.org/10.7150/ijbs.9224 | N/A | 2934 compounds supplied at 10 mM in DMSO; 2762 screened. Provided by the J.O. Liu laboratory. |
| Chemical compound, drug | Lomitapide | Aegerion Pharmaceuticals | Cat#AEGR-733 | MTP inhibitor; positive control at 5 µM on every screening plate |
| Chemical compound, drug | Enoxolone (18β-glycyrrhetinic acid) | Cayman Chemical | Cat#11845; Lot#0505871-11; Lot#619606 | Primary lead compound; screen hit re-purchased for validation and all secondary studies. Tested at 8–0.0625 µM. |
| Chemical compound, drug | BI6015 | Cayman Chemical | Cat#12032 | HNF4⍺ antagonist |
| Chemical compound, drug | BIM5078 | Cayman Chemical | Cat#12031 | HNF4⍺ antagonist |
| Chemical compound, drug | DiI-labeled human LDL | Thermo Fisher Scientific | Cat#L3482 | Migration standard loaded on every native-PAGE gel |
| Chemical compound, drug | NanoLuciferase inhibitor | Promega | Cat#CS1576A01 | Positive control at 400 nM in NanoLuciferase enzymatic activity assays (Figures 4C and 5C) |
| Chemical compound, drug | 3-Methylcholanthrene | Enzo Life Sciences | Cat#BML-GR239-0010; Lot#5111807 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Acetaminophen | Cayman Chemical | Cat#10024; Lot#0489606-25 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Cetrimonium bromide | Sigma-Aldrich | Cat#PHR2688; Lot#LRAC4761 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Cyproterone | Santa Cruz Biotechnology | Cat#sc-278913; Lot#62519 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Calcipotriene | Cayman Chemical | Cat#10009599; Lot#0493709-30 | Primary screen hit re-purchased for validation; secondary characterization in Figure 3C |
| Chemical compound, drug | Cinnamon oil | Sigma-Aldrich | Cat#W229202; Lot#MKCH6102 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Cytidine 5′-monophosphate | Cayman Chemical | Cat#29340; Lot#0574850-1 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Danazol | Cayman Chemical | Cat#16471; Lot#0467035-15 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Danthron | Sigma-Aldrich | Cat#D108103; Lot#WXBC6772V | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Demecarium bromide | United States Pharmacopeia | Cat#1169001; Lot#G0G185 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Doxycycline | Cayman Chemical | Cat#14422; Lot#0459117-85 | Primary screen hit re-purchased for validation; secondary characterization in Figure 3D, E |
| Chemical compound, drug | Emodin | Sigma-Aldrich | Cat#E7881; Lot#04K35051 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Fenbendazole | Cayman Chemical | Cat#19687; Lot#0484070-15 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Fendiline; fendiline hydrochloride | Tocris Bioscience | Cat#6407; Lot#IA/206580 | Single stock used for both entries; fendiline was tested twice in the primary screen (Figure 2) |
| Chemical compound, drug | Ferron | Sigma-Aldrich | Cat#55370; Lot#BCBW9936 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Gentian violet | Sigma-Aldrich | Cat#C6158; Lot#MKBW7836V | Primary screen hit re-purchased for validation; lethal at all doses tested (Supplementary file 1C) |
| Chemical compound, drug | Hydroxyurea | Cayman Chemical | Cat#23725; Lot#0517543-13 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Ketoprofen | Cayman Chemical | Cat#10006661; Lot#0533033-20 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Medroxyprogesterone acetate | Cayman Chemical | Cat#23664; Lot#0569166-4 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Maleic acid | Sigma-Aldrich | Cat#M0375; Lot#SLBZ5070 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | NADIDE | Sigma-Aldrich | Cat#N1511; Lot#SLBZ5280 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Nabumetone | Cayman Chemical | Cat#20251; Lot#0599492-1 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Pergolide mesylate | Cayman Chemical | Cat#26085; Lot#0539883-2 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Pimethixene maleate | Supelco | Cat#P4545; Lot#117F0852 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Piperacillin sodium | Cayman Chemical | Cat#20766; Lot#0592623-4 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Pomiferin | Sigma-Aldrich | Cat#PH002052 | Primary screen hit re-purchased for validation; secondary characterization in Figure 3A |
| Chemical compound, drug | Prochlorperazine dimaleate | Cayman Chemical | Cat#20742; Lot#0531409-8 | Primary screen hit re-purchased for validation; secondary characterization in Figure 3H, I |
| Chemical compound, drug | Reserpine | Sigma-Aldrich | Cat#83580; Lot#WXBD4843V | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Riboflavin tetrabutyrate | Combi-Blocks | Cat#QE-8262; Lot#A50116 | Primary screen hit re-purchased for validation; secondary characterization in Figure 3B |
| Chemical compound, drug | Ricobendazole | Cayman Chemical | Cat#21880; Lot#0502730-9 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Strophanthin K | Sigma-Aldrich | Cat#S355445; Lot#B02648050 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Sulconazole | Cayman Chemical | Cat#23800; Lot#0518706-2 | Primary screen hit re-purchased for validation; increased B-lp levels (Supplementary file 1C) |
| Chemical compound, drug | Triptonide | Sigma-Aldrich | Cat#SMB00325; Lot#SLBX3901 | Primary screen hit re-purchased for validation studies (Supplementary file 1C) |
| Chemical compound, drug | Thiethylperazine malate | United States Pharmacopeia | Cat#1658008; Lot#R057V0 | Primary screen hit re-purchased for validation; secondary characterization in Figure 3F, G |
| Chemical compound, drug | Thonzonium bromide | Cayman Chemical | Cat#31253; Lot#0591856-1 | Primary screen hit re-purchased for validation; increased B-lp levels (Supplementary file 1C) |
| Chemical compound, drug | Verteporfin | Sigma-Aldrich | Cat#129497-78-5; Lot#111385 | Identified as a direct NanoLuciferase inhibitor (Supplementary file 1C). |
| Commercial assay, kit | Nano-Glo Luciferase Assay System | Promega | Cat#N1110 | NanoGlo buffer and substrate; used for all luminescence assays and LipoGlo electrophoresis imaging |
| Commercial assay, kit | Direct-zol RNA Microprep Kit | Zymo Research | Cat#R2060 | Total RNA extraction from pools of five larvae |
| Commercial assay, kit | TruSeq Stranded mRNA Library Prep Kit | Illumina | Cat#20020595 | Libraries prepared from ~500 ng total RNA |
| Commercial assay, kit | TruSeq RNA CD Index Plate | Illumina | Cat#20019792 | 8x8 indexing |
| Commercial assay, kit | GoTaq DNA Polymerase | Promega | Cat#M3001 | Genotyping PCR for the rdu14 allele |
| Commercial assay, kit | cOmplete EDTA-free Protease Inhibitor Cocktail | Roche | Cat#11873580001 | Component of the larval homogenization buffer |
| Software, algorithm | FIJI (ImageJ) v2.0.0 | National Institutes of Health | RRID:SCR_002285 | Image rotation and cropping, standard-length measurement, native-PAGE plot profiles |
| Software, algorithm | R | R Foundation for Statistical Computing | RRID:SCR_001905 | Fold change and SSMD calculation, statistical testing, and plotting |
| Software, algorithm | DESeq2 v1.42 | Bioconductor | RRID:SCR_015687 | Differential expression analysis |
| Software, algorithm | sva | Bioconductor | RRID:SCR_012836 | Batch correction of 8 hpt RNAseq samples |
| Software, algorithm | topGO | Bioconductor | RRID:SCR_014798 | Gene ontology analysis |
| Software, algorithm | nf-core/rnaseq v3.11.1 | Ewels et al., 2020; https://doi.org/10.1038/s41587-020-0439-x | DOI:10.1038/s41587-020-0439-x | Adapter trimming, rRNA filtering, and mapping to GRCz11 with Ensembl 110 annotation |
| Software, algorithm | Enrichr | Chen et al., 2013; https://doi.org/10.1186/1471-2105-14-128 | RRID:SCR_001575 | Gene set enrichment analysis of enoxolone-responsive genes |
| Software, algorithm | Custom analysis code | This paper | Source code 1 | All R scripts used for screen analysis, statistics, and figure generation |
Additional files
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Supplementary file 1
Tables assocaited with JHDL data, analysis, hits, RNAseq and downstream analysis.
(A) Raw data from JHDL primary screen. (B) Analyzed summary data from JHDL primary screen. (C) List of hits from primary screen with results of secondary screening validation studies. (D) Full RNAseq results at all time points following enoxolone treatment. (E) Differentially expressed genes at each time post enoxolone treatment. (F) Gene ontology at each time post enoxolone treatment. (G) Overlap of enoxolone-responsive differentially expressed genes with HNF4α and HNF4γ knockout datasets.
- https://cdn.elifesciences.org/articles/105314/elife-105314-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/105314/elife-105314-mdarchecklist1-v1.docx
-
Source code 1
Scripts and associated raw data for analysis of all figures throughout manuscript.
- https://cdn.elifesciences.org/articles/105314/elife-105314-code1-v1.zip
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A whole-animal phenotypic drug screen identifies suppressors of atherogenic lipoproteins
eLife 14:RP105314.
https://doi.org/10.7554/eLife.105314.3
