Fig 1.
Chemical structures of test compounds.
Chemical structures of (A) the natural THR ligand triiodothyronine, T3, (B) sobetirome, GC-1, (C) resmetirom, MGL-3196, (D) VK2809, and VK2809A, which is produced by CYP3A-mediated cleavage of VK2809 after first pass intrahepatic activation.
Table 1.
THRα/β EC50 values and selectivity of test compounds.
Fig 2.
Differential gene expression in Huh-7 cells and PHH resulting from treatment with THR agonists.
(A) Illustration of the in vitro, hepatic cell-based differential gene expression assay design. (B) THRB and THRA RNA levels were quantified by RT-qPCR in HepG2 (n = 3), Huh-7, (n = 3), and PHH (n = 5) cells. Mean RQ values ± SEM are reported with means annotated within the bars. (C) Huh-7 cells were treated with increasing doses of T3 (n = 2), GC-1 (n = 2), or MGL-3196 (n = 2) for 24 hrs. ANGPLT4, CPT1A, and DIO1 RNA levels were quantified by RT-qPCR and dose-response curves were generated for each gene-compound combination. Mean EC50 values (red bar) and individual replicate EC50 values (black symbols) are reported. (D) Huh-7 cells were treated with increasing doses of T3 (black), GC-1 (red), MGL-3196 (green), VK2809A (solid blue), or VK2809 (dashed blue) for 24 hrs. CPT1A RNA levels were quantified by RT-qPCR. Representative mean RQ values at each compound concentration and fitted dose-response curves are reported. (E) PHH were treated with increasing doses of T3 (black), GC-1 (red), MGL-3196 (green), VK2809A (solid blue), or VK2809 (dashed blue) for 24 hrs. THRSP RNA levels were quantified by RT-qPCR. Representative mean RQ values at each compound concentration and fitted dose-response curves are reported. (F) EC50 values for every test compound were calculated from dose-response curves generated from the TR-FRET THRβ, luciferase (Luc) reporter THRβ, Huh-7 differential gene expression (RQ), and PHH RQ assays (data reported in Tables 1 and 2). Mean EC50 values ± SEM are reported.
Table 2.
Differential gene expression assay EC50 values in Huh-7 cells and PHH.
Fig 3.
Modulation of serum lipid levels after a single-dose treatment of MGL-3196 or T3 in HFD fed rats.
(A) Illustration of in vivo study design. (B) After two weeks of ND or HFD consumption and before compound treatment, total cholesterol levels of all animals were measured. Animals were then randomized into treatment groups: ND: Vehicle (n = 20); HFD: Vehicle (n = 20), 5.0 mg/kg MGL-3196 (n = 12), 1.5 mg/kg MGL-3196 (n = 20), 0.5 mg/kg MGL-3196 (n = 6), and 0.5 mg/kg T3 (n = 6). Total cholesterol level means ± SEM are reported. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA and the mean of each group was compared to the mean of every other group; ‘a’ is statistically significant from ‘b’ with P < 0.01. (C) LDL-C was measured in the same animals described in B). LDL-C level means ± SEM are reported. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests and the mean of each group was compared to the mean of every other group; ‘a’ is statistically significant from ‘b’ with P < 0.01. (D) The rats described above were then dosed once with their assigned treatments. Twenty-four hrs later, total cholesterol levels of all animals were measured. Results are presented as percent change between pre-dose and post-dose total cholesterol levels of individual animals. Percent change means ± SEM are reported with mean values annotated within the bars. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests and the mean of each group was compared to the mean of the HFD fed, vehicle-control group; ****P < 0.0001. (E) LDL-C measurements were obtained from the same rats described in D). Results are presented as percent change between pre-dose and post-dose LDL-C levels of individual animals. Percent change means ± SEM are reported with mean values annotated within the bars. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests and the mean of each group was compared to the mean of the HFD fed, vehicle-control group; *P < 0.05, ****P < 0.0001. (F) Triglyceride measurements were obtained from the same rats described in D). Results are presented as percent change between pre-dose and post-dose triglyceride levels of individual animals. Percent change means ± SEM are reported with mean values annotated within the bars. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests and the mean of each group was compared to the mean of the HFD fed, vehicle-control group; ***P < 0.001, ****P < 0.0001. (G) Plasma compound concentration in MGL-3196-treated animals was determined using LC-MS/MS. Results are presented as the mean plasma AUC0-24hrs with mean values annotated above the bars.
Fig 4.
Modulation of liver gene expression after single-dose treatment of MGL-3196 or T3 in HFD fed rats.
(A) After two weeks of consuming a HFD, animals were dosed once with vehicle (n = 6), 5.0 mg/kg MGL-3196 (n = 3), or 1.5 mg/kg MGL-3196 (n = 3). Four hrs later, the animals were sacrificed and liver Cpt1a, Dio1, Me1, and Thrsp RNA levels were quantified by RT-qPCR. Results are presented as expression relative to the expression levels in vehicle-control rats. Mean RQ values ± SEM are reported. (B) Animals were treated as in A) except that they were sacrificed 24 hrs post-dosing and gene expression analysis was conducted in the same manner. Results are presented as expression relative to the expression levels in vehicle-control rats. Mean RQ values ± SEM are reported. (C) The rats described in Fig 3 were sacrificed 24 hrs after compound treatment and liver Dio1 RNA levels were quantified by RT-qPCR. Results are presented as expression relative to the expression levels in HFD fed, vehicle-control rats. Mean RQ values ± SEM are reported with mean values annotated within the bars. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests on the ΔΔCt values. The mean of each group was compared to the mean of the HFD fed, vehicle-control group; **P < 0.01, ****P < 0.0001. (D) Liver Me1 expression was quantified in the same rats as described in C) and in the same manner. Results are presented as expression relative to the expression levels in HFD fed, vehicle-control rats. Mean RQ values ± SEM are reported with mean values annotated within the bars. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests on the ΔΔCt values. The mean of each group was compared to the mean of the HFD fed, vehicle-control group; *P < 0.05, **P < 0.01, ****P < 0.0001.