In vitro activity: MGL-3196 is a highly potent and selective agonist of the thyroid hormone receptor β (THR-β) with EC50 value of 0.21 μM. MGL-3196 is 28-fold selective for THR-β over THR-α in a functional assay.

Kinase Assay: The ligand binding domain (amino acids 148–410) of THR-β (H6-THR-β) and the ligand binding domain (amino acids 202–461) of THR-α (H6-THR-α) were cloned into an E. coli expression vector pET28a (Novagen, Milwaukee, WI) that contained a N-terminal hexa His sequence. The resulting recombinant hexa His tagged proteins were produced in E. coli BL21(DE3) cells. Cells were grown in Terrific Broth (in-house prepared medium of Bacto tryptone (3.3%, w/v), Difico yeast extract (2.0%, w/v), and NaCl (0.5%, w/v)) using shake flasks with a 24 h induction in 0.2 mM IPTG at 25 °C, harvested, and lysed with five volumes of buffer A (0.05 M Tris, 0.3 M NaCL, 1%W/V betaine, 0.01 M imidazole, 0.02 M β-mercaptoethanol, pH 8.0). Lysozyme (1.0 mg/mL, Sigma) and Complete protease inhibitor cocktail (Roche Diagnostics Gmbh) were added to slurry, and the solution was sonicated for 1 min five times at 4 °C. The suspension was centrifuged in a Ti45 Beckmann rotor for 2 h at 127 300 RCF, and the supernatant was loaded onto NI_ NTA agarose (Quigen 30210) column. After a washing with buffer A, H6-TRβ or H6-TRα was eluted with buffer A containing 0.25 M imidazole.

THR-β/RXR/GRIP1 Coactivator Peptide Recruitment Assay: An amount of 30 μL of H6-THR-β (50 nM) in 50 mM Hepes, pH 7.0, 1 mM DTT, 0.05% NP40, and 0.2 mg/mL BSA (binding buffer) was mixed with an equal volume of EE-RxRα (50 nM) in binding buffer. An amount of 6 μL of T3 (0–14.8 μM) or test compound (0–1.2 mM) in DMSO was then added and the solution incubated at 37 °C for 30 min. Then 30 μL of biotin-GRIP1 peptide (biotin-Aca-HGTSLKEKHKILHRLLQDSSSPVDL-CONH2) (100 nM) in 30 μL of binding buffer plus 5% DMSO was added and the solution incubated at 37 °C for 30 min. An amount of 30 μL of solution containing 12 nM europium-conjugated anti hexa His antibody and 160 nM APC-conjugated streptavidin in 50 mM Tris, pH 7.4, 100 mM NaCl, and 0.2 mg/mL BSA was added, and the solution was incubated at 4 °C overnight. An aliquot (35 μL/sample) was transferred to 384-well black microtiter plates. The HTRF signal was read on the Victor 5 reader (PerkinElmer Life and Analytical Sciences).

C57Bl/6J-Diet-Induced Obese (DIO) Mice Study: All animal studies were done according to IACUC approved protocols. Six week old C57Bl/6J mice were placed on a high fat diet for 34 weeks. At day 0, 9 mice per group were treated daily doses by gavage with vehicle (2% Klucel LF, 0.1% Tween 80 in water) or 0.3, 1, 3, or 10 mg/kg 53 (MGL-3196) for 23 days. In a parallel study, at day 0, 9 mice per group were treated with daily doses of vehicle (Dulbecco’s phosphate buffered saline, pH adjusted to 9.0 with 1 N NaOH) or 10, 30, or 100 μg/kg T3. Body weight and food intake were monitored during the study. BMD and body composition assessments were made on day 22. On day 23 at necropsy, organ weights were obtained for determination of organ weight and blood samples were assessed for cholesterol and other chemistry parameters.