Journal: Molecular Metabolism

Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

doi: 10.1016/j.molmet.2023.101832

Figure Lengend Snippet: Small molecule inhibitors targeting Them1 activity identified by high-throughput screening. Reactions were performed in 384-well microplates with recombinant His-tagged human Them1 (Them1; 125 nM), myristoyl (C 14 )-CoA (C 14 -CoA; 25 μM) and compounds (12.5 μM), and incubated for 60 min at 22 °C. (A) Z′ factors were calculated from reactions containing no enzyme [(+) control] or no C 14 -CoA [(−) control; 25 μM]. Plate ID indicates the unique number assigned for each plate in the compound library. (B) Scatter plot for the normalized percent inhibition (NPI) of 360,705 library compounds (yellow dots [(+) control)]; green dots [(−) control] and gray dots [compounds (12.5 μM)]). (C) Representative 384-well microplate from the high-throughput screen. Compounds were considered as potential inhibitors based on a NPI ≥ 30%. Negative values are attributable to the absorbance of a minority of compounds. The Z′ factor is shown. (D) Representative compounds that exhibited inhibition of Them1 activity (open circles) or exceeded the maximum IC 50 screening criteria of 20 μM (closed circles). Data represent mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

Article Snippet: Primary brown adipocytes cultured from C57BL/6 mice were seeded at 2,000 per well into white 384-well transparent bottom opaque microplates (GreinerBio One International AG; Kremsmünster, Austria) pre-coated with rat tail collagen (Sigma Aldrich; St. Louis, MO) at 22 °C and treated for 48 h with DMSO (Control) or compounds (250 μM–7.7 nM) in growth medium.

Techniques: Activity Assay, High Throughput Screening Assay, Recombinant, Incubation, Control, Drug discovery, Inhibition

Journal: Molecular Metabolism

Article Title: High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

doi: 10.1016/j.molmet.2023.101832

Figure Lengend Snippet: START domain-dependent inhibition of Them1 activity by freshly synthesized compound U1 and novel structural derivatives. Scaffold modifications of freshly synthesized compound U1 and novel structural derivatives ( U6 – U17 ). Reactions were performed in 384-well microplates with recombinant His-tagged human Them1 (Them1; 125 nM), myristoyl-CoA (C 14 -CoA; 25 μM) and compounds, and incubated for 60 min at 22 °C. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, error bars are contained within the symbol sizes.

Article Snippet: Primary brown adipocytes cultured from C57BL/6 mice were seeded at 2,000 per well into white 384-well transparent bottom opaque microplates (GreinerBio One International AG; Kremsmünster, Austria) pre-coated with rat tail collagen (Sigma Aldrich; St. Louis, MO) at 22 °C and treated for 48 h with DMSO (Control) or compounds (250 μM–7.7 nM) in growth medium.

Techniques: Inhibition, Activity Assay, Synthesized, Recombinant, Incubation