Journal: bioRxiv

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: 10.1101/2022.10.15.512369

Figure Lengend Snippet: (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

Article Snippet: Cultures of E. coli Shuffle T7 competent cells (New England Biolabs, Ipswich, MA) transformed with pET19b-Them1 plasmid were grown to an A 600 of ∼ 0.5 – 0.7 in terrific broth followed by induction of recombinant Them1 by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

Techniques: Activation Assay, Activity Assay, Recombinant, Purification, SDS Page, Incubation, Inhibition

Journal: bioRxiv

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: 10.1101/2022.10.15.512369

Figure Lengend Snippet: (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

Article Snippet: Cultures of E. coli Shuffle T7 competent cells (New England Biolabs, Ipswich, MA) transformed with pET19b-Them1 plasmid were grown to an A 600 of ∼ 0.5 – 0.7 in terrific broth followed by induction of recombinant Them1 by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

Techniques: Recombinant, Purification, SDS Page

Journal: bioRxiv

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: 10.1101/2022.10.15.512369

Figure Lengend Snippet: Pre-dialysis reactions were performed with recombinant His-tagged human Them1 (Them1) and myristoyl (C14)-CoA. Reactions subjected to an overnight dialysis were incubated with C14-CoA. ( A ) Time course of Pre- and Post- dialysis reactions for the indicated timepoints. ( B ) Pre- and Post-reactions consisting of compounds U1 , W1 or Ebselen [(-) control]. Each graph is representative of four independent experiments. ( C ) Left panels , recombinant His-tagged mouse Them1 was purified from BL21 (DE3) E. coli competent cells on a HisTrap HP column. Upper right panel , SDS-PAGE of recombinant mouse Them1. Right panel , saturation curve of recombinant mouse Them1 (125 nM) and C14-CoA reactions (black dots) was used to determine steady state kinetic constants. ( D ) All reactions were performed with either recombinant mouse Them1 or recombinant human Them1. Acot activities quantified as IC 50 values are shown. Data represent the mean ± s.e.m. of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes. ***, P<0.001; Pre- ( U1 ) vs. Post- ( U1 ); ###, P<0.001; Pre- ( W1 ) vs. Post- ( W1 ).

Article Snippet: Cultures of E. coli Shuffle T7 competent cells (New England Biolabs, Ipswich, MA) transformed with pET19b-Them1 plasmid were grown to an A 600 of ∼ 0.5 – 0.7 in terrific broth followed by induction of recombinant Them1 by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

Techniques: Recombinant, Incubation, Purification, SDS Page

Journal: bioRxiv

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: 10.1101/2022.10.15.512369

Figure Lengend Snippet: (A) Schematic showing Them1-mediated de-activation of myristoyl (C14)-CoA into myristic acid plus CoA. Free CoA liberated by Them1 activity covalently binds to a non-fluorescent detection reagent, generating a fluorescent product. (B) Recombinant His-tagged human Them1 (Them1) purified from Shuffle T7 competent cells on a HisTrap HP column (Black line). Upper right panel , SDS-PAGE of Them1 (67 kD). (C) Recombinant Them1 and C14-CoA (25 μM) reactions (black dots). (D) Reactions consisting of Them1 and C14-CoA (white dots, 0 μM; black dots, 25 μM) were incubated for the indicated timepoints. (E) Saturation curve of Them1 and C14-CoA reactions (black dots) were used to determined steady state kinetic constants. (F) A pilot small-molecule screen was performed using the library of pharmacologically active compounds (LOPAC, 12.5 μM, 1,056 compounds) with Them1 and C14-CoA as substrate. Compounds were considered as potential inhibitors when Them1 activity was inhibited based on a normalized percent inhibition (NPI) β 30. A representative plate with Z’ factor and 2 compounds fulfilling the inhibitor criteria are shown. Negative values are attributable to the absorbance of a minority of compounds. (G) Reproducibility of the pilot small molecule screen was assessed by NPI values of compounds over 2 independent days. Correlation between d 1 and 2 is indicated by the purple line; R 2 = 0.93. Data represent the mean (s.e.m.) of triplicate determinations. Where not visible, standard error bars are contained within the symbol sizes.

Article Snippet: These plasmids were transformed into E. coli strains BL21 (DE3) (New England Biolabs; Ipswich, MA) or Shuffle T7 competent cells (New England Biolabs, Ipswich, MA), grown to an A 600 of 0.5 – 0.7 in terrific broth and then induced by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

Techniques: Activation Assay, Activity Assay, Recombinant, Purification, SDS Page, Incubation, Inhibition

Journal: bioRxiv

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: 10.1101/2022.10.15.512369

Figure Lengend Snippet: (A-G) Acot isoform panels : Left panels , recombinant Acot isoform enzymes including type I [Acot1, Acot2] and type II [Acot9, Acot12 and Acot13], as well as a truncated Them1 either containing only the 2 thioesterase domains but lacking the START domain (Them1-ΔSTART) or the START domain but lacking the 2 thioesterase domains (START) were purified from Shuffle T7 or BL21 (DE3) E. coli competent cells on a HisTrap HP column unless otherwise specified. Recombinant START was further purified using a Superdex 75 column. Recombinant Acot9 was purified on a MBPTrap HP column. Upper right panel , SDS-PAGE of recombinant Acot isoform enzymes. Right panel , saturation curves of recombinant Acot isoform enzymes and myristoyl (C14)-CoA reactions (black dots) were used to determine steady state kinetic constants. Where not visible, standard error bars are contained within the symbol sizes. ( H ) Table , steady state kinetic constants (K m , V max , K cat and K cat /K m ) were determined as described in the text. Data represent the mean (s.e.m.) of triplicate determinations.

Article Snippet: These plasmids were transformed into E. coli strains BL21 (DE3) (New England Biolabs; Ipswich, MA) or Shuffle T7 competent cells (New England Biolabs, Ipswich, MA), grown to an A 600 of 0.5 – 0.7 in terrific broth and then induced by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C.

Techniques: Recombinant, Purification, SDS Page