Journal: PLoS ONE

Article Title: Interaction of the N -(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode

doi: 10.1371/journal.pone.0142711

Figure Lengend Snippet: Structures of the compounds are shown in Panels A-C: A, Flu-AM1; B, Ibu-AM5; C, ibufenac-AM1. The asterisks show the chiral centres. In Panels D-F, the inhibition of 0.5 μM [ 3 H]AEA hydrolysis in rat brain homogenates by the compounds is shown. Data are means ± SEM (when not enclosed by the symbols), N = 3 for the enantiomers of D, Flu-AM1; E, Ibu-AM5 and F, racemic Ibu-AM5 and Ibufenac-AM1.

Article Snippet: K i and α values were obtained using the enzyme kinetics mixed model inhibition curve fitting algorithm available in the GraphPad Prism programme.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Interaction of the N -(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode

doi: 10.1371/journal.pone.0142711

Figure Lengend Snippet: Species-dependent inhibition of FAAH by the enantiomers of Flu-AM1 and Ibu-AM5.

Article Snippet: K i and α values were obtained using the enzyme kinetics mixed model inhibition curve fitting algorithm available in the GraphPad Prism programme.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Interaction of the N -(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode

doi: 10.1371/journal.pone.0142711

Figure Lengend Snippet: Panels A and B show the time-dependencies of ( R )-Flu-AM1 and URB597, respectively. The data are means ± SEM, N = 3. In Panel C, homogenates (at 20-fold normal strength) were preincubated with either vehicle, 2, 4 or 6 μM ( R )-Flu-AM1 for 60 min. Aliquots were then diluted 20-fold and assayed for FAAH activity. These are shown as 2 →0.1, 4 →0.2 and 6 →0.3. Concomitantly, ( R )-Flu-AM1 was added to vehicle-preincubated aliquots to give concentrations of 0.1, 0.2 and 0.3 μM (representing free concentrations after a 20- fold dilution), 2, 4 and 6 μM final concentrations. The panel shows the data as % of corresponding control (means ± SEM, N = 3). For a fully reversible compound, the inhibition seen in the yellow bars (i.e. following the dilution) should be lower than in the purple bars (the inhibition seen at the undiluted concentrations) but equal to the green bars (the free concentrations after the dilution).

Article Snippet: K i and α values were obtained using the enzyme kinetics mixed model inhibition curve fitting algorithm available in the GraphPad Prism programme.

Techniques: Activity Assay, Control, Inhibition

Journal: PLoS ONE

Article Title: Interaction of the N -(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode

doi: 10.1371/journal.pone.0142711

Figure Lengend Snippet: ( R )-Flu-AM1 and B. carprofen. Samples were incubated for 18 h with inhibitor and substrate (0.5 μM [ 3 H]AEA). The concentrations of lysates used were FAAH wt , 0.04 μg/assay, FAAH T488A , 0.4 μg/assay, empty vector 0.36 μg/assay. Shown are means ± SEM, N = 3–6. The pI 50 values, with corresponding IC 50 and n H values in brackets were: ( R )-Flu-AM1: FAAH wt , 5.08±0.03 (8.3 μM; n H = 1.02±0.07); FAAH T488A , 4.55±0.07 (28 μM; n H = 1.47±0.37), FAAH wt + empty vector, 5.10±0.02 (8.0 μM; n H = 0.99±0.06). Carprofen: FAAH wt , 4.43±0.02 (37 μM, n H = 3.00±0.31); FAAH T488A , 4.32±0.04 (48 μM, n H = 2.43±0.37), FAAH wt + empty vector, 4.50±0.01 (32 μM; n H = 4.28±0.41. Note that for carprofen and wild-type FAAH, the analyses suggested that a curve with a maximum inhibition of 82–91% fitted the data better than a curve with 100% maximum inhibition, and these values have been used here.

Article Snippet: K i and α values were obtained using the enzyme kinetics mixed model inhibition curve fitting algorithm available in the GraphPad Prism programme.

Techniques: Incubation, Plasmid Preparation, Inhibition