Black optiplate 96f

The assay
was run in 96-well microplates (Black OptiPlate-96F; PerkinElmer,
Massachusetts, USA) in a total reaction volume of 200 μL. hNAAA protein preparation (4.0 μg) was preincubated
for 30 min with various concentrations of test compounds or vehicle
control (DMSO 5%) in 100 mM citrate/phosphate buffer (pH 4.5) containing
3.0 mM DTT, 0.1% NP40 0.1%, 0.05% BSA, 150 mM NaCl. N-(4-Methyl-2-oxo-chromen-7-yl)-hexadecanamide (PAMCA) was used as
a substrate (2.0 μM), and the reaction was carried out for 50
min at 37 °C. Fluorescence was measured with an EnVision 2014
Multilabel Reader (PerkinElmer, Massachusetts, USA) using an excitation
wavelength of 355 nm and emission of 460 nm. IC₅₀ values
were calculated by nonlinear regression analysis of log[concentration]/inhibition
curves using GraphPad Prism 5 (GraphPad Software Inc., CA, USA) applying
a standard slope curve fitting. The reported IC₅₀ values
are the mean of at least three independent experiments performed in
three technical replicates.

The fluorescence
assay to measure FAAH activity was performed in 96-well black plates
(Black OptiPlate-96F; PerkinElmer, Massachusetts, USA): 2.5 μg
of hFAAH membrane preparation was preincubated for
50 min at 37 °C in 190 μL of assay buffer (50 mM Tris HCl
pH 7.4, 0.05% fatty acid free BSA), with 5 μL of inhibitor or
5 μL of DMSO to measure FAAH total activity. The background
(no activity) samples were prepared using 190 μL of assay buffer
without hFAAH and 5 μL of DMSO. The reaction
was then started by the addition of 5 μL of substrate (AMC Arachidonyl
Amide, A6855, Merck) dissolved in DMSO and used at a final concentration
of 800 nM. The reaction was carried out for 45 min at 37 °C,
and fluorescence was measured with an EnVision 2014 Multilabel Reader
(PerkinElmer, Massachusetts, USA) (excitation wavelength 355 nm/emission
wavelength 460 nm). The concentration causing half-maximal inhibition
(IC₅₀) was determined by nonlinear regression analysis
of the log[concentration]/response curves generated with mean replicate
values using a four-parameter Hill equation curve fitting with GraphPad
Prism 5 (GraphPad Software Inc., CA, USA). The reported IC₅₀ values are the mean of at least three independent experiments performed
in three technical replicates.

The fluorescence assay
to measure FAAH activity was performed in 96-well black plates (Black
OptiPlate-96 F; PerkinElmer, Massachusetts, USA): 2.5 μg of
human FAAH-1 membrane preparation was preincubated for 50 min at 37
°C, in 190 μL of assay buffer (50 mM Tris-HCl pH 7.4, 0.05%
Fatty acid-free BSA) with 5 μL of inhibitor or 5 μL DMSO
to measure FAAH total activity. The background (no activity) samples
were prepared using 190 μL of assay buffer without human FAAH-1
and 5 μL of DMSO. The reaction was then started by the addition
of 5 μL of substrate (AMC arachidonoyl amide, A6855 Merck) dissolved
in DMSO and used at a final concentration of 800 nM. The reaction
was carried out for 45 min at 37 °C and fluorescence was measured
with the EnVision 2014 Multilabel Reader (PerkinElmer, Massachusetts,
USA) (excitation wavelength, 355 nm/emission wavelength, 460 nm).
The concentration causing half-maximal inhibition (IC₅₀) was determined by non-linear regression analysis of the Log[concentration]/response
curves generated with mean replicate values using a four-parameter
Hill equation curve fitting with GraphPad Prism 5 (GraphPad Software
Inc., CA, USA).