Prism v 2

Data generated was statistically evaluated by Student’s t-test and 2-way ANOVA using the statistical analyses package of GraphPad prism (V2.0).

Binding assays were carried out by using plasma membranes (12 μg/well)
expressing human dopamine D₁R (Perkin Elmer). Membranes
were incubated in a Multiscreen FC 96-well plate (Millipore) with
0.7 nM [³H]SCH23390 (Perkin Elmer) and increasing concentrations
of the test compound in assay buffer (50 mM Tris–HCl, 5 mM
MgCl₂; pH 7.4) for 1 h at 27 °C. After this time,
the well content was filtered through a Millipore manifold and membranes
were washed four times with assay buffer. The plate was dried and
radioactivity was measured in a Microbeta Trilux liquid scintillation
reader (Perkin Elmer). Non-specific binding was determined in the
presence of 1 μM (+)-butaclamol. Data were normalized to the
percentage of specific binding and fitted to a 4-parameter logistic
equation using with Prism v2.1 software (GraphPad Inc). For the calculation
of the affinity ratio of DA in the presence of the allosteric modulators,
data were fitted according to the equations reported by Lazareno and
Birdsall.⁴⁴

Functional studies at human D₁ and D₂ receptors were carried out in endogenously expressed human D₁R in the SK-N-MC cell line (ATCC), and recombinant human D₂R stably transfected in an in-house CHO cell line. Cells were
thawed and seeded into a black 96-well plate (1 × 10⁴ cells/well for SK-N-MC cell line, 5 × 10³ cells/well
for CHO cell line) in Opti-MEM containing 500 μM IBMX (3-isobutyl-1-methylxanthine).
Test compounds were added to the cells and incubated for 15 min at
25 °C for D₁R, and for 5 min at 37 °C for D₂R. After this time, dopamine was added to the corresponding
wells and incubated for additional 15 min at 25 °C for D₁R, and 10 min at 37 °C for D₂R. Next, treated
cells were lysed (for the D₂R assay, cells were previously
treated with 10 μM forskolin for 5 min at 37 °C) and the
cAMP concentration was measured by HTRF, using a kit from Cisbio.
HTRF was read in a Tecan M1000 Genius Pro reader (λ_excitation = 320 nm, λ_emission = 620 and 665 nm, 30 flashes),
and data were normalized to the dopamine maximum effect and fitted
to a 4-parameter logistic equation by using Prism v2.1 software (GraphPad
Inc).