TABLE 2

5-HT and SERT inhibitors competitively inhibit [3H]DASB and [3H]S-citalopram binding to hSERT For the analysis using Cheng-Prusoff relationship, the IC50 values for radioligand displacement were determined for each unlabeled ligand by coincubation with multiple concentrations of radioligand in at least three separate experiments. These data were pooled to assess the relationship between observed IC50 value and (([A*]/Kd) + 1). Linear regression coefficients (r2) and equilibrium dissociation constants (KB) for 5-HT and SERT inhibitors, as determined from the observed slopes, are presented. For analysis of competitivity by the Scatchard method, the apparent Kd values for [3H]DASB and [3H]S-citalopram binding were determined from the slope of the Scatchard plots in presence of test compound at two concentrations, 1 log unit apart (see 5-HT example in Fig. 4). The Ki of the inhibitor was calculated using the equation Ki = [Inhibitor]/(concentration ratio – 1), in which the concentration ratio was calculated from the apparent Kd value of [3H]DASB or [3H]S-citalopram binding in the presence and the absence of the inhibitor. The average and the standard deviation of the two Ki values derived using the two concentrations of 5-HT or SERT inhibitors are presented. For these analyses, the equilibrium binding affinities of the radioligands, Kd values, were found to be 0.14 and 1.2 nM for [3H]DASB and [3H]S-citalopram, respectively.


Radioligand

Unlabeled Ligand

r2

Cheng-Prusoff Slope (KB)

Scatchard Ki
nM
[3H]DASB 5-HT 0.99 7800 ± 160 3356 ± 1027
DASB 0.87 0.13 ± 0.01 0.09 ± 0.02
S-Citalopram 0.78 1.17 ± 0.07 1.08 ± 0.04
R-Citalopram 0.77 20.8 ± 1.30 42.7 ± 13.7
Paroxetine 0.98 0.03 ± 0.001 0.07 ± 0.05
Clomipramine 0.99 0.06 ± 0.003 0.04 ± 0.01
Fluvoxamine 0.77 1.84 ± 0.19 5.98 ± 1.54
Imipramine 0.89 2.06 ± 0.09 4.88 ± 4.00
Duloxetine 0.99 0.06 ± 0.001 0.06 ± 0.01
Venlafaxine 0.93 13.7 ± 0.65 17.14 ± 3.94
β-CIT 0.97 0.28 ± 0.01 0.44 ± 0.19
Indatraline 0.91 0.32 ± 0.01 0.24 ± 0.03
Cocaine 0.96 400 ± 2 211 ± 117
[3H]S-Citalopram 5-HT 0.96 3300 ± 130 2028 ± 54.6
DASB 0.81 0.19 ± 0.01 0.16 ± 0.02
S-Citalopram 0.88 1.13 ± 0.04 1.08 ± 0.10
Imipramine 0.89 3.90 ± 0.21 5.38 ± 1.80
Duloxetine 0.86 0.15 ± 0.01 0.13 ± 0.02

Venlafaxine
0.96
25.2 ± 0.10
21.52 ± 1.52