Fig 1.
Synthesis scheme of multifunctional antidepressants and reference compounds.
(A) Reference substances: overview of clinically active antidepressants (1, 2, 3, 4, 5); (B) Synthesis scheme of azidobupramine: a) NaN3, H2O, RT, 1h. b) NaNO2, 10% HCl c) KOH, MeOH, 60°C, 15h. d) NaH, ClCH2CH2CH2N(CH3)2 (10), 0–60°C, toluene. e) (Boc)2O, DMAP, ACN, RT, 2h. f) TsCl, Et3N, DCM, 0°C. g) NaHMDS, toluene, -78°C to 70°C, 6h. h) 20% TFA, DCM, RT, 4h. i) 1-bromo-3-butyne, K2CO3, KI, 60°C, 12h, acetone.
Table 1.
Equilibrium affinity constants pKi ±SEM for MSBA and pIC50 ±SEM for RBA and corresponding binding affinities (Ki) of 17 to SERT, NET, and DAT; affinity constants were measured using two different technical approaches based on mass spectrometry (MSBA), and based on classical radioligand binding assays (RBA); each data point represents the average value of three independent experiments, each performed in triplicates.
Table 2.
Equilibrium affinity constants (pIC50 ±SEM) and corresponding binding affinities (Ki) of 17, 15, 11, and the antidepressants paroxetine, imipramine (1), desipramine (2), clomipramine (3) and lofepramine (5) at rSERT; the conversion of IC50 to Ki values was carried out according to Cheng-Prusoff while using the actual [3H]-citalopram concentration for each calculation [44]; each data point represents the average value of at least eight independent experiments.
Fig 2.
Binding parameters of 17 and reference compounds for rSERT.
(A) Saturation analysis using 1 μg membrane homogenate per reaction from recombinant rSERT expressing cells with [3H]-citalopram as radio-ligand; the maximal number of binding sites (Bmax±SEM) and equilibrium dissociation constants for the radioligand (Kd±SEM) were calculated by means of non-linear regression analysis (Bmax 77.2±1.8 pmol/mg; Kd 1.8±0.2 nM) and verified after Scatchard transformation (inset: Bmax 80.3±1.5 pmol/mg; Kd 2.1±0.4 nM); each data point represents the average value (±SEM) of 12 independent experiments. (B) 0.5 nM [3H]-citalopram was used for competition experiments; dilutions were prepared from 17, 15, 11, and paroxetine, clomipramine, imipramine, desipramine and lofepramine; each data point represents the average value of at least eight independent experiments.
Fig 3.
Analysis of 17 for PAL and CuAAC.
Membrane fractions were prepared from tetracycline induced (TETi) and non-induced T-REx-SERT cells; differential expression of rSERT was verified by Western blot analysis (lower panel); while the CuAAC-mediated rhodamine-labelling reaction was carried out in all conditions, 17-rSERT complexes are only visualized if rSERT is expressed, 17 is present and the reaction was exposed to UV-light (upper panel, lane5); the blot displayed is a representative example of five independent experiments. The full, uncropped original images of the fluorescence and Western blot analyses are provided in S1 and S2 Figs.
Fig 4.
Competition experiments of azidobupramine (17) binding at enriched rSERT by paroxetine (PAR) and mirtazapine (MRT).
Membrane preparations from rSERT expressing cells were incubated with 17 in the absence or presence of PAR or MRT. (A) After PAL and CuAAC-mediated fluorescent labelling, proteins were separated by SDS-PAGE and fluorescence was monitored; expression of rSERT was verified by Western blot analysis (lower panel). (B) PAR leads to a 60% decrease in binding signal (***p < 0.001) while MRT has no effect; for data analysis one way ANOVA followed by Bonferroni adjustment was applied; each box-plot represents the average of 12 independent experiments, each performed in triplicates.
Fig 5.
Analysis of azidobupramine (17) binding to rSERT in living cells.
Living rSERT expressing cells were exposed to 17, in the presence or absence of PAR followed by PAL-reaction, still in living cells; immediately afterwards we performed cell disruption, membrane purification, affinity enrichment and CuAAC-mediated rhodamin labelling. (A) Relative change of 17-specific binding to rSERT depending on the presence of the competitor PAR; Wilcoxon-Mann-Whitney test was applied, ***p <0.001; each box-plot represents the average of 20 independent experiments, each performed in triplicates. (B) Representative fluorescence scans (lanes 1–3) and corresponding Coomassie-stained gels (lanes 1’-3’); lanes 3 and 3’ show control eluates from affinity gels that were not loaded with protein material.