Fig. 1: Effect of mutations in the S1 and the allosteric site on vilazodone affinity.

a A top view of allosterically bound vilazodone (orange) to SERT’s extracellular vestibule (purple ribbons) acquired from the previous cryo-EM structure²⁵ (PDB ID: 7LWD). The interacting side chains are shown (purple)²⁵. b, c The half-maximal inhibitory concentration (IC₅₀) of vilazodone for SERT WT and mutants located at the allosteric site (b), the S1 site, or the combined double mutants (c). The IC₅₀ values were derived from whole-cell [³H]5-HT uptake competition experiments performed on intact COS-7 cells transiently expressing SERT WT or mutants. Of the investigated mutants, only I172M, N177V, F341V and S438T showed a significant change in IC₅₀ values compared to SERT WT. The statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison post-hoc test (****, p < 0.0001). Pairwise comparisons revealed adjusted p values as follows: WT vs. R104K (p > 0.9999), WT vs. Q332A (p = 0.5968), WT vs. F335A (p = 0.5233), WT vs. E493N (p = 0.7618), WT vs. E494Q (p = 0.317), WT vs. Y495A (p = 0.748), WT vs. F556A (p = 0.9991), WT vs. P561G (p > 0.9999), WT vs. Y579A (p = 0.5291), WT vs. Y95F (p = 0.8672), WT vs. I172M (p < 0.0001), WT vs. N177V (p < 0.0001), WT vs. F341V (p < 0.0001), WT vs. S438T (p < 0.0001), WT vs. Y95F-F556A (p = 0.9997), WT vs. I172M-F556A (p = 0.0961), WT vs. S438T-F556A (p = 0.9411). Bars represent mean ± SD (error bars) of log[IC₅₀] values from n independent experiments performed in triplicate; individual dots denote the replicate values. For F335A, E493N, Y495A, P561G, Y95F, I172M, N177V, F341V, Y95F-F556A, I172M-F556A and S438T-F556A n = 3 experiments were performed. For WT, R104K, Q332A, E494Q n = 4 experiments were performed. For F556A and Y579A n = 5 experiments were performed.