The antidepressant sertraline targets intracellular vesiculogenic membranes in yeast

Abstract

Numerous studies have shown that the clinical antidepressant sertraline (Zoloft) is biologically active in model systems, including fungi, which do not express its putative protein target, the serotonin/5-HT transporter, thus demonstrating the existence of one or more secondary targets. Here we show that in the absence of its putative protein target, sertraline targets phospholipid membranes that comprise the acidic organelles of the intracellular vesicle transport system by a mechanism consistent with the bilayer couple hypothesis. On the basis of a combination of drug-resistance selection and chemical-genomic screening, we hypothesize that loss of vacuolar ATPase activity reduces uptake of sertraline into cells, whereas dysregulation of clathrin function reduces the affinity of membranes for sertraline. Remarkably, sublethal doses of sertraline stimulate growth of mutants with impaired clathrin function. Ultrastructural studies of sertraline-treated cells revealed a phenotype that resembles phospholipidosis induced by cationic amphiphilic drugs in mammalian cells. Using reconstituted enzyme assays, we also demonstrated that sertraline inhibits phospholipase A(1) and phospholipase D, exhibits mixed effects on phospholipase C, and activates phospholipase A(2). Overall, our study identifies two evolutionarily conserved membrane--active processes-vacuolar acidification and clathrin-coat formation--as modulators of sertraline's action at membranes.

Figure 1.—

Summary of dose–response experiments and cross-resistance test. For all panels the strains are color coded as follows: BY4716, black; CUP5^R46I, red; and SWA2^C603STOP, blue. Strains were treated with sertraline (A), chlorpromazine (B), or cycloheximide (C). Small-molecule drug concentration is given as log [M]. All strains were grown at 30°. Experimental OD₆₀₀ values were normalized to OD₆₀₀ values of untreated isogenic controls. Data points are the average of quadruplicate OD₆₀₀ measurements. Points were fit with a variable slope sigmoidal dose–response equation. Error bars indicate standard deviation.

Figure 2.—

Epistasis analysis of cup5 swa2 and cup5 tfp1 double mutants. (A) IC₅₀ values normalized by the wild-type tetratype of three spores derived from a representative tetrad. The three spores are color coded as follows: cup5, red; swa2 and tfp1, blue; and cup5 swa2 and cup5 tfp1, purple. (B) Time course of cup5 swa2 double mutant (purple circles) and the wild-type strain (black circles). Both strains were treated with sertraline and grown at 30°. Data points are the average of six replicates. Error bars indicate standard deviation.

Figure 3.—

Biphasic effects of sertraline and chlorpromazine on mutants with dysregulated clathrin function. For all panels the strains are color coded as follows: BY4716, black; CHC1^E292K, blue; and chc1-521, red. Strains were treated with sertraline (A), chlorpromazine (B), or cycloheximide (C). Small-molecule drug concentration is given as log [M]. All strains were grown at 30°, except as indicated. Experimental OD₆₀₀ values were normalized to OD₆₀₀ values of untreated isogenic controls. Data points are the average of quadruplicate OD₆₀₀ measurements. Points were fit with a variable slope sigmoidal dose–response equation. Error bars indicate standard deviation.

Figure 4.—

Kinetic time-course experiments with select sert^R strains grown in YPD supplemented with 0 μm (blue circles), 7.5 μm (green circles), or 30 μm (red circles) sertraline reveal two modes of resistance. (A) Baseline and sertraline-treated growth curves of the wild-type BY4716. (B) Baseline and sertraline-treated growth curves of the CUP5^R46I mutant. (C) Baseline and sertraline-treated growth curves of the CHC1^E292K mutant. (D) Baseline and sertraline-treated growth curves of the chc1-521 temperature-sensitive mutant. Cells were grown in 96-well plates and incubated with shaking at 30° in a TECAN plate reader except for D, which was grown at 37°. The amount 10⁴ sec equals ∼2.8 hr. All OD₅₉₅ measurements represent the average of six experimental replicates. Error bars indicate standard deviation.

Figure 5.—

Single-cell viability assay shows that sertraline is irreversibly cytotoxic. The percentage of colony-forming units is plotted as a function of the duration of sertraline treatment in minutes. Data correspond to BY4716 (blue circles), CHC1^E292K (red circles), and CUP5^R46I (green circles).

Figure 6.—

Transmission electron micrographs reveal loss of vacuoles. (A) Representative thin section of a field of untreated wild-type BY4716 cells. (B) Representative thin section of a field of sertraline-treated wild-type BY4716 cells. Bar, 2.0 μm.

Figure 7.—

Transmission electron micrographs reveal sertraline-induced phospholipidosis. Wild-type BY4716 cells in mid-log phase were treated with 120 μm for 1 hr. (A) Thin section of a sertraline-treated cell containing multilamellar bodies. (B) Thin section of a sertraline-treated cell illustrating change in vacuolar contents and accumulation of inclusions. (C) High magnification image of multilamellar body indicated by asterisk in A. (D) High magnification image of distended vacuole-like structure containing undigested autophagosomes indicated by asterisk in B. (E) High magnification image of a representative crescent-shaped multilamellar array encapsulated by an autophagosome. Scale bars are included in each panel.

Figure 8.—

Effects of sertraline and chlorpromazine on enzymatic activity of a panel of phospholipases. Data corresponding to sertraline treatment is shown in blue circles; chlorpromazine treatment is shown in red triangles. (A) Dose–response curve of phospholipase A1 activity. (B) Dose–response curve of phospholipase A2 activity. (C) Dose–response curve of phospholipase C activity. (D) Dose–response curve of phospholipase D activity. All data points are normalized to baseline enzymatic activity in untreated wells and represent the mean of five experimental replicates. Error bars indicate standard deviation.