Antifungal activity of selective serotonin reuptake inhibitors attributed to non-specific cytotoxicity

Sir, In two recent publications, Lass-Flörl et al.1,2 report that a number of selective serotonin reuptake inhibitors (SSRIs) exhibit fungicidal activity against a spectrum of medically important opportunistic fungal pathogens, including species of Candida and Aspergillus. This appears to be an exciting observation and is one we have investigated with interest because sertraline, a Pfizer SSRI, features in these articles. SSRIs exert their pharmacological action through inhibition of the sodium-dependent serotonin transporter (5-HTT). The findings of Lass-Flörl et al.1,2 are intriguing, as there is no fungal orthologue of 5-HTT. It is therefore unlikely that SSRIs exert their antifungal activity through their known pharmacological target. To investigate this hypothesis, we selected sertraline, eight close analogues and three other marketed SSRIs to cover a wide range of potencies against 5-HTT and determined their MICs against a variety of fungi using NCCLS3 methods (Table 1). In agreement with Lass-Flörl et al.,1,2 sertraline displayed broad-spectrum antifungal activity (6.3–25 mg/L) and was also fungicidal against the panel of species tested (data not shown). Paroxetine and fluoxetine were also found to have antifungal activity, being most potent against the hypersusceptible Candida albicans strain DSY1204, which has a number of drug-efflux pumps deleted.4 However, we discovered that CP-51973, CP-50723 and CP-52002, the enantiomer and two diastereoisomers of sertraline, respectively, had essentially identical antifungal activity against the panel of species tested to sertraline itself, despite their reduced potency against 5-HTT. When analysed by the method of ordinary least squares (OLS) there was no significant correlation between the IC50 (half maximum inhibitory concentration) of the tested compounds against 5-HTT, and the MIC against fungal species (for instance, r2 = 0.12 for DSY1204). These data suggest that the antifungal activity of sertraline is unlikely to be a consequence of inhibiting 5-HTT. Interestingly, we observed a relationship between the antifungal potency of SSRIs and the degree of lipophilicity (OLS r2 = 0.57 between cLogP and MIC for DSY1204, where cLogP is the logarithm of the calculated partition coefficient between n-octanol and water), with the most potent antifungal compound being the most lipophilic. We often find that lipophilic compounds exhibit cytotoxic characteristics at high concentrations, a phenomenon also observed by others (e.g. Chen et al.5), suggesting that toxicity rather than a specific inhibitory mechanism may be driving the antifungal activity of SSRIs. To investigate this hypothesis, we determined the cytotoxic activity of each compound following 72 h exposure to a human epithelial cell line, HeLa, using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] cell viability assay6 (Table 1, CC50 value). All compounds tested were cytotoxic at concentrations lower than their MIC. This apparent fungal resilience is a phenomenon we observe regularly for toxic compounds and can be attributed, at least in part, to the fungal cell wall and the activity of drug efflux pumps. Consistent with our hypothesis, we observed a good correlation between the CC50 (half maximum cytotoxic concentration) of SSRIs and their antifungal activity, with the most cytotoxic compounds having the most potent antifungal properties (OLS r2 = 0.62 for DSY1204). Furthermore, of particular significance to the conclusion of the present study was the similarity of the CC50s for sertraline, its enantiomer and two distereoisomers, which reflects their observed antifungal properties and suggests again a non-specific, toxic mechanism of action. As discussed by Lass-Flörl et al.,1,2 sertraline reaches steady-state plasma levels of 50 µg/L, a concentration significantly lower than that required for antifungal activity. The present study indicates that the antifungal effect of SSRIs is mediated through a non-specific mechanism related to the lipophilicity of the agents, and is associated with significant toxic effects on human cells at concentrations required for activity against fungal pathogens. Therefore, based on the results of the present study, it is our opinion that SSRIs will neither prove useful chemotherapeutic mediators of opportunistic fungal infections nor provide useful leads for the development of more potent antifungal agents with a specific mechanism of action. *Corresponding author. Tel: +44-1304-644856; Fax: +44-1304-651819; E-mail: Tim_Young@Sandwich.Pfizer.com In vitro antifungal, cytotoxic and 5-HTT activity of sertraline, eight close analogues and other selected SSRIs aMICs were determined at 24 h by the NCCLS M27-A microdilution method at an inoculum of 103–104 organisms per well.3 bCytotoxicity (CC50) was determined in the HeLa cell line using the MTS assay after 72 h exposure of compound to the cells.6 cPotency for 5-HTT was determined by either (i) displacement of [3H]sertraline binding, essentially as described previously,7 or (ii) [3H]serotonin uptake by a HEK 293 cell line expressing human 5-HTT essentially as described previously.8 dThe logarithm of the calculated partition coefficient between n-octanol and water, cLogP, represents molecular hydrophobicity. In vitro antifungal, cytotoxic and 5-HTT activity of sertraline, eight close analogues and other selected SSRIs aMICs were determined at 24 h by the NCCLS M27-A microdilution method at an inoculum of 103–104 organisms per well.3 bCytotoxicity (CC50) was determined in the HeLa cell line using the MTS assay after 72 h exposure of compound to the cells.6 cPotency for 5-HTT was determined by either (i) displacement of [3H]sertraline binding, essentially as described previously,7 or (ii) [3H]serotonin uptake by a HEK 293 cell line expressing human 5-HTT essentially as described previously.8 dThe logarithm of the calculated partition coefficient between n-octanol and water, cLogP, represents molecular hydrophobicity.