A Selective Serotonin Reuptake Inhibitor, a Proton Pump Inhibitor, and Two Calcium Channel Blockers Inhibit Candida albicans Biofilms

Abstract

Biofilms formed by the human fungal pathogen Candida albicans are naturally resistant to many of the antifungal agents commonly used in the clinic. We screened a library containing 1600 clinically tested drug compounds to identify compounds that inhibit C. albicans biofilm formation. The compounds that emerged from the initial screen were validated in a secondary screen and then tested for (1) their abilities to disrupt mature biofilms and (2) for synergistic interactions with representatives of the three antifungal agents most commonly prescribed to treat Candida infections, fluconazole, amphotericin B, and caspofungin. Twenty compounds had antibiofilm activity in at least one of the secondary assays and several affected biofilms but, at the same concentration, had little or no effect on planktonic (suspension) growth of C. albicans. Two calcium channel blockers, a selective serotonin reuptake inhibitor, and an azole-based proton pump inhibitor were among the hits, suggesting that members of these three classes of drugs or their derivatives may be useful for treating C. albicans biofilm infections.

Keywords: Candida albicans; Pharmakon 1600 compound library; antimicrobial resistance; biofilm disruption; biofilm inhibition; biofilms; drug repurposing; high-throughput screens; therapeutics.

Figure 1

High-throughput screening of the Pharmakon 1600 compound library for the ability to inhibit C. albicans biofilm formation. (a) Overview of the adherence inhibition optical density biofilm assay used in these experiments. Compounds were included at a concentration of 10 µM during the 90-min adherence step but not in the 24-h growth step of in vitro biofilm formation. (b) Comparisons of the differences from the mean, in units of standard deviation, for each of the 1600 compounds in the two replicate assays. The 43 candidate hits that were pursued further are indicated in red. The 27 other hits that were not pursued, which consisted of a mixture of well-known antifungal agents, compounds intended for topical use, and compounds with high toxicity values (<100 mg/kg), are indicated in blue. All other compounds are indicated in black. On each axis, the dotted lines indicate no difference from the mean and the dashed lines indicate a threshold of two standard deviations below the mean. (c) Subset of the statistically significant hits at 40 µM from the adherence inhibition optical density biofilm assay. Mean OD₆₀₀ readings with standard deviations are shown, significant differences from the DMSO solvent control as determined by Welch’s t-test (two-tailed, assuming unequal variance) with the Bonferroni correction are indicated for α = 0.05 (*) and α = 0.01 (**).

Figure 2

Thirteen candidate compounds inhibited biofilm formation by themselves or in combination with one or more known antifungal agents. (a) Overview of the experimental setup for the sustained inhibition optical density biofilm assay used for these experiments. Compounds were included during both the 90-min adherence step and the 24-h growth step of in vitro biofilm formation. (b) Subset of the statistically significant hits from the stand-alone sustained inhibition optical density biofilm assay. Mean OD₆₀₀ readings with standard deviations are shown, significant differences from the DMSO solvent control as determined by Welch’s t-test (two-tailed, assuming unequal variance) with the Bonferroni correction are indicated for α = 0.05 (*) and α = 0.01 (**). Although a single repeat is shown, the indicated threshold was met by all of the repeats of each compound shown. (c) Subset of the statistically significant hits from the combination sustained inhibition optical density biofilm assays with caspofungin. For each compound, the wells with caspofungin (+ caspofungin) are indicated in yellow and wells without caspofungin (– caspofungin) are indicated in red. Mean OD₆₀₀ readings with standard deviations are shown; significant differences from the compound without caspofungin control (e.g., chloroxine, − caspofungin), as determined by Welch’s t-test (two-tailed, assuming unequal variance) with the Bonferroni correction, are indicated for α = 0.05 (*) and α = 0.01 (**). Significant differences from the caspofungin without compound control (e.g., DMSO, + caspofungin), as determined by Welch’s t-test (two-tailed, assuming unequal variance) with the Bonferroni correction, are indicated for α = 0.05 (#) and α = 0.01 (##). In panels b and c, data within a chart are taken from the same plate on the same day. (d) Venn diagram illustrating the degree of overlap between the combination sustained inhibition screens with amphotericin B, caspofungin, and fluconazole.

Figure 3

Fourteen candidate compounds disrupted mature biofilms by themselves or in combination with the known antifungal agent caspofungin. (a) Overview of the disruption optical density biofilm assay used for these experiments. In brief, the media was removed after the 24-h growth step and fresh media containing the compound was added, after which biofilms were grown in vitro for an additional 24 h. (b) Subset of the statistically significant hits from the stand-alone disruption optical density biofilm assay. Mean OD₆₀₀ readings with standard deviations are shown, significant differences from the DMSO solvent control as determined by Welch’s t-test (two-tailed, assuming unequal variance) with the Bonferroni correction are indicated for α = 0.05 (*), α = 0.01 (**), or mixed results (&). Although a single repeat is shown, the indicated significance threshold was met by all of the repeats of each compound shown with the exception of mefenamic acid. In that case, one of the four repeats did not pass either significance threshold while the remaining three repeats passed at α = 0.01. (c) Subset of the statistically significant hits from the combination disruption optical density biofilm assays with caspofungin. For each compound, the wells with caspofungin (+ caspofungin) are indicated in yellow and the wells without caspofungin (− caspofungin) are indicated in red. Mean OD₆₀₀ readings with standard deviations are shown; significant differences from the compound without caspofungin control (e.g., nisoldipine, − caspofungin), as determined by Welch’s t-test (two-tailed, assuming unequal variance) with the Bonferroni correction, are indicated for α = 0.05 (*) and α = 0.01 (**). Significant differences from the caspofungin without compound control (e.g., DMSO, + caspofungin), as determined by Welch’s t-test (two-tailed, assuming unequal variance) with the Bonferroni correction are indicated for α = 0.05 (#) and α = 0.01 (##). In both panels b and c, the data within a chart are all taken from the same plate on the same day.

Figure 4

A number of compounds had effects in only a subset of the biofilm assays. (a) Compounds with an effect by themselves at 40 µM in the adherence inhibition, sustained inhibition, or disruption optical density biofilm assays are indicated. In total, 14 compounds had an effect in at least one of these three assays. (b) Compounds with an effect in either the stand-alone or the combination versions of the sustained inhibition or disruption optical density biofilm assays are indicated. In total, 18 compounds had an effect in at least one of these four assays.