CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms

Abstract

Multidrug resistance (MDR) transporters such as ATP Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased multidrug resistance transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of CDR transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 represents a unique example of a medicinal chemistry-based conversion of chemical scaffold from MDR-sensitive to MDR-resistant and, hence, active against fungi that have developed resistance to clinically used antifungals such as the azoles.

Introduction

Phenothiazines are one of the oldest classes of drugs with derivatives of this class being used to treat a wide range of diseases from parasitic infections to nausea to psychosis (1). One of the features of the phenothiazine class of molecules is that it readily crosses the blood-brain-barrier (1). Accordingly, most of the phenothiazine-derived drugs currently in use are for the treatment of CNS-related conditions. Clinically used phenothiazines have also been studied for repurposing to other indications with anti-cancer and anti-infective applications being amongst the most widely investigated (2).

The potential of phenothiazines such as trifluoperazine, thioridazine and fluphenazine as antifungal agents has been investigated for many years (3), including work from our group (4). An important dose-limiting toxicity of these drugs is their antipsychotic and sedative CNS activity. As part of our efforts in this area, we reported a fluphenazine derivative, CWHM-974 (Fig. 1), with increased antifungal activity against fungal pathogens such as C. albicans and C. neoformans as well as reduced affinity for dopamine and histamine receptors (4).

Figure 1. Chemical structures of fluphenazine and CWHM-974.

Fluphenazine, the parent drug of CWHM-974, has poor activity against C. albicans with a minimum inhibitory concentration (MIC) of 64 μg/mL while CWHM-974 is 8-fold more active (MIC; 8 μg/mL). Fluphenazine is a strong inducer of ATP-Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins such as CDR1 and MDR1 in C. albicans and, as such, is frequently used to study C. albicans the regulation of multidrug transporter expression (5, 6). One of the important features of CWHM-974 is that it is active against a C. albicans strain that is fluconazole-resistant due to increased expression of CDR1 and MDR1 (4). We, therefore, were interested to determine if the increased anti-C. albicans activity of CWHM-974 relative to fluphenazine was because of altered interactions with the multidrug transporters or their regulators. Specific questions were: 1) does CWHM-974 induce pump expression? 2) Is CWHM-974 less susceptible to efflux pump-mediated resistance? 3) does CWHM-974 inhibit efflux pump function?

Here, we show that both CWHM-974 and fluphenazine induce the ABC transporters CDR1 and CDR2 and that CWHM-974 is a more potent inducer than fluphenazine. We also found that fluphenazine is susceptible to multi-drug transporter mediated resistance while CWHM-974 is not. Both fluphenazine and CWHM-974 antagonize fluconazole in a Tac1/Cdr-dependent manner, indicating that CWHM-974 is unlikely to inhibit transporter function. To our knowledge, the CWHM-974 is one of the few examples of a molecule in which relatively small structural modifications significantly reduced susceptibility to multidrug transporter-mediated resistance.

Results

Fluphenazine and CWHM-974 induce distinct patterns of efflux drug expression.

Fluphenazine is a well-studied inducer of CDR1 expression in C. albicans (6, 7). We confirmed those observations (Fig. 2A) and found that CWHM-974 also induces robust CDR1 expression. Concentrations of the two molecules for these experiments were normalized to their respective MIC value and, thus, CWHM-974 leads to ~2.5-fold higher CDR1 expression compared to fluphenazine. Both molecules also induce the expression of CDR2 (Fig. 2A) but only CWHM-974 induces MDR1 expression. Thus, CWHM-974 appears to be a more potent and more general inducer of drug ABC/MDR expression than the fluphenazine parent compound.

Figure 2. Fluphenazine and CWHM-974 induce distinct CDR1/2 and MDR1 transporter expression patterns.

The expression of the indicated genes was assayed using quantitative RT-PCR of RNA isolated from the indicated strains after 2 hr exposure of logarithmic phase cells to fluphenazine (FZN, 50 μg/mL), CWHM-974 (6.25 μg/mL), DMSO carrier alone (1%). The expression levels were normalized to DMSO treated cells and data are mean fold-change from triplicate experiments with the error bars indicating the standard deviation. Statistical significance was determined using 2-way ANOVA with Dunnett’s correction for multiple comparisons and defined as an adjusted p value <0.05. Statistically significant differences from the SN250 reference are indicated by an asterisk (*). Effect of Tac1 and Mrr2 on expression of CDR1 (A), CDR2 (B), and MDR1 (C).

Previous work has shown that fluphenazine-induced expression of CDR1 and CDR2 is dependent on the transcription factor Tac1 (6, 7). As shown in Fig. 2A, deletion of TAC1 reduces both CDR1 and CDR2 expression in cells exposed to either fluphenazine or CWHM-974. However, CWHM-974-induced CDR1 expression remains significantly elevated relative to untreated cells in the tac1ΔΔ strain. Gain-of-function alleles of MRR1 also increase expression of CDR1 (8) and, therefore, we asked if Mrr2 contributes to CWHM-974 induced expression. To test this, we constructed a tac1ΔΔ mrr2ΔΔ double mutant and compared CWHM-974-induced expression in this strain to the two single mutants. In the presence of fluphenazine (Fig. 2A), CDR1 expression did not differ from WT in the mrr2ΔΔ mutant and the tac1ΔΔ mrr2ΔΔ mutant did not differ from tac1ΔΔ. In contrast, CDR1 expression was modestly reduced in the mrr2ΔΔ mutant exposed to CWHM-974 while it was returned to the level of untreated cells in the tac1ΔΔ mrr2ΔΔ double mutant. These genetic interaction data strongly indicate that CWHM-974 induces CDR1 expression through both Tac1 and Mrr2 while fluphenazine activates expression almost exclusively through Tac1.

Tac1 is also required for CDR2 induction for both fluphenazine and CWHM-974 (6, 7). Curiously, while the mrr2Δ mutant has no effect on fluphenazine-induced CDR2 expression (Fig. 2B), it dramatically accentuates CWHM-974 induced CDR2 expression. The increased CDR2 expression in the mrr2ΔΔ mutant is completely abolished by the deletion of TAC1. This observation suggests that Mrr2 negatively modulates CWHM-974 activation of Tac1 either directly or indirectly.

A similar phenomenon was observed with CWHM-974 induction of MDR1 expression (Fig. 2C). In this case, however, deletion of TAC1 leads to a 4-fold increase in MDR1 expression relative to the wildtype strain. Deletion of MRR2 also increases CWHM-974-induced expression of MDR1. The level of MDR1 expression induced in the tac1ΔΔ mrr2ΔΔ double mutant expression is, however, slightly reduced relative the tac1ΔΔ, suggesting that these two transcription factors may make some contribution to the positive regulation of MDR1. However, the expression is still 2-fold higher than wildtype indicating that other factors must regulate MDR1 expression. Taken together, these data indicate that the fluphenazine derivatives induce multiple efflux pumps through a complex inter-related network of regulators. Our CWHM-974 data also indicate compensatory interplay between CDR1/2 expression and MDR1 expression that is mediated by Mrr1/Tac1 and an unidentified regulator of MDR1 expression. Finally, these results indicate that the improvement in anti-C. albicans activity of CWHM-974 is not due to reduced induction of ABC/MFS multidrug transporter proteins.

The antifungal activity of CWHM-974 is required for induction of CDR1 expression

The antifungal activity of fluphenazines and other phenothiazines is due in part to inhibition of calmodulin-dependent processes (4, 8). In contrast, the molecular mechanism by which these molecules induce multidrug resistance transporter expression is not clear. Fluphenazine has reduced antifungal activity and reduced potency with respect to CDR1 expression relative to CWHM-974, suggesting that there may be a correlation between these two properties. We, therefore, asked if the antifungal activity of the fluphenazine derivatives was required to induce CDR1 expression. Oxidation of the S atom in the phenothiazine core structure blocks the ability of the molecule to bind calmodulin (8) and we previously showed that oxidation of the phenothiazine antipsychotic thioridazine abolished its antifungal activity (4). We synthesized the sulfoxide of CWHM-974 (894) and found that it had no antifungal activity at concentrations up to the limit of its solubility (Fig. 3A). 894 also did not induce CDR1 expression at the highest soluble concentrations (Fig. 3B). This observation suggests that antifungal activity is required for fluphenazine-induced CDR1 expression and further supports the notion that the potency of antifungal activity correlates with the extent of CDR1 expression in the fluphenazine series. One explanation for these observations is that CDR1 expression is in response to the physiologic changes caused by inhibition of the antifungal target of fluphenazine/CWHM-974. A second possibility is that the receptor that recognizes multidrug transporter inducing molecules shares structural features with the antifungal target of the fluphenazines and, therefore, is subject to a similar structure-activity relationship.

Figure 3. S-oxidation of CWHM-974 eliminates antifungal activity and ability to induce CDR1.

A. Chemical structures of CWHM-974, CWHM-894, and fluphenazine (FZN) along with their MIC values against C. albicans. B. CDR1 expression of SN250 cells exposed to CWHM-974 (128 μg/mL) compared to DMSO carrier solvent alone (1%).

The antifungal activity of fluphenazine but not CWHM-974 is modulated by the interactions of Tac1, Mrr2, and Cdr1.

Next, we asked whether the mutants of the Tac1/Mrr2/Cdr system contributed to the antifungal susceptibility of C. albicans to fluphenazine or CWHM-974. As shown in Table 1, single deletion mutants of TAC1, MRR2, and CDR1 did not show significant difference in susceptibility to either fluphenazine or CWHM-974 under modified CLSI microbroth dilution conditions (temperature 37°C rather than 35°C). Double and triple mutants involving Tac1, Mrr2, and Cdr1 had no significant effect on the MIC of CWHM-974. In contrast, the tac1ΔΔ cdr1ΔΔ mutant was 4-fold more susceptible to fluphenazine. The fluphenazine MIC of neither the tac1ΔΔ mrr2ΔΔ nor the mrr2ΔΔ cdr1ΔΔ mutant was significantly different than WT but the tac1ΔΔ mrr2ΔΔ cdr1ΔΔ triple mutant had the lowest fluphenazine MIC (4 μg/mL; 16-fold reduction from WT). These data indicate that fluphenazine is highly sensitive to the Tac1-Mrr2-Cdr multidrug transporter while CWHM-974 is not. This strongly suggests that the structural modifications made to CWHM-974 block the ability of these transporters to modify their activity, particularly since the modification increase its potency as an inducer of this drug transporter system.

Table 1.

Strain	974		FLU		FICI	FZN		FLU		FICI
	MICA	MICC	MICA	MICC		MICA	MICC	MICA	MICC
SN250	8	1	0.5	16	32.125	64	8	0.5	4	8.125
tac1Δ	4	1	0.5	1	2.25	32	4	0.5	0.25	0.625
mrr2Δ	8	2	0.5	16	32.25	64	8	0.5	2	4.125
cdr1Δ	8	4	0.25	1	4.5	64	4	0.25	0.5	2.0625
tac1Δcdr1Δ	8	1	0.25	0.5	2.125	16	4	0.25	0.125	0.75
mrr2Δcdr1Δ	8	4	0.25	2	8.5	64	4	0.25	0.5	2.0625
tac1Δmrr2Δ	4	0.5	0.25	1	4.125	32	8	0.25	0.125	0.75
tac1Δmrr2Δcdr1Δ	4	0.5	0.25	0.5	2.125	4	0.5	0.25	0.5	2.125

Fluphenazine and CWHM-974 induce Tac1-Cdr1-dependent fluconazole antagonism in C. albicans

The combination of fluphenazine and fluconazole is antagonistic against C. albicans (9, 10), presumably by induction of CDR1/2, although that has not been experimentally confirmed. To test this hypothesis further, we first asked whether the combination of fluconazole and fluphenazine induced CDR1 in an additive or synergistic manner. As shown in Fig. 4A, expression of CDR1 is higher in C. albicans cells treated with fluconazole and either fluphenazine or CWHM-974 than in cells treated with either drug alone. The expression of CDR1 in the presence of both fluconazole and fluphenazine is equal to the product of the expression levels of each drug alone, indicating an additive effect. The expression of CDR1 is, however, less than the product of the expression observed in cells treated with either fluconazole or alone CWHM-974, indicating that the expression may be reaching saturation. This observation is consistent with a model in which all three molecules induce expression of CDR1 through a common mechanism or pathway.

Figure 4. Fluphenazine and CWHM-974 induce CDR1 expression but are not antagonistic.

A. Fluconazole and fluphenazines combine to increase CDR1 expression relative to single drug treatments. SN250 cells were treated with both fluconazole and either FZN or CWHM-974 at concentrations 2-fold below their respective FIC values (Table 1) for 2 hr before being processed for quantitative RT-PCR. All treatments showed statistically significant changes from untreated SN250 cells. B. Representative western blot of Cdr1 expression in the presence of the indicated concentrations of FZN and CWHM-974. C. Quantitative analysis with bars indicating the mean and error bars the standard deviation of duplicate western blots. The treated cells are statistically different than untreated but there is no statistical difference between FZN and CWHM-974. D. FIC analysis of the interaction of FZN and CWHM-974 with fluconazole in C. glabrata.

Consistent with previous work (9, 10), both fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans by checkerboard analysis (Table 1). Interestingly, both fluphenazine and CWHM-974 strongly inducing the expression of CDR1 (Fig. 4B&C). However, there is no antagonism between either fluphenazine or CWHM-974 and fluconazole in C. glabrata (Fig. 4D). In C. albicans (Table 1), CWHM-974 treatment causes a 32-fold increase in the fluconazole MIC while the MIC of fluconazole is 8-fold higher in combination with fluphenazine, suggesting that the higher expression of CDR1 in the presence of CWHM-974 and fluconazole translates to lower susceptibility. In the presence of fluconazole, the MIC of the fluphenazine and CWHM-974 is reduced 8-fold, suggesting that inhibition of Erg11 increases the activity of the phenothiazines. Thus, the antagonism, as analyzed by this checkerboard approach, is driven solely by the increase in fluconazole MIC.

Next, we determined the effect of tac1ΔΔ, cdr1ΔΔ, and tac1ΔΔ cdr1ΔΔ mutations on the increase in fluconazole MIC induced by the fluphenazine derivatives. As shown in Table 1, deletion of either TAC1 or CDR1 return the combination fluconazole MIC to within a 2-fold dilution of that observed for fluconazole alone. The strain lacking both TAC1 and CDR1 has a combination fluconazole MIC identical to that of wild type treated with fluconazole alone. Thus, the induction of antagonism by fluphenazine and CWHM-974 can be explained by activation of the Tac1-Cdr1 axis. Phenothiazines have been shown to inhibit efflux pump function in other settings (10), raising the possibility that the increased anti-C. albicans activity of CWHM-974 may be due to its ability to inhibit Cdr1. If that were the case, then CWHM-974 would induce CDR1 but there would be no antagonism with fluconazole. Consequently, these data are inconsistent with multidrug transport protein inhibition contributing to the improved activity of CWHM-974 against C. albicans relative to fluphenazine.

Discussion

Multidrug transport mediated resistance is one of the most clinically important mechanisms of antifungal drug resistance because it reduces susceptibility to the azole class of molecules in multiple fungal pathogens including Candida spp and Cryptococcus spp (12). Identifying new drugs that are not susceptible to these mechanisms is critical to improving the overall efficacy of antifungal chemotherapy. A wide range of non-antifungal drugs and bioactive small molecules induce the expression of ABC and MFS transporters in fungi (9, 13). Of these one of the most widely studied is fluphenazine, particularly with respect to ABC transporter expression in C. albicans. As part of a repurposing project aimed at the medicinal chemistry-based optimization of phenothiazine antifungal activity (4), we synthesized a derivative of fluphenazine (CWHM-974) with reduced affinity for dopamine and histamine receptors. CWHM-974 also showed improved activity against C. albicans relative to the parent fluphenazine. This improved activity was also observed for strains that are fluconazole resistant due to increased expression of CDR and MFS transporters, suggesting that CWHM-974 may not be susceptible to ABC/MFS mediated resistance mechanisms.

The genetic analysis described provides strong support for this hypothesis. First, we have confirmed that fluphenazine induces the expression of CDR1 and CDR2 and that the increased expression of these transporters is responsible for its poor antifungal activity against C. albicans. Second, CWHM-974 is a more potent inducer of CDR1/2 but mutants lacking these genes or with dramatically reduced expression of CDR1/2 show no change in susceptibility compared to WT. Third, CWHM-974 and fluphenazine potently induce Tac1-Cdr1-mediated fluconazole antagonism, indicating that CWHM-974 is unlikely to interfere indirectly or directly with the function of this resistance pathway to a significant extent. We are unaware of another example whereby structural modification of an antifungal small molecule that is susceptible to CDR/MFS mediated resistance leads to abrogation of that resistance.

At this point, it is not clear how replacement of the alkyl chain linking the phenothiazine core to the piperazine moiety of fluphenazine with a larger, benzyl linker causes this dramatic change in sensitivity to the CDR resistance mechanism. The simplest explanation is that, as proposed in the literature (9, 13), fluphenazine is a substrate for the efflux system while the benzyl-modified CWHM-974 is not. Additional work will be required to identify the structural and biochemical basis for the ability of CWHM-974 to evade CDR-mediated resistance mechanisms.

Our genetic interaction analysis of the Tac1-Mrr2-CDR system with respect to fluconazole susceptibility has led to observations that warrant emphasis. First, deletion of multiple regulators, with or without deletion of CDR1, had almost no effect on the susceptibility of those strains to fluconazole. Second, and consistent with this finding, fluconazole alone is a relatively weak inducer of CDR1 expression. Consequently, fluconazole is not a sufficiently strong inducer of CDR1 expression to mediate its own resistance. Fluphenazine, on the other hand, induces sufficient CDR1 and CDR2 expression to mediate its own resistance. In addition, fluphenazine resistance can be mediated by either CDR1 or CDR2 or possibly other transporters since the tac1ΔΔ mrr1ΔΔ cdr1ΔΔ mutant is more susceptible than the single mutants or the tac1ΔΔ mrr1ΔΔ double mutant. In contrast to fluphenazine-induced, fluphenazine resistance, fluphenazine/CWHM-974-induced, fluconazole resistance is completely derived from Tac1-dependent expression of CDR1.

Finally, it is worth noting that neither fluphenazine nor CWHM-974 show antagonism with fluconazole in C. glabrata despite inducing a 10-fold increase in CDR1 expression (Fig. 4B–D). C. glabrata strains with elevated CDR1 expression by virtue of Pdr1 gain-of-function mutants are resistant to fluconazole compared to strains with WT alleles (14). Indeed, pdr1^R376W has a nearly identical 10-fold increase Cdr1 protein levels as that observed with fluphenazine and CWHM-974 exposure (14). Consistent with that increased expression, the pdr1^R376W mutant has reduced susceptibility to fluconazole compared to the WT strain (14). Based on the similar levels of CDR1 expression, one would expect fluphenazine and CWHM-974 to induce antagonism. The lack of antagonism between the fluphenazines and fluconazole, however, suggests that fluphenazine and CWHM-974 have additional effects on fluconazole susceptibility that counter the increased expression of CDR1.

In summary, we have found that the fluphenazine derivative CWHM-974 displays increased activity against C. albicans because it is less susceptible to CDR/MFS mechanisms of resistance. Efforts to further optimize the antifungal activity and therapeutic index of this promising, repurposed scaffold are ongoing.

Materials and methods

Strains, media and reagents.

All yeast strains were in the SN background and maintained on yeast peptone (2%) dextrose (YPD) plates after recovery from 25% glycerol stocks stored at −80°C. Transcription factor mutants tac1ΔΔ and mrr2ΔΔ were obtained from the Homann deletion collection provided by the Fungal Genetics Stock Center (15). The cdr1ΔΔ, tac1ΔΔ mrr2Δ and tac1ΔΔ mrr2ΔΔ cdr1ΔΔ mutants (see Table S1 for strains) were generated using transient CRISPR methods described by Huang et al. The mutants were confirmed using PCR analysis of the marker junctions and lacked the targeted ORF by PCR analysis; see Table S2 for primers used for these analyses. Yeast media were prepared using recipes described in Homann et al (15). Fluconazole and fluphenazine were obtained from Sigma-Aldrich. CWHM-974 was synthesized as previously reported and was greater than 95% pure.

Quantitative RT-PCR analysis

The indicated strains were pre-cultured overnight in YPD at 30°C and then back-diluted to a density between 0.9 and 0.15 OD₆₀₀. The resulting cultures were incubated for 3 hr at 30°C (OD₆₀₀ ~ 0.3–0.4). The strains were exposed to DMSO carrier solvent (final concentration 1%), fluphenazine (50 μg/mL), CWHM-974 (6.25 μg/ml) or fluconazole (16 μg/mL) for 2 hr at 30°C. The cells were harvested and frozen at −80°C prior to isolation of RNA using the MasterPure Yeast RNA purification kit. 500ng of RNA was used for cDNA synthesis with iScript cDNA synthesis kit. The resulting cDNA was diluted 1:5 processed using the qRT-PCR with iQ SYBR Green Supermix with 0.20 μM primers (See Table S2 for PCR primers) qRT-PCR was performed on the BioRad CFX Connect using a 3-step amplification with 54°C annealing temperature and melt curve analysis. Gene expression was normalized to ACT1 and analyzed using the ΔΔCt method. The experiments were performed in triplicate and differences in expression as analyzed by 2-way ANOVA with Dunnett’s correction for multiple comparisons with significance limit defined as an adjusted p value < 0.05.

Minimum inhibitory concentration and fractional inhibitory concentration determinations

Minimum inhibitory concentrations (MIC) and fractional inhibitory concentrations (FIC) were determined using modification of the CLSI conditions. All yeasts were cultured overnight in 3 mL YPD at 30°C, then washed twice in sterile PBS. Two-fold serial dilutions of each compound were prepared in RMPI+MOPS pH 7 (Gibco RPMI 1640 with L-glutamine and 0.165M MOPS) with DMSO as carrier solvent (final concentration 1%), then 1 × 10³ cells were added per well. Plates were incubated at 37°C for 24 h and the MIC/FIC values were determined visually as the lowest concentration with a clear well (fluphenazine and CWHM-974) or with a distinct reduction from DMSO only well. Assays were performed in duplicate or triplicate.

Synthesis of 10-[(p-{[4-(2-hydroxyethyl)-1-piperazinyl]methyl}phenyl)methyl]-2-(trifluoromethyl)-5λ⁴-5-phenothiazinone (SLU-10894):

To a stirred solution of 2-(4-(4-((2-(trifluoromethyl)-10H-phenothiazin-10-yl)methyl)benzyl)piperazin-1-yl)ethanol (CWHM-974; 0.1 mmol) in MeOH (1 mL) was added a solution of NaIO₄ (0.1 mmol) in MeOH containing one drop of conc. HCl was added dropwise. The reaction mixture was then stirred at room temperature for 15–20 min. The reaction turned dark red. It was then quenched by the addition of a few drops of water and extracted with EtOAc (2×25 mL). The combined organic layer was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to afford the crude. The crude was then purified by reverse-phase HPLC (5–100 % CH₃CN/H₂O + 0.1 % formic acid). The pure fractions were then lyophilized to afford the titled compound as a white solid (11.2 mg, 22%). ¹H NMR (400 MHz, DMSO) δ 8.27 (d, J = 7.7 Hz, 1H), 8.07 (d, J = 6.2 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.65 – 7.55 (m, 2H), 7.49 (d, J = 8.4 Hz, 1H), 7.40–7.34 (m, 1H), 7.24 (d, J = 7.9 Hz, 2H), 7.11 (d, J = 7.9 Hz, 2H), 5.76 (s, 2H), 4.33 (br s, 1H), 3.46 (s, 2H), 3.40 (s, 2H), 2.44 – 2.21 (m, 10H); HPLC purity >96%. LCMS m/z 516 [M+H]⁺. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₇H₂₈F₃N₃O₂S 516.1932, found 516.1933.

Western blot analysis of C. glabrata Cdr1

C. glabrata ATCC 2001 was used for the analysis of the induction of Cdr1 expression under different drug conditions (14). Cultures of C. glabrata were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) medium at 30°C. Overnight cultures were diluted to an OD₆₀₀ of 0.2 in fresh YPD, grown at 30°C to an OD₆₀₀ of 0.4, split between three flasks, and treated separately with either fluconazole (16 μg/ml), fluphenazine (32 μg/ml), or 974 (4 μg/ml). Cell samples were acquired immediately prior to splitting the initial cultures and at the 1 and 2 hours after drug treatment. Proteins were extracted as previously described (Shahi et al., 2010), resuspended in urea sample loading buffer (8M urea, 1% 2-mercaptoethanol, 40 mM Tris-HCL pH 8.0, 5% SDS, bromophenol blue), and incubated at room temperature overnight. Protein extracts were resolved in an ExpressPlus 4–12% gradient gel (GenScript), electroblotted to a nitrocellulose membrane, blocked with 5% nonfat dry milk, and probed with an anti-Cdr1 polyclonal antibody (Vu et al., 2019) and an anti-alpha-tubulin monoclonal antibody (12G10; Developmental Studies Hybridoma Bank at the University of Iowa). For detection and quantification of blotted proteins, secondary Li-Cor antibodies, IRD dye 680RD goat anti-rabbit and IRD dye 800LT goat anti-mouse, were used in combination with the Li-Cor infrared imaging system (application software version 3.0) and Image Studio Lite software (Li-Cor). Cdr1 protein levels were normalized to tubulin and compared to the pretreatment condition. Measurements represent the result of two technical replicates each from two biological replicates.