1 Antifungal phenothiazines: optimization, characterization of

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Antifungal phenothiazines: optimization, characterization of mechanism and modulation of neuroreceptor activity Marhiah C Montoya, Louis DiDone, Richard Heier, Marvin Meyers, and Damian Krysan ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00157 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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ACS Infectious Diseases

Antifungal phenothiazines: optimization, characterization of mechanism, and modulation of neuroreceptor activity. Running Title: Antifungal phenothiazines Marhiah C. Montoya1, Louis DiDone2, Richard F. Heier3, Marvin J. Meyers3,4, and Damian J. Krysan2,5 Clinical and Translational Science Institute, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 146421; Departments of Pediatrics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 146422; Department of Pharmacology & Physiology, Saint Louis University School of Medicine, 1402 South Grand Blvd, St. Louis, MO 631043; Department of Chemistry, 1402 South Grand Blvd Saint Louis University, St. Louis, MO 631034; Departments of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 146425. Corresponding Authors: Marvin J. Meyers M132 Schwitalla Hall, Saint Louis University School of Medicine, 1402 South Grand Blvd, St. Louis, MO 63104 Email: [email protected] Tele: 314-977-5197 Damian J. Krysan 2040 ML, 200 Hawkins Drive, University of Iowa, Iowa City, IA 52242

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Email: [email protected] Tele: 319-335-3066. FAX: 319-356-7147


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New classes of antifungal drugs are an urgent un-met clinical need. One approach to the challenge of developing new antifungal drugs is to optimize the antifungal properties of currently used drugs with favorable pharmacologic properties, so-called drug or scaffold repurposing. New therapies for cryptococcal meningitis are particularly important given its world-wide burden of disease and limited therapeutic options. We report the first systematic structure-activity study of the anti-cryptococcal properties of the phenothiazines. We also show that the antifungal activity of the phenothiazine scaffold correlates well with its calmodulin antagonism properties and, thereby, provides the first insights into the mechanism of its antifungal properties. Guided by this mechanism, we have generated improved trifluoperazine derivatives with increased anticryptococcal activity and, importantly, reduced affinity for receptors that modulate undesired neurological effects. Taken together, these data suggest that phenothiazines represent a potentially useful scaffold for further optimization in the search for new antifungal drugs.

Key Words: Cryptococcus neoformans, Candida, antifungal, phenothiazine, repurposing



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Invasive fungal infections are one of the most difficult challenges in modern infectious disease medicine.1 Currently, only three classes of antifungal drugs are available for the treatment of invasive fungal infections: 1) azole ergosterol inhibitors; 2) polyene ergosterol binding agents; and 3) echinocandin glucan synthase inhibitors.2 The relative paucity of antifungal agents and the slow rate of development would not be an issue if the outcomes of invasive fungal infections were good. Unfortunately, the mortality rates for many invasive fungal infections remain unacceptably high while some infections with some organisms are essentially untreatable using current drugs. Globally, the development of a new therapy for cryptococcal meningitis (CM) is one of the most urgent un-met clinical needs facing the field of medical mycology.3 Recent, epidemiological data indicate that as many as 200,000 cases occur each year world-wide and lead to 180,000 deaths.4 The vast majority of CM occurs in patients living with HIV/AIDS and Cryptococcus is the most common cause of meningitis in some highly endemic areas. For many patients in these regions, the symptoms of CM are what lead to the diagnosis of HIV and, accordingly, patients must first survive CM in order to take advantage of the spectacular advances in HIV therapy. The gold standard therapy for CM is the combination of amphotericin B (AMB), a polyene, and 5-flucytosine (5FC), an adjunctive drug that is not useful as monotherapy. The superiority of this combination relative to AMB monotherapy was established by a landmark clinical trial in 2013.5 In resource-rich regions of the world, where this combination is readily available, the majority of patients survive CM; mortality rate 10%.6 Unfortunately, AMB can only be administered intravenously, has significant toxicities, and, thus, is not feasible in many resource limited regions with the highest burden of disease.7 Furthermore, 5FC is simply not available in resource limited regions; additionally, it also has significant hematologic toxicities that limit its use in resource-rich regions as well. The alternative therapy in resource-limited 4 ACS Paragon Plus Environment

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regions is fluconazole. The advantages of fluconazole are: 1) oral bioavailability; 2) low cost; and 3) low toxicity. The disadvantage is that fluconazole is much less effective than AMB/5FC with mortalities of 55-70% observed in many resource-limited regions.7 The primary reason that fluconazole is less effective than the AMB plus 5FC combination is that fluconazole is fungistatic while AMB/5FC is fungicidal.8 Clinical studies have established that early fungicidal activity within the CNS correlates with good clinical outcome. Consequently, new CM therapies must have fungicidal activity and must penetrate the blood-brain-barrier. In order to be useful to patients in regions of the world with the highest burden of disease, any new agent must also have oral bioavailability.3 These are relatively stringent requirements that many agents would not meet. Since the pharmacological properties of a new cryptococcal therapy were particularly crucial, we have pursued a “repurposing” approach with the notion of identifying previously developed drug scaffolds with the proven ability to penetrate the CNS and which are fungicidal against Cryptococcus. As part of a previously reported screening campaign focused on a library of FDA-approved drugs, we identified phenothiazine antipsychotic drugs such as thioridazine and trifluoperazine as molecules that satisfy the pharmacological criteria as potential anti-cryptococcal agents in that they are orally bioavailable and access the CNS.9 The activity of phenothiazines against C. albicans and C. neoformans was first characterized thirty years ago by Eilam et al.10 In addition, Spitzer et al. identified trifluoperazine in a screen for FDA-approved drugs that were synergistic with fluconazole against S. cerevisiae, C. albicans, C. neoformans, and C. gattii.11 More recently, a number of groups have shown that phenothiazines have activity against medically important molds such as Zygomycetes and Aspergillus.12 In general, the in vitro activities of the phenothiazines have been at concentrations higher than can be safely achieved in humans and mammalian models of fungal infection. The exception to this trend is trifluoperazine which was shown by Eilam et al 5 ACS Paragon Plus Environment


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to prolong survival in murine models of disseminated candidiasis and cryptococcosis.10 Phenothiazines concentrate in the brain and achieve concentrations in the microgram/mL range in humans.13 Thus, the activity of the phenothiazines reported to date would need to be improved or, alternatively, side-effect profiles improved before they could be used to treat fungal infections. To our knowledge, however, no systematic structure-activity studies or medicinal chemistry-based antifungal optimization of the trifluoperazine scaffold has been reported. Here, we report the first structure-activity relationship (SAR) analysis of the antifungal activity of previously synthesized phenothiazines as well as a set of synthetic trifluoperazine derivative. We demonstrate that phenothiazine derivatives with reduced binding to neurotransmitter receptors retain and, indeed, show improved anti-cryptococcal activity relative to trifluoperazine. In addition, these derivatives have improved activity against drug-resistant C. albicans as well. Finally, we provide evidence that calmodulin inhibition plays an important role in the mechanism of phenothiazine antifungal activity. Results C. neoformans SAR for commercially available phenothiazines. A wide range of phenothiazines are currently or have been used as drugs for a variety of medical indications, most typically as anti-psychotic medication. The two most commonly used phenothiazine antipsychotics are thioridazine (1) and trifluoperazine (9). The C. neoformans MIC values have been previously determined for each of these and are shown in Table 1 for reference. We repeated the testing with our reference strain and obtained values similar to those previously reported: specifically, thioridazine (1) and trifluoperazine (8) gave MIC values (16 µg/mL) identical those previously reported.10 Related phenothiazine drugs and other analogs (1-12) were purchased to develop an initial SAR. Promazine (2), chlorpromazine (3), triflupromazine (4), 5 and 6 are based on a common structural core and differ only at the R2 substituent. The former three drugs 2-4 are two- to four-fold less potent than thioridazine. The methyl (5) and 6 ACS Paragon Plus Environment

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ethyl (6) ketones show some diverging SAR with the latter being four-fold more potent, on par with thioridazine. A clearer R2 SAR pattern emerges in head-to-head comparisons of R2 = chloro versus trifluoromethyl groups. For example, prochloroperazine-trifluoperazine (7,8) and perphenazine-flupenazine (9,10) pairs demonstrate a two- to four-fold improvement in MIC potency for trifluoromethyl over chloro substitution. Fluphenazine (10) is the most potent approved drug with a MIC of 8 µg/mL. A modest eight-fold range in MIC values over this small sampling indicates that the R2 position has an effect on potency but likely could be further optimized. Additionally, none of the previously used phenothiazine drugs nor any of the commercially available analogs are active enough to suggest that they could be directly repurposed as anti-cryptococcal agents. Table 1. Commercial phenothiazine C. neoformans SAR

Compound

X

Thioridazine, THZ (1)

S

Promazine (2) Chlorpromazine (3) Triflupromazine (4) 5 6

10

R

R

C. neoformans MIC (ug/mL)

ClogP

CNS MPO

SMe

16

5.47

2.49

H

64

3.93

3.87

Cl

25

4.54

3.26

CF3

32

4.81

2.99

COCH3

64

3.49

4.50

COCH2CH3

16

4.19

3.80

2

S



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Compound Prochlorperazine (7) Trifluoperazine, TFP (8) Perphenazine (9) Fluphenazine (10)

X

R10

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R2

C. neoformans MIC (ug/mL)

ClogP

CNS MPO

Cl

64

4.38

3.36

CF3

16

4.66

2.85

Cl

16

3.69

4.10

CF3

8

3.97

3.59

S

S

11

SO

SMe

≥128

4.10

3.70

12

O

Cl

8

5.08

2.87

Effect of the central ring heteroatom on C. neoformans activity. The sulfur atom of the phenothiazine ring is susceptible metabolic oxidation in vivo. The corresponding sulfoxide has been shown to decrease affinity for calmodulin by over 3 orders of magnitude.14 Previously, we have shown that phenothiazines bind to C. neoformans calmodulin in vitro and disrupt calmodulin function in C. neoformans cells, supporting the notion that calmodulin represents a target that contributes to the antifungal activity of this class of molecules.9,15 From this analysis, we predicted that sulfoxide would be a poor antifungal. Consistent with that expectation, the sulfoxide derivative of thioridazine (11) had no antifungal activity at the limit of solubility (MIC ≥ 128). This observation further supports the correlation between anti-calmodulin activity and anti-cryptococcal activity. It also indicates that an approach to improving the pharmacology of the scaffold may be to modify this part of the molecule to eliminate this metabolic liability. A simple exchange of sulfur for oxygen would eliminate this metabolic liability. Therefore, we obtained phenoxazine 12 from a commercial source to test whether the sulfur was important for anti-cryptococcal activity. Phenoxazine 12, which also inhibits the kinase Akt


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in mammalian systems, has anti-cryptococcal activity that is similar to the sulfur containing analogs, indicating that this substitution may represent an option if sulfur oxidation proves to limit in vivo efficacy.16 We note that the closest Akt analog in the C. neoformans genome is the kinase Sch9; however, it is not essential and, thus, is unlikely to the sole molecular target responsible for the antifungal activity of 12. Phenoxazines also bind to, and inhibit, the function of calmodulin with a similar SAR to phenothiazines.13 Effect of alkyl amine linker chain length on anti-cryptococcal activity. The anti-calmodulin activity of the phenothiazine and phenoxazine class has been optimized toward a variety of goals (14, 17). One of the consistent themes of those studies is that the length of the alkyl chain linking the amine moiety to the heterocyclic core has a modest but consistent effect on calmodulin antagonism with improving potency as the length of the linker increases.14 Analysis of the bovine calmodulin-trifluoperazine crystal structure, suggests that the basic amine sidechain could play a role in binding to the structurally related CnCAM1 via contact with conserved acidic residues near the solvent surface of the binding pocket.18 Based on these considerations, a series of analogs (14a-e) were synthesized from 2(trifluoromethyl)-10H-phenothiazine (13) to ascertain the importance of the position of the basic amine over a range of two- to six-carbon atom linkers (Scheme 1). Starting with phenothiazine 13, direct alkylation with 2-N-pyrrolidinylethyl tosylate furnished 14a. Analogs with longer chains (14b-e) were obtained using an approach similar to that of Madrid et al. by first alkylation with dibromoalkanes followed by displacement with pyrrolidine.19



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Scheme 1. Synthesis of TFP analogs. Reagents: (a) 2-(pyrrolidin-1-yl)ethyl tosylate, NaH, DMF; (b) BrCH2(CH2)nBr, NaH, DMF; (c) amine, KI, K3PO4, DMF; (d) 1,4bis(bromomethyl)benzene, NaH, DMF.

Consistent with our expectations, analogs with longer chain basic amines were more potent anti-cryptococcal molecules (Table 2). Similarly, the modest two- to four-fold improvement in MIC values is consistent with the effects of chain length variation reported for in vitro calmodulin affinity with analogous compound series.14,17 These modest effects on potency and affinity are also consistent with a generalized electrostatic effect on binding rather than a highly specific interaction between the molecule and CnCAM1. Alternatively, these results may also merely reflect the fact that the CnCAM1 binding pocket is quite large with multiple potential binding sites.20


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Table 2. Phenothiazine C. neoformans SAR

Compound

n

C. neoformans MIC (µg/mL)

ClogP

CNS MPO Score

14a

1

32

5.15

2.71

14b

2

32

5.21

2.56

14c

3

16

5.73

2.18

14d

4

8

6.18

1.89

14e

5

8

6.62

1.59

Effect of alkyl amine linker chain on anti-cryptococcal activity and CNS receptor binding affinities. The anti-psychotic properties of phenothiazine drugs are well known. An ideal anticryptococcal agent will retain the CNS penetrating properties of these drugs while disposing of the CNS effects. Phenothiazines exert many of their antipsychotic properties via the dopamine and serotonin receptors. Madrid et al explored the binding affinities of a set of TFP analogs for the dopamine receptors (D1, D2 and D3) and serotonin receptors (5-HT1A, 5-HT2A, and 5-HT2C) with the goal of retaining the anti-tuberculosis activity and reducing the neuroleptic activity.19 While most of the derivatives they evaluated had strong binding affinities for these six receptors, a few compounds were unable to displace radiolabeled ligands at 10 µM, demonstrating the potential for elimination of the CNS side effects. The reported dopamine- and serotonin-sparing compounds all contained a dibenzyl linker between the phenothiazine core and the basic amine. Using an approach similar to that of Madrid (Scheme 1), we prepared a series of compounds



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(17a-g) to determine if such compounds might retain antifungal properties.19 To our delight, these compounds were as potent against C. neoformans as the most potent phenothiazine drugs. Basic amines 17a-b,d-f all have MIC values under 10 µg/mL with 17e being the most potent compound we have evaluated in this series to date (MIC 4 µg/mL; 8 µM). Morpholine 17c, a less basic amine, did not have significant antifungal activity, supporting the expected requirement for the presence of a strongly basic amine in the side chain. Table 3. Phenothiazine C. neoformans SAR


pKa

ClogP

CNS MPO Score

17a

8

9.48

7.11

1.71

17b

8

9.38

6.80

1.78

17c

> 64

7.02

6.18

2.31

17d

8

8.45

6.24

2.21

17e

4

8.23

5.55

2.33

Compound

R


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Compound

17f

R


pKa

ClogP

CNS MPO Score

16

9.08

7.74

1.75

Based on the work of Madrid et al., compounds 8 and 17a-c, are not expected to be CNS-sparing.19 However, Madrid et al reported that 17d and 17f exhibited 0% ligand displacement for five of the six receptors reported and 50% binding affinity at 10 µM (Table 4).19 Gratifyingly, 17d and 17e exhibited a significant reduction in dopamine binding affinity relative to TFP (≥20-fold less potent against all 5 dopamine receptors vs. compound 8). 17d and 17e exhibited a more modest selectivity enhancement versus the serotonin receptors (2- to 30-fold less potent vs. compound 8). In particular, 5-HT2A, 5-HT2B and 5-HT2C stand out as being problematic with 17d and 17e having sub-micromolar binding affinity against these three receptors. Generally speaking, however, the benzyl side chain chemotype featured by 17d and 17e tends to give a significant reduction in affinity for both the dopamine and serotonin receptors.



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Table 4. CNS Receptor Binding Profile.

Receptor D1 D2 D3 D4 D5 5-HT1A 5-HT1B 5-HT1E 5-HT2A 5-HT2B 5-HT2C 5-HT3 5-HT5a 5-HT6

Radioligand Binding Assay IC50 (nM)1 8 17d 17e 44 1157 1,403 541 993 >10,000 29 235 2,307 416 >10,000 >10,000 70 1,477 1,336 670 1,242 >10,000 1,210 >10,000 >10,000 >10,000 >10,000 >10,000 11 183 325 112 243 347 63 127 464 581 >10,000 >10,000 1,795 >10,000 >10,000 235 1,102 2,470

1

Compounds with 10,000 nM.

These results illustrate the challenge to elimination of neurotransmitter receptor binding affinity completely. These data do show, however, that it is possible to reduce affinity for receptors that modulate the antipsychotic properties of the phenothiazine scaffold. It is interesting to note that the SAR of these compounds for Mycobacterium tuberculosis (Mtb) differs from that for C. neoformans. Antitubercular potency of compounds 17a, 17d and 17f were previously reported by Madrid et al.19 All three compounds were less potent (>10, 15, and >20 µg/mL, respectively) than TFP (6-12 µg/mL) against Mtb. This suggests that the molecular targets responsible for antimycobacterial activity are distinct from those responsible for antifungal activity. The molecular target for TFP in Mtb is known to be a complex II NADH dehydrogenase.21 Although fungi has complex II NADH dehydrogenases, inhibition of this process does not appear to contribute to the antifungal activity of our series of phenothiazines. Specifically, the activity of molecules that interfere with mitochondrial respiration in yeast is increased in the presence of non-fermentable carbon sources such as glycerol. The MIC of the


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molecules in our series is identical in glucose and glycerol media (17d: MICglucose = 8µg/mL; MICglycerol = 8µg/mL). This observation effectively rules out complex II as a target of the phenothiazines in C. neoformans. Further supporting the notion that the SAR for anti-Mtb and antifungal activities do not correlate, the compounds with improved antitubercular potency did not have significantly improved CNS-sparing profiles. In contrast, our most potent anticryptococcal compounds (17d and 17e) also have decreased affinity for CNS receptors. Assessment of calculated physiochemical parameters. Calculated physiochemical parameters are often used to predict the potential of a given compound to be successfully developed as an oral drug. Lipinski’s Rule of Five highlights the importance of lipophilicity in drug design wherein compounds with ClogP < 5 are projected to be more likely to be orally bioavailable.22

We have calculated the ClogP values for all of the

compounds tested herein (Tables 1-3). With the exception of THZ, all of the CNS drugs tested have ClogP values between 3 and 5. It is gratifying to note that antifungal activity is not directly tied to increased ClogP values as demonstrated by fluphenazine (10), one of the more potent antifungal compounds which has a lower ClogP value of 3.97. Compounds 14a-e do show a correlation between antifungal activity and ClogP, but this may be due to proper positioning of the basis amine side chain for interacting with the molecular target (see below). The antifungal potency of compounds 17a-e does not correlate with ClogP as the most potent compound in this series 17e has the lowest ClogP value. Wager and coworkers at Pfizer have developed a scoring function that predicts assessment of potential of compounds to be developed as CNS drugs.23 They have termed this scoring function CNS multiparameter optimization (CNS MPO). CNS MPO is an algorithm made up of lipophilicity (ClogP), calculated distribution coefficient at pH 7.4, molecular weight, topological surface area, number of hydrogen bond donors and the pKa of the most basic center. Utilization of CNS MPO score may be a very useful tool to assist in the optimization of



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phenothiazine derivatives for cryptococcal meningitis which will require both oral and CNSdruglike properties. CNS MPO scores range from 0 to 6 with 74% of marketed CNS drugs exhibiting scores >4. Using this algorithm, we determined the CNS MPO score of the compounds tested herein (Tables 1-3). The phenothiazine CNS drugs (1-10) exhibited MPO scores of 2.49-4.50. Compounds 14a-e show an inverse correlation between antifungal potency and CNS MPO score, likely due to increasing ClogP values. However, compounds designed for reduced CNS neurological activity such as 17d and 17e have CNS MPO scores (2.21 and 2.33, respectively) comparable to thioridazine (2.49) and TFP (2.85).

Improved potency anti-cryptococcal phenothiazine derivatives also have improved activity against Candida albicans The phenothiazine derivatives reported in the literature have been much less active against C. albicans, typically >4-fold higher MICs.10,11 We wondered whether the derivatives with improved anti-cryptococcal activity would also be more active against C. albicans. As shown in Table 5, 17d and 17e both have improved activity (16 µg/mL) against the standard reference C. albicans strain SC5314 as compared to trifluoperazine (>32 µg/mL). We also tested these two molecules against clinical C. albicans isolates that are resistant to fluconazole. The set of strains represent isolates from the same patient during therapy with fluconazole.24 As shown in Table 5, these strains have increasing fluconazole MIC as mutations in both efflux pump and Erg11, the target of fluconazole, accumulate. The MIC for 17d and 17e were 8 µg/mL for all isolates despite an increase in fluconazole MIC by over 400-fold. Phenothiazine derivatives have been previously shown to inhibit efflux pump function in S. cerevisiae.25 Therefore, we asked if 17d and 17e would act synergistically with fluconazole in the efflux pump-expressing resistant isolates. We, therefore, performed checkerboard fractional inhibitory concentration assays using a combination of fluconazole and 17d or 17e. The combination of both 17d and 17e with fluconazole reduced the lowest concentration to inhibit growth by 2-fold 16 ACS Paragon Plus Environment

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relative to each drug alone. This observation indicates an additive interaction and, consequently, neither 17d nor 17e modulated the fluconazole susceptibility of the resistant strains. Although we cannot rule out efflux pump inhibition by 17d/e, the lack of a microbiologically significant effect of the molecules on fluconazole susceptibility suggests that any such inhibition is minimal. Overall, these data provide the first indications that the phenothiazine scaffold has reasonably good activity against C. albicans and, importantly, indicates that these phenothiazine derivatives are not subject to efflux pump-mediated resistance mechanisms.

Table 5. Antifungal activity of 17d/e against fluconazole resistant-C. albicans FLUa MIC 17d MIC 17e MIC MORb (µg/mL) (µg/mL) (µg/mL)

Entry

Strain

1

SC5314

0.25

1

16

16

2

TWO7229

0.25

1

8

8

3

TWO7230

2

2

8

8

4

TWO7241

16

2, 3

8

8

5

TWO7243

128

2, 3

8

8

a

Fluconazole (FLU); data are from reference 24. MOR: Mechanism of resistance; 1) None; 2) Elevated efflux pump expression; 3) Elevated ERG11 expression;

b

Calmodulin inhibition correlates with the anti-cryptococcal activity of the phenothiazine scaffold. One of the best characterized and most widely exploited activities of the phenothiazine scaffold is its ability to inhibit the function of the calcium-dependent protein calmodulin. Calmodulin functions by binding to other proteins in a calcium-dependent fashion and modulates their activity. Examples of well-characterized calmodulin targets are calmodulin17 ACS Paragon Plus Environment


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dependent protein kinases, phosphodiesterases, and the phosphatase calcineurin. We have previously shown that phenothiazines such as thioridazine and trifluoperazine bind to C. neoformans calmodulin using thermal shift assays.9 We have also shown that these same molecules inhibit the activity of calmodulin in live cells by assessing the effect of the molecules on the nuclear localization of the calmodulin-calcineurin-dependent transcription factor Crz1.26,27 We, therefore, hypothesized that the antifungal activity of the phenothiazine derivatives may correlate with calmodulin antagonism. Since a wide range of phenothiazines have been evaluated for in vitro calmodulin antagonism, we utilized these literature values and plotted the MIC for the phenothiazine derivatives against their IC50 for calmodulin interaction (Fig. 1A).14 Although the sample size for this set is small, the correlation is quite good (Pearson correlation; R2 = 0.94). To further test this hypothesis, we evaluated the ability of the increased potency phenothiazines 17d and 17e to block calmodulin-dependent localization of Crz1. To do so, we used a C. neoformans strain containing a GFP-tagged Nop1, a nucleolar marker, and Crz1 fused to mCherry.26,27 Upon shifting ambient temperature to 37oC, Crz1 translocates to the nucleus (for images please see supplementary material Figure S12). We and others have previously validated this assay.26,27 Calmodulin inhibitors (tamoxifen and trifluoperazine) and 18 ACS Paragon Plus Environment

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calcineurin inhibitors (FK506) reduce nuclear localization of Crz1.26,27 For example, subinhibitory concentrations of trifluoperazine (8 µg/mL) fail to affect Crz1 localization while higher concentrations reduce localization significantly (Fig. 1B). Consistent with our hypothesis, the two most active antifungal phenothiazines also inhibit Crz1 nuclear localization at lower concentrations than trifluoperazine. As noted above calmodulin has many binding partners in the cell and the ability of the trifluoperazine analogs to inhibit calcineurin-dependent processes is used here as a reporter of calmodulin antagonism; it is unlikely that the antifungal effects of these drugs are only due to calmodulin-calcineurin axis inhibition. However, these data indicate that optimization of calmodulin activity could be used to guide the development of more potent derivatives.

Discussion Phenothiazines are one of the oldest examples of a drug scaffold, having been initially discovered as having anti-plasmodial activity by Paul Erhlich in the 19th century. The scaffold has proven to one of the most versatile in pharmacology and derivatives have made contributions to psychiatry, infectious diseases, and other areas. As part of the surge in drug repurposing efforts, derivatives of phenothiazines have emerged in the fields of tuberculosis therapy, cancer chemotherapy, and the treatment of neurodegenerative diseases.28 The antifungal activity of the phenothiazines was initially described in the 1930s and has surfaced in the literature sporadically over the intervening years. Because of the ability of phenothiazines to cross the blood-brain barrier, we became interested in this scaffold in the context of a search for new approaches to the treatment of cryptococcal meningitis. Toward that end, we have carried out the first SAR of the phenothiazine scaffold with respect to antifungal activity and have generated derivatives of the antipsychotic trifluoperazine with improved antifungal activity against not only C. neoformans but also C. albicans. Importantly, these derivatives have activity



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against reference clinical strains and strains that are resistant to fluconazole, one of the most important drugs for the treatment of candidiasis. The modifications that led to improved antifungal activity were guided, in part, by previous work aimed at improving the calmodulin antagonism of the phenothiazine scaffold.14,17 Although it seemed likely that the calmodulin antagonist activity of the phenothiazines contributed to its antifungal activity, the data to support that assertion were preliminary. Thus, our observation that trifluoperazine derivatives with increased antifungal activity also have increased ability to block calmodulin-dependent process in the yeast cell provides strong experimental support for calmodulin inhibition as an important mechanism of action for this structural class. It is important to note, however, that many cellular processes appear to be susceptible to phenothiazine inhibition.28 With this caveat in mind, the identification of a molecular target that can be used to guide optimization of a desired activity is one of the most important reasons for characterizing a scaffold’s mechanism of action. As such, these data put us in an excellent position to further optimize the antifungal activity of the scaffold. Calmodulin is an important calcium sensor in eukaryotic cells and affects the function of target proteins that function in a wide range of cellular processes.29 At first glance, may not seem to be an attractive antifungal drug target due to host toxicity concerns. Interestingly, calmodulin has been targeted for a variety of processes, mostly related to cancer therapy as both an anti-proliferative agent and as an adjuvant to overcome tumor resistance. The rationale for these efforts stems from the fact that phenothiazines-based calmodulin inhibitors have little activity against quiescent mammalian cells while having potent anti-proliferative activity. For example, phenothiazines have little effect on normal lymphocytes but are potently cytotoxic against leukemic cells.30 This selectivity is likely the reason behind the fact that the dose limiting side effect of clinically used phenothiazines is sedation and not cytotoxicity despite concentrations of drug sufficient to inhibit calmodulin.


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Calcineurin is a well-characterized calmodulin target; is involved in T-cell activation; and is the target of an important class of immunosuppressive drugs, e.g., cyclosporine.29 Inhibition of T-cell function through calmodulin modulation in HIV patients with critically reduced CD4 Tcell counts may also seem contra-indicated during treatment of an opportunistic infection. However, a recent clinical trial on the timing of antiretroviral therapy (ART) in the setting of cryptococcosis found that initiation of ART during cryptococcal therapy reduced survival relative to delayed ART.31 Follow-up studies indicate that the higher mortality of early ART is due to increased macrophage and T-cell activation, suggesting that recovery of T-cell function is detrimental during the acute CEM.31 This is consistent with an earlier study which found that transplant patients who developed cryptococcosis while receiving calcineurin-based immunosuppression were more likely to survive than those receiving other types of immunosuppressive drugs.32 Based on these data, non-toxic levels of calmodulin inhibition in the host that may accompany treatment with PTZ-based anti-cryptococcal agents should not adversely affect the host-response to the cryptococcal infection and, indeed, may be beneficial by blunting adverse T-cell activation during the initial, acute phase of therapy. The most significant pharmacologic liability to the phenothiazine scaffold is its neuroleptic and sedative activity.13,19 The most important drivers of these effects are modulation of dopamine and serotonin receptors.19 If this scaffold is to lead to a new class of clinically useful antifungal drugs, then its neurologic activity will need to be reduced while maintaining or improving the antifungal activity. Previously, Madrid et al. undertook a similar approach to improving the anti-tuberculosis activity of phenothiazines and, concurrently, reducing activity toward dopamine and serotonin.19 We were pleased to see that the molecules described by Madrid et al. retained antifungal activity and were actually among the most potent in our series.19 Furthermore, we synthesized a novel derivative based on the Madrid series and were similarly pleased that it is the most active molecule in our set and has consistently ~10100-fold reduced affinity for dopamine and serotonin receptors while retaining physiochemical 21 ACS Paragon Plus Environment


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properties similar to known CNS-penetrating drugs. Taken together, these data suggest that further exploration of the phenothiazine scaffold may lead to new antifungal drugs with favorable pharmacological properties and activity against strains resistant to currently used drugs.

Methods Yeast strains and general microbiological methods. C. albicans strain SC5314 was obtained from Gus Haidaris (Rochester). Cryptococcus neoformans strains H99 and XW252 (H99, MATα, CRZ1-mCherry::NEO, GFP-NOP1::NAT) were gifts of Joseph Heitman (Duke University).26,27 C. albicans clinical isolates TWO7229, TWO7230, TWO7241, and TWO7243 were obtained from Ted White (University of Missouri-Kansas City).24 Yeast media was prepared using standard recipes. All yeast incubations were done at 30oC unless otherwise indicated. In vitro antifungal susceptibility assays.

Antifungal susceptibility testing was performed

using the protocols described in the document CLSI M27-A3.26 The reported MIC values represent the highest value for at least three independent biological replicates performed in technical triplicate. Checkerboard fractional inhibitory concentration assays were performed as previously described using the same medium and conditions as the CLSI M27-A3 protocol.26 Crz1 Nuclear Translocation assays. XW252 was grown overnight for 16 hours in YPD medium at 30°C shaking and subsequently diluted to an OD600 ~0.1 in fresh YPD medium.26,27 The cells were cultivated grown at 25°C to OD600 ~ 0.5. Phenothiazine or DMSO was added to the culture and immediately placed in a water bath at the appropriate temperature for 15 minutes. Samples were fixed in 4% formaldehyde for 15 minutes, harvested by centrifugation and re-suspended in 50 µL of 40 mM potassium phosphate/1.2 M sorbitol buffer. Images were captured using Nikon ES80 epi-fluorescence microscope visualized with a CoolSnap CCD 22 ACS Paragon Plus Environment

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camera and NIS-Elements Software. Quantitative analysis was performed by visually inspecting images for co-localization of Crz1-mCherry with Nop1-GFP. The data represent the mean of at least two biological replicates for which at least 100 cells were counted. Error bars indicate standard deviation. Acknowledgements: This work was supported by NIH grants 1R01AI091422 (to DJK) and, in part, 1R01AI097142 (to DJK). In addition, MCM was supported by Burroughs Wellcome Fund grants BWF1014095 and TL1TR002000. Receptor binding profiles was generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. Supporting Information: Supplementary Experimental Methods: Detailed experimental procedures for the synthesis of all compounds. Chemical compound characterization data for all compounds are provided. Supplementary Figure 1: Fluorescence microscopy images corresponding to Crz1 localization data depicted in the Figure of main text.

References 1. Brown, G.D., Denning, D.W., and Levitz, S.M. (2012) Tackling human fungal infections. Science 336, 647.



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25. Kolaczkowski, M., Michalak, K., and Motohashi, N. (2003) Phenothiazines as potent modulators of yeast multidrug resistance. Int J Antimicrob Agents. 22, 279-283 26. Butts, A., Koselny, K., Chabrier-Roselló, Y., Semighini, C.P., Brown, J.C., Wang, X., Annadurai, S., DiDone, L., Tabroff, J., Childers, W.E. Jr., Abou-Gharbia, M., Wellington, M., Cardenas, M.E., Madhani, H.D., Heitman, J., and Krysan D.J. (2014) Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF-hand proteins and synergize with fluconazole in vivo. mBio 5, e00765. doi: 10.1128/mBio.00765-13. 27. Chow, E.W., Clancey, S.A., Billmyre, R.B., Averette, A.F., Granek, J.A., Mieczkowsk, P., Cardenas, M.E., and Heitman J. (2017) Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans. PLoS Genet. 13, e1006667. doi: 10.1371/journal.pgen.1006667. 28. Ohlow, M.J., and Moosmann, B. (2011) Phenothiazine: the seven lives of pharmacology’s first lead structure. Drug Disc. Today 16, 119-131. 29. Yáñez, M., Gil-Longo, J., and Campos-Toimil, M. (2012) Calcium-binding proteins. Adv Exp Med Biol. 740:461-82. 30. Zhelev, Z., Ohba, H., Bakalova, R., Hadjimitova, V., Ishikawa, M., Shinohara, Y., and Baba Y. (2004) Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal cells. Cancer Chemother. Pharmacol. 53:267275. 31. Boulware, D.R., Meya, D.B., Muzoora, C., Rolfes, M.A., Huppler Hullsiek, K., Musubire, A., Taseera, K., Nabeta, H.W., Schutz, C., Williams, D.A., Rajasingham, R., Rhein, J., Thienemann, F., Lo, M.W., Nielsen, K., Bergemann, T.L., Kambugu, A., Manabe, Y.C., Janoff, E.N.,



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Figure 1 190x254mm (96 x 96 DPI)

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1 Antifungal phenothiazines: optimization, characterization of

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