Repurposing drugs to fast-track therapeutic agents for the treatment of cryptococcosis

Many infectious diseases disproportionately affect people in the developing world. Cryptococcal meningitis is one of the most common mycoses in HIV-AIDS patients, with the highest burden of disease in sub-Saharan Africa. Current best treatment regimens still result in unacceptably high mortality rates, and more effective antifungal agents are needed urgently. Drug development is hampered by the difficulty of developing effective antifungal agents that are not also toxic to human cells, and by a reluctance among pharmaceutical companies to invest in drugs that cannot guarantee a high financial return. Drug repurposing, where existing drugs are screened for alternative activities, is becoming an attractive approach in antimicrobial discovery programs, and various compound libraries are now commercially available. As these drugs have already undergone extensive optimisation and passed regulatory hurdles this can fast-track their progress to market for new uses. This study screened the Screen-Well Enzo library of 640 compounds for candidates that phenotypically inhibited the growth of Cryptococcus deuterogattii. The anthelminthic agent flubendazole, and L-type calcium channel blockers nifedipine, nisoldipine and felodipine, appeared particularly promising and were tested in additional strains and species. Flubendazole was very active against all pathogenic Cryptococcus species, with minimum inhibitory concentrations of 0.039–0.156 μg/mL, and was equally effective against isolates that were resistant to fluconazole. While nifedipine, nisoldipine and felodipine all inhibited Cryptococcus, nisoldipine was also effective against Candida, Saccharomyces and Aspergillus. This study validates repurposing as a rapid approach for finding new agents to treat neglected infectious diseases.


INTRODUCTION
Cryptococcal meningitis is a devastating fungal disease that disproportionately affects the poorest, most resource-limited, underdeveloped regions of the world, where the health burden is extremely high.Those who are most at risk are patients who are various pathogenic fungal species to determine the spectrum of activity.Flubendazole was the most potent compound and was found to specifically target Cryptococcus species.Nifedipine, nisoldipine and felodipine demonstrated antifungal activity towards Cryptococcus species and to varying degrees towards other yeast and mould pathogens.Our findings highlight the value of repurposing strategies to rapidly screen drug libraries that can reveal new classes of antifungal agents for further development.

METHODS Strains
Fungal strains used in this study are listed in Tables 1 and 2. Strains were maintained as glycerol stocks stored at -80 C. Strains were grown on sabouraud dextrose agar (SDA) for 48 h at 30 C before use.

Primary drug screening
We evaluated all 640 FDA-approved compounds in the Enzo library for efficacy against C. deuterogattii strain R265.In the primary screen, each compound was tested once at 10 mg/mL and once at 40 mg/mL using 96-well flat-bottom microtitre plates.Amphotericin B was included as a standard antifungal control.Untreated cells and  2008b).Briefly, an isolated colony of C. deuterogattii strain R265 from a 2-day old plate was inoculated into 1 mL of Yeast Nitrogen Broth (YNB, pH 7; Sigma-Aldrich, St. Louis, MO, USA).A working cell suspension was prepared at 1 Â 10 6 to 5 Â 10 6 cells/mL by cell counting using a haemocytometer, and was diluted 1:100.Final cell concentrations were 0.5 Â 10 3 to 2.5 Â 10 3 cells/mL with a final volume of 200 mL per well.Plates were incubated at 37 C (humidified) without agitation for 72 h.Initial 'hit' candidates were defined as compounds that visibly inhibited the growth of C. deuterogattii.
To identify compounds with the greatest potential for repurposing as novel antifungal agents, compounds that complied with the following were excluded from further assessment: (i) compounds that were active only at concentrations greater than 10 mg/mL; (ii) compounds that were known antineoplastic agents or had otherwise unwanted adverse profiles; and (iii) currently established antifungal agents or non-antifungal agents with antifungal activity that had already been described and assessed at the time of testing.

Broth microdilution test
The in vitro antifungal activities of 'hit' compounds were re-evaluated using the stocks supplied in the library as well as from independently sourced stocks, and testing was extended to additional pathogenic fungal species (Tables 1 and 2).Drug compounds were serially diluted twofold in YNB medium (final concentrations: 0.078125-40 mg/mL).Amphotericin B was included as an antifungal control for reference.Niguldipine, a member of the same class of 1,4-dihydropyridines (calcium channel blocker (CCB)) that had been identified as a non-'hit' compound in the screen was included as a negative control for the CCB series.Fungal cell suspensions were prepared following the CLSI standard method for broth dilution antifungal susceptibility testing of yeasts (Clinical and Laboratory Standards Institute (CLSI), 2008b) or filamentous fungi (Clinical and Laboratory Standards Institute (CLSI), 2008a).Plates were incubated without agitation at 35 or 37 C (humidified) for 48 h for non-Cryptococcus species or 72 h for Cryptococcus species.The minimum inhibitory concentration (MIC) was determined as the lowest drug concentration at which a compound visibly inhibited growth (100% for FLB, MEB, BEN, NIF, NISO, FELO, NIG, AMB and 5FC; 80% for FLC, ITZ and VRZ).To obtain the minimum fungicidal concentration (MFC), 30 mL from each dilution well at the MIC concentration and higher was spread onto SDA plates.The MFC was defined as the lowest drug concentration that yielded three or fewer colonies after 72 h incubation (Espinel-Ingroff et al., 2002).All microdilution assays were tested in technical duplicates with at least two biological replicates unless otherwise specified.
FICI values were calculated as: All drug combinations were tested against C. deuterogattii strain R265 and C. neoformans strain H99 as per CLSI guidelines.All plates were incubated at 37 C and inhibition was read visually at 72 h.

Time-kill assay
Cryptococcus neoformans strain H99 was grown overnight to exponential phase in YNB media, shaking (180 rpm) at 37 C. Cells were adjusted to 1 Â 10 6 cells/mL using a haemocytometer and sub-cultured in fresh YNB media.Three hours post sub-culturing, two 50 mL aliquots were taken.One culture was not treated, while the other was treated with 0.06 mg/mL flubendazole (1.5 Â MIC).Cultures were returned to the shaking incubator and monitored for cell growth at 2, 3, 4, 5, 6, 9 and 24 h post-treatment.At each time point, each culture was sampled in technical duplicates, serially diluted in milli-Q water and spread onto SDA plates.All plates were incubated at 37 C and observed at 48 h and 72 h for viability counts.

Primary drug screening identifies non-antifungal compounds with anti-cryptococcal activity
The preliminary screening of the Enzo library revealed a diverse variety of compounds that inhibited the growth of C. deuterogattii strain R265 (Fig. 1; Fig. S1; Table S1).
A significant proportion (15%) of 'hits' from this initial screen were antineoplastic drugs.This antifungal affect may be attributed to the cytotoxic nature of these compounds since cryptococcal and humans cells are eukaryotic and may share cellular structures and processes that result in non-selective toxicity.For this reason, this group of drugs were not pursued as potential antifungal candidates.Using the selection criteria described in the "Methods" section, 16 of the remaining compounds identified in the primary screen were short-listed for further evaluation by MIC assays against C. deuterogattii.
Antifungal activity was confirmed for ten compounds using drug stocks sourced from the library.The most potent compounds (MICs < 5 mg/mL) were aripiprazole, nisoldipine, oxatomide, flubendazole, nifedipine and anethole-trithione.Lack of commercial availability restricted further evaluation to flubendazole, an anthelmintic drug (Fig. 2), and nisoldipine and nifedipine, which are antihypertensive CCBs (Fig. 3).
Felodipine and niguldipine, which are CCBs structurally related to nifedipine and nisoldipine, were also included in the assay.

In vitro spectrum of antifungal activity of CCBs and flubendazole
To investigate the spectrum of antifungal activity, MICs were determined for nisoldipine, nifedipine, felodipine, niguldipine and flubendazole against nine additional strains from four fungal species (Table 1).MIC assays using independently sourced drug stocks confirmed the MICs established for C. deuterogattii strain R265 and demonstrated a diverse range of antifungal activities against other fungal strains and species (Table 1 an emerging fungal pathogen, with an MIC of 20 mg/mL.In contrast, nisoldipine had higher and more variable MICs against different Cryptococcus species (5-40 mg/mL) and was the only drug that had activity against all nine fungal species.Niguldipine was not effective at the highest tested concentration against the fungal strains in this assay, while felodipine demonstrated activity against the Cryptococcus species with MICs between those of nifedipine and nisoldipine, but was only active against Cryptococcus species.

Antifungal activities of flubendazole
To test whether flubendazole is broadly active across Cryptococcus species, we extended the study to encompass all members of the C. gattii species complex, as well as the major molecular genotypes of C. neoformans (Table 2; Table S2), including veterinary, clinical and environmental isolates from various geographic locations.Flubendazole inhibited all isolates of both Cryptococcus species at consistently low concentrations (MIC 0.039-0.156mg/mL).Furthermore, for isolates where fluconazole MIC data were available, flubendazole was found to be equally effective regardless of fluconazole susceptibility (Table 2; Table S2).
To explore the inhibitory effect of flubendazole on Cryptococcus cells, cell viability was assessed using a time-kill assay and determining the MFC in a microwell format.Analysis by time-kill curve over 24 h demonstrated that Cryptococcus cells exposed to flubendazole had slowly declining growth for up to 9 h post-treatment prior to loss of viability (Fig. 4; Table S3).Microbroth dilution assays generated MFC values that were similar to the respective MIC values (MFC 0.039-0.156mg/mL; Table 2).To explore interactions with current, commonly used antifungals, flubendazole and the other benzimidazole agents mebendazole and benomyl was tested in combination with amphotericin B, fluconazole, itraconazole, voriconazole and 5-flucytosine against C. deuterogattii and C. neoformans in a checkerboard assay.All combinations produced additive/indifferent results and no synergistic or antagonistic interactions were seen (Table 3; Tables S4-S5).

DISCUSSION
Despite the major health problems caused by cryptococcal infections, there is a lack of new antifungal drugs.Promising, albeit limited, research has demonstrated the potential for repurposing drugs and the implications of this approach for treating cryptococcal infections have been highlighted (Butts et al., 2013;Dehdashti et al., 2013;Joffe et al., 2017;Rabjohns et al., 2013;Rhein et al., 2016;Samantaray et al., 2016;Spitzer et al., 2011).Most C. neoformans cells in logarithmic growth phase were treated with 0.06 mg/mL flubendazole and incubated at 37 C with shaking.The growth of treated cells tracked the untreated control cells for 3-4 h post-treatment and began to slow thereafter.Viability declined markedly by 9 h and did not recover by 24 h.Growth curve was generated using Prism 7 (Version 7.0b; GraphPad Software, La Jolla, CA, USA).
Full-size  DOI: 10.7717/peerj.4761/fig-4 Table 3 Fractional inhibitory concentration index values of flubendazole, mebendazole and benomyl in combination with various antifungals against C. neoformans H99 and C. deuterogattii R265.studies have screened the Prestwick Chemical Library (n = 1,120), the Library of Pharmaceutically Active Compounds (n = 1,280) and the National Institutes of Health clinical collection (n = 727).Within the last decade, these drug screening studies have led to the discovery of multiple non-antifungal compounds with antifungal activities, which were often novel and fungicidal (Butts et al., 2013;Dehdashti et al., 2013;Joffe et al., 2017;Rabjohns et al., 2013;Samantaray et al., 2016;Spitzer et al., 2011).Some have demonstrated the ability to target intracellular C. neoformans in macrophages and cryptococcal biofilms and to act synergistically with the widely-used drug fluconazole (Butts et al., 2013;Dehdashti et al., 2013;Joffe et al., 2017;Rabjohns et al., 2013;Samantaray et al., 2016;Spitzer et al., 2011).However, these studies have focused on C. neoformans with only one reporting on C. deuterogattii strain R265 (Spitzer et al., 2011).There can be significant differences in target specificity among closely related genera and species of pathogenic yeasts (Chong et al., 2010;Spitzer et al., 2011).Thus, while C. neoformans and C. deuterogattii are sibling species, drugs may work differently between them.

Cryptococcus species
Here we report the first screening of a 640-compound FDA-approved library against C. deuterogattii strain R265, with subsequent testing of additional pathogenic cryptococci (encompassing an extensive list of C. neoformans species and the entire C. gattii species complex) and other fungi.

Repurposing CCBs as antifungal agents
Calcium channel blockers, widely used in the treatment of hypertension, are broadly divided into dihydropyridines and non-dihydropyridines classes (Catterall, 2011).Our screen identified nifedipine, felodipine and nisoldipine (Table 1; Fig. 3) as having antifungal activity against Cryptococcus.These are all 1,4-dihydropyridines and therefore present as intriguing leads for a potential new class of antifungal drugs.Members of the 1,4-dihydropyridine CCB group share a core phenyl group attached to a 1,4-dihydropyridine that has two ester side groups (Fig. 3).Variations in side groups may account for the diverse antifungal activities observed, as compounds with the bulkier side groups (lacidipine and niguldipine) were ineffective against C. deuterogattii.Nifedipine, felodipine, nisoldipine and niguldipine target L-type calcium channels-one of five types of voltage-gated calcium channels (VGCCs) designated L-, N-, P/Q-, R-and T-type (Catterall, 2011).In the model yeast Saccharomyces cerevisiae, Cch1 and Mid1 are homologs of the mammalian VGCCs, and L-type CCBs are known to inhibit fungal growth via a Cch1-Mid1 protein channel complex located in the plasma membrane (Prole & Taylor, 2012;Teng et al., 2008).Cch1 acts as the calcium channel and has homologs in almost all fungi, while Mid1 is a regulatory subunit that interacts with Cch1 to mediate calcium influx (Hong et al., 2013).Hence, it appears likely that the antifungal effects of CCBs involve the Cch1-Mid1 calcium channel.
Our findings support a growing body of evidence suggesting a crucial role of calcium regulation and calcium channels in determining the growth and survival of yeasts under stressful conditions (Hong et al., 2010;Liu et al., 2006;Peiter et al., 2005).In C. neoformans, Cch1 facilitates calcium entry with Mid1 when there is a depletion of intracellular calcium (Hong et al., 2010).In the presence of the calcium chelator BAPTA, the growth of Ácch1 mutants is markedly diminished at elevated temperature (25-38.5 C), a key virulence factor that is critical for human infection (Liu et al., 2006).Cch1 and Mid1 also appear linked to the regulation of intracellular redox under cell stress, as S. cerevisiae Ácch1 and Ámid1 (single-and double-knockout) mutants are sensitive to cold stress (10 C) and iron toxicity due to a compromised ability to cope with oxidative stress (Peiter et al., 2005).Cell growth can be re-established following supplementation with either 10 mM calcium or reduced glutathione (an antioxidant), indicating that a functional Cch1-Mid1 complex is essential for stress tolerance (Peiter et al., 2005).
As well as possessing antifungal properties, CCBs have also been shown to act synergistically with fluconazole against fluconazole-resistant and -susceptible strains of C. albicans (Liu et al., 2016).Interestingly, CCBs alone or in combination with fluconazole do not significantly alter the gene expression level of CCH1 and MID1 (Liu et al., 2016) suggesting that either the cell is not sensing a disruption to calcium homeostasis, or CCBs act on targets other than calcium channels.Recently, a screen of the Prestwick Chemical Library Ò of FDA-approved small molecules identified fendiline hydrochloride, a non-dihydropyridine CCB, as having unique activity to potentiate phagosomal killing of intracellular C. neoformans in host macrophages (Samantaray et al., 2016).
Although it has a very different structure to the antifungal CCBs identified here, fendiline is an L-type CCB, suggesting this class of CCBs may be the most promising for antifungal drug development.Furthermore, the antifungal activity of L-type CCBs in combination with standard antifungals have been characterised in C. albicans and Aspergillus (Afeltra et al., 2004;Liu et al., 2016).As hypertensive agents, L-type CCBs are currently administered in long term use with relatively minimal side effects (Chrysant, 2003).Of clinical relevance, some CCBs including nifedipine and nisoldipine can traverse the blood-brain barrier and may therefore be able to treat cryptococcal meningitis (Gaggi, Cont & Gianni, 1993).We therefore suggest L-type CCBs are promising leads for repurposing and redevelopment as potential new antifungal agents.

Repurposing flubendazole as an anti-cryptococcal agent
Flubendazole is an anthelmintic drug belonging to a family of benzimidazole carbamates.First introduced in 1980, it is currently used to treat parasitic gastrointestinal worm infections in humans and animals (Fig. 2) (Spagnuolo et al., 2010).Flubendazole acts by binding to and inhibiting the polymerisation of tubulin, a cytoskeletal protein that polymerises to form microtubules (Chatterji et al., 2011;Cruz & Edlind, 1997;Spagnuolo et al., 2010).Microtubules play major roles in various cell processes including division, motility, adhesion and intracellular transport (Chatterji et al., 2011).Benzimidazoles have been found to act selectively against fungal tubulin and not mammalian tubulin, possibly due to differences in their b-tubulin sequence (Cruz & Edlind, 1997).
Benzimidazoles were originally developed as agricultural fungicides prior to their use as anthelmintic drugs (Horton, 2000).While the antifungal activities of benzimidazoles and their potential to be developed as anti-cryptococcal agents have been known for over two decades (Patent US5434163) (Cruz, Bartlett & Edlind, 1994;Del Poeta et al., 1998;Edlind & Cruz, 1995), no further development as antifungal agents has occurred since 1998 (Del Poeta et al., 1998).Our study indicated that flubendazole is fungicidal at extremely low concentrations against C. neoformans and C. deuterogattii (Table 2).Our findings show that flubendazole equally inhibits pathogenic and environmental isolates of Cryptococcus, as well as strains that are resistant to fluconazole.This indicates that flubendazole may be an effective treatment against genetically and physiologically diverse Cryptococcus species and strains.As resistance to fluconazole is frequently induced during prolonged treatment and has been associated with clinical failure in Cryptococcus-infected patients (Bicanic et al., 2006;Jarvis et al., 2014;Smith et al., 2015), flubendazole may have promise as a second-line therapy when fluconazole fails, particularly in resource-poor regions where there are often no suitable alternatives.
Flubendazole interferes with normal cell growth as early as 3 h post-treatment and continues to render treated Cryptococcus cells unviable (Fig. 4), and in a microwell format with 72 h incubation the MFC values were either the same or one increment higher than the respective MIC values (Table 2).This indicates that flubendazole is a fungicidal drug, and with the correct clinical administration, it could effectively clear fungal burden.As high fungal burden is a strong determinant for mortality and overall poor clinical outcomes, and currently amphotericin B is the only fungicidal agent that is effective against cryptococcosis, this reinforces the potential utility of flubendazole as an anti-cryptococcal agent (Jarvis et al., 2014).It is noted however, that unlike amphotericin B, flubendazole does not act synergistically with other standard antifungal agents, consistent with previously published data (Table 3) (Joffe et al., 2017).
The current findings support a recent screening of 727 compounds in the National Institute of Health Clinical Collection (Joffe et al., 2017).Flubendazole and three related anti-helminthic benzimidazoles (mebendazole, albendazole and triclabendazole; sharing the benzimidazole scaffold) were among 17 compounds active against C. neoformans (Joffe et al., 2017).However, that study focused on mebendazole on the basis that the drug is able to penetrate the blood-brain barrier.Low MIC and MFC values were reported, and efficacy against biofilms and against cryptococcal cells internalised in macrophages were shown, along with additive activity when combined with amphotericin B (Joffe et al., 2017).Flubendazole may mirror the antifungal capabilities observed for mebendazole as they share structural similarities, and there is evidence that like mebendazole, flubendazole can traverse the blood-brain barrier (Te ´llez-Giro ´n et al., 1984) making these compounds favourable candidates for the treatment of cryptococcal meningitis.However, as flubendazole is formulated to treat gastrointestinal worms, it is not yet known whether it would be able to reach therapeutic concentrations in the brain required to elicit an antifungal effect.
The precise mechanism underlying the antifungal activity of flubendazole is unclear, however it is thought that it targets b-tubulin in C. neoformans (Cruz & Edlind, 1997).The kill-curve demonstrated initial growth after treatment followed by declining viability and death at 9 h, which is consistent with a mode of action that works by interfering with cell growth and division.In Echinococcus granulosus, a tapeworm parasite, flubendazole has been found to disrupt a wide spectrum of cellular homeostatic mechanisms, including energy metabolism and calcium homeostasis.In the latter it appears to increase cytosolic calcium from extracellular and intracellular stores (Cumino, Elissondo & Denegri, 2009), demonstrating an interesting overlap with the CCBs outlined above.
Overall, flubendazole (and more generally the benzimidazole scaffold) may be a promising starting point for the treatment of cryptococcal disease.This view is further encouraged by a lack of evidence of serious adverse side effects in patients undergoing anti-helminthic treatment and in mice experimentally treated with flubendazole (Ceballos et al., 2009;Feldmeier et al., 1982;Lasse `gue et al., 1984;Te ´llez-Giro ´n et al., 1984).In preliminary cryptococcal meningitis studies, flubendazole demonstrated fungicidal activity in a hollow fibre infection model and efficacy in vivo, reducing fungal burden in both a rabbit and mice model (Nixon et al., 2018).However, whether flubendazole can accumulate in the brain to concentrations that are clinically achievable and able to elicit an antifungal effect, and whether this can be sustained in the absence of adverse reactions, remains unknown and requires more extensive in vivo testing.

CONCLUSION AND PERSPECTIVES
This study, and others like it, demonstrate the utility of drug repurposing to discover novel agents for the treatment of neglected diseases like cryptococcosis.L-type CCBs and anti-helminthic benzimidazoles have emerged in different drug screens and appear particularly promising as both are registered for use in other applications, are well tolerated and appear able to traverse the blood-brain barrier.Phenotypic screening approaches have repeatedly demonstrated utility, ensuring that identified drug candidates possess the desired phenotype of inhibiting growth of the pathogen prior to further mechanistic investigations.In addition to serendipitously finding molecules or compounds that can be used directly in the clinic, repurposing strategies may identify related classes of compounds with activity and thus provide a scaffold for subsequent medicinal chemistry to initiate lead drug discovery programs.
Currently, it costs billions of dollars for a drug to successfully obtain FDA-approval and reach market.To help alleviate this financial risk and provide an incentive, there are moves by regulatory agencies in the United States and Europe to encourage academic and pharmaceutical research to bring new antimicrobials to market.For example, the GAIN Act (Generating Antibiotic Incentives Now) grants companies manufacturing a 'qualified infectious disease product' an additional period of market exclusivity, with or without a patent, to ensure a better return on their investment.This applies to the repurposing of existing drugs with new indications, and should encourage pharmaceutical companies to develop some of these newly identified agents as therapeutics for fungal diseases, which are urgently required in global health.

Figure 4
Figure4Time-kill assay of C. neoformans strain H99 treated with flubendazole.C. neoformans cells in logarithmic growth phase were treated with 0.06 mg/mL flubendazole and incubated at 37 C with shaking.The growth of treated cells tracked the untreated control cells for 3-4 h post-treatment and began to slow thereafter.Viability declined markedly by 9 h and did not recover by 24 h.Growth curve was generated using Prism 7 (Version 7.0b; GraphPad Software, La Jolla, CA, USA).Full-size  DOI: 10.7717/peerj.4761/fig-4 amphotericin B; FLC, fluconazole; ITZ, itraconazole; VRZ, voriconazole; 5FC, 5-flucytosine.Data shown were produced from two independent replicates.

Table 1
Minimum inhibitory concentrations of flubendazole and a series of calcium channel blockers against Cryptococcus, Saccharomyces, Candida and Aspergillus species.
Notes:MIC ranges are given for compounds with variable results.MICs greater than 40 mg/mL (shaded in grey) indicate where an MIC was not obtained at the highest testing concentration.FLB, flubendazole; NIF, nifedipine; NIG, niguldipine; FELO, felodipine; NISO, nisoldipine.a MICs obtained from four biological replicates each with technical duplicates.

Table 2
Minimum inhibitory and minimum fungicidal concentrations of flubendazole against Cryptococcus species from varied sources.
Notes:a USA, United States of America; FLU, fluconazole.bFLC (fluconazole) MIC data obtained from Chong et al. (2010) and Lai et al. (2016).c Pie chart of drug compound classes that inhibited the growth of C. deuterogattii strain R265 at 10 mg/mL.A total of 54 compounds inhibited the growth of C. deuterogattii strain R265 at 10 mg/mL.