High Efficiency Drug Repurposing for New Antifungal Agents

: There has been a persistent


Introduction
• Antifungal drug repurposing is the repositioning process of non-antifungal, marketed drugs (previously approved for treating other diseases) to treat fungal infections, where the modes of action, cellular targets or safety of the drugs are already identified (Stylianou et al. 2014).While drug repurposing has become a viable approach to accelerate new antifungal drug development, this strategy still requires highly sensitive screening systems.• The antioxidant system of fungi is a potential target of antifungal agents (Smits andBrul 2005, Jager andFlohe 2006).Certain natural compounds, such as derivatives of benzoic acid or sulfur-containing compounds, can be redoxactive and thus inhibit fungal growth by interfering with cellular redox homeostasis/antioxidant system (Guillen andEvans 1994, Jacob 2006).• Antifungal chemosensitization is an intervention strategy, in which coapplication of a certain natural or synthetic compound, viz., chemosensitizer, with a commercial drug augments the efficacy of the drug co-applied (Kim et al., 2012).While a chemosensitizer does not necessarily have antifungal potency, chemosensitization can lead to: (a) the augmentation of antifungal efficacy of commercial drugs co-applied; (b) overcoming fungal resistance to commercial antifungal drugs; and also (c) enhanced inhibition of mycotoxin production by fungi, such as aflatoxigenic Aspergillus parasiticus (Kim et al., 2014).
• The yeast Saccharomyces cerevisiae is a useful model system for the identification of antifungal drugs and their molecular targets in view that: (1) the genome of S. cerevisiae has been sequenced and well annotated (Saccharomyces Genome Database, www.yeastgenome.org),(2) S. cerevisiae gene deletion mutant collections (~6,000 mutants) have proven to be very useful for determining drug mechanism of action (Parsons et al, 2004;Norris et al, 2013;Lee et al, 2014), and (3) many genes in S. cerevisiae are orthologs of genes of fungal pathogens including Aspergillus sp.(Kim et al, 2005).• Using the model yeast S. cerevisiae bioassay, we previously identified several chemosensitizers, which target cellular antioxidant or cell wall integrity systems (See next slide for examples).• In this in vitro study, we tried to develop a high-efficiency drug repurposing strategy for effective control of fungal pathogens.We selected two drugs (aspirin, bithionol) previously investigated, and concentrated on targeting the oxidative stress response system of fungi with redox-active chemosensitizers, viz., 2-isopropyl-5-methylphenol (Thymol), 4-isopropyl-3-methylphenol (Structural analog of thymol) and 3,5-dimethoxybenzaldehyde. • The susceptibility of fungi to the candidate drug (Bithionol) could be enhanced by co-applying with redox-active chemosensitizers.Bithonol also mitigated fludioxonil tolerance of Aspergillus fumigatus antioxidant signaling mutants.

Results and discussion
Examples)of)repositioned)drugs)possessing)antifungal)activities.) We chose aspirin and bithionol as representative drugs for further investigation.
• Bithionol is a halogenated anti-infective agent that is used against trematode and cestode infestations.This drug inhibits human soluble adenylyl cyclase (Kleinboelting et al. 2016).
• Octyl gallate (OG) was used as a positive control for antifungal bioassay.The mechanism of antifungal action of OG was previously determined as: (a) interrupting the lipid bilayer-protein interface in fungal cells, and (b) functioning as a pro-oxidant (redox-active oxidative stressor), thus triggering cytotoxicity in fungi (Kim et al. 2018).

Fungal signaling system as a target:
Meanwhile, oxidative signaling systems, such as mitogen-activated protein kinase (MAPK) signaling pathway, have been served as effective antifungal targets for redoxactive drugs or compounds (Kim et al. 2012) (Next page).www.aspergillusgenome.orgwww.yeastgenome.orgMAPK Aspergillus fumigatus is a causative agent of the highly debilitating human invasive aspergillosis, where the sakA and mpkC genes encode MPAKs in A. fumigatus.A. fumigatus sakAΔ is an osmotic/oxidative stress sensitive MAPK mutant, while the mpkCΔ is a MAPK mutant of the polyalcohol sugar utilization system (Xue et al. 2004;Reyes et al. 2006).We previously determined that both mutants were highly susceptible to redoxactive reagents such as amphotericin B, itraconazole or natural phenolics compared to the wild type strain (Kim et al. 2011(Kim et al. , 2012)).

2-isopropyl-5-methylphenol (Thymol) 4-isopropyl-3-methylphenol
Bithionol + 2-Isopropyl-5-methylphenol (Thymol): Results showed that antifungal activity of bithionol was greatly enhanced in the presence of thymol (chemosensitizer; a chemical probe targeting fungal antioxidant system), while that of aspirin was almost not affected, indicating "drug-chemosensitizer specificity" exists for the enhancement of antifungal activity.Results also showed that A. fumigatus MAPK mutants were more susceptible to the treatment compared to the wild type, indicating increased susceptibility of antioxidant mutant to the application of redox-active agents, such as thymol (Test concentrations-Aspirin & Bithionol: 32 to 1,024 µM; OG: 1 & 5 mM).Bithionol + 4-Isopropyl-3-methylphenol (4I3M; Thymol analog): We found the level of bithionol activity was enhanced further when the structural analog of thymol, viz., 4I3M, was co-applied as a chemosensitizer.For example, antifungal activity of bithionol was enhanced at much lower concentration of bithionol (32 to 128 µM), and the sizes of zone of inhibition were also larger than that with thymol.Therefore, results indicated that 4I3M could be more effective chemosensitizer to bithionol.As observed in thymol, the activity of aspirin was almost not affected (Test concentrations-Aspirin & Bithionol: 32 to 1,024 µM; OG: 1 & 5 mM).
Results indicated 4I3M negatively affects both cellular ion and redox homeostasis in fungi.Similar results were also observed with thymol in a previous study (Kim et al. 2012), indicating 4I3M and thymol share analogous cellular targets in fungi.
Test in Aspergillus parasiticus, a mycotoxigenic fungus producing hepatocarcinogenic aflatoxins (Bithionol + Thymol or 4I3M): Similar results were obtained in A. parasiticus 2999 strain, where thymol exhibited higher activity comparing to its analog 4I3M.Also, the sizes of zone of inhibition were generally smaller than that observed in A. fumigatus, indicating "strain specificity" also exists for the We investigated the effect of other types of chemosensitizer for the enhancement of bithionol activity.Results showed that antifungal activity of bithionol was also increased when co-applied with 3,5-D.3,5-D was also shown to negatively affect cellular antioxidant system, such as superoxide dismutases or glutathione reductase (Kim et al. 2011).As observed in thymol/4I3M, antifungal activity of aspirin was almost not affected.In general, the level of the enhancement of bithionol activity with 3,5-D was lower than that with thymol/4I3M (Test concentrations-Aspirin & Bithionol: 32 to 1,024 µM; OG: 1 & 5 mM).Test in the mycotoxigenic Aspergillus parasiticus (Bithionol + 3,5-Dimethoxybenzaldehyde): Similar results were obtained in A. parasiticus 5862 strain, where the sizes of zone of inhibition were generally smaller than that observed in A. fumigatus, further indicating "strain specificity" for the efficacy of "bithionol + 3,5-dimethoxybenzaldehyde" treatment (Test concentrations-Aspirin & Bithionol: 32 to 1,024 µM; OG: 1 & 5 mM).Currently, antifungal drug repurposing is underway using the following scheme: Overcoming Fludioxonil Tolerance of Aspergillus fumigatus MAPK Mutants by Bithionol: Fludioxonil is a commercial phenylpyrrole fungicide, which triggers abnormal and excessive stimulation of the antioxidant MAPK signaling system (Kojima et al. 2004).This abnormal activation of MAPK system triggers cellular energy deprivation via metabolic shifts from normal fungal growth to exhaustive oxidative stress defense.Therefore, application of fludioxonil prevents the growth of fungal pathogens.However, fungi having mutations in components of upstream signaling system, viz., antioxidant MAPK signaling pathway, can escape fludioxonil toxicity (Kojima et al. 2004).
As shown in the figure (next page), A. fumigatus MAPK mutants sakAΔ and mpkCΔ were tolerant to fludioxonil (50 μM), thus developed radial growth on potato dextrose agar (PDA), whereas the growth of wild type was completely inhibited.However, co-application of sub-fungicidal concentration of bithionol (125 μM) with fludioxonil (50 μM) effectively prevented fungal tolerance to fludioxonil, thus achieving complete inhibition of the growth of MAPK mutants.Comprehensive determination of the efficacy of bithionol as an antifungal drug warrants future in-depth study.Bithionol overcomes fludioxonil resistance of Aspergillus fumigatus MAPK mutants (%, Radial growth rate): • High sensitivity antifungal screening method was investigated by incorporating redox-active chemosensitizers (chemical probes) and antioxidant mutants of A. fumigatus.
• Thymol, 4I3M or 3,5-D can be used as potent chemosensitizers to enhance antimycotic activity of the repurposed drug bithionol, while the efficacy of the other drug aspirin was almost not affected, indicating "chemosensitizer -drug specificity" exists.
• While similar enhancement of antifungal efficacy was also observed in the mycotoxigenic A. parasiticus, the level of sensitivity of this species was not comparable to that in A. fumigatus, thus indicating "strain specificity" also exists during chemosensitization.
• In summary, current data could be used for achieving high-efficiency, largescale repositioning of marketed drugs with no known antifungal activities as new antifungal drugs, which can reduce costs, abate resistance, alleviate negative side effects associated with current antifungal treatments. •

Repurposed drug examples: PubMed
search in the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) by using the key words "Drug Antifungal Repositioning" (Search date: May 31, 2018) retrieved 70 articles.We re-evaluated the content of the retrieved articles for their relevance to drug screening, and examples are as shown below.