Drug Repurposing Strategies in the Discovery of Antifungal Agents

The morbidity and mortality caused by invasive fungal infections is increasing across the globe due to developments in transplant surgery, the use of immunosuppressive agents, and the emergence of drug-resistant fungal strains, which has led to a challenge in terms of treatment due to the limitations of three classes of drugs. Hence, it is imperative to establish effective strategies to identify and design new antifungal drugs. Drug repurposing is an effective way of expanding the application of existing drugs. In the last years, various existing drugs have been shown to be useful in the prevention and treatment of the invasive fungi. In this review, we summarize the currently used antifungal agents. In addition, the most up to date information on the effectiveness of existing drugs with antifungal activity is discussed. Moreover, the antifungal mechanisms of existing drugs are highlighted. These data will provide valuable knowledge to stimulate further investigation and clinical application in this field.


Introduction 1
Fungal infection has become a significant event leading to over 1.5 million deaths annually 2 worldwide [1]. To date, the most common fungal infections related to human mortality and 3 morbidity are caused by Cryptococcus, Candida, and Aspergillus [2]. The impact of mycoses has 4 increased due to developments in transplant surgery, chemoradiotherapy, hemodialysis, and the use 5 of immunosuppressive agents, especially in patients with immunodeficiency disorders, with an 6 estimated mortality ranging from 35 % to 45 % [3]. Hence, antifungal therapy represents a 7 challenging problem for clinicians. In addition, the limited number of antifungal agents in the clinic 8 can induce side-effects and a great number of drug-resistant or multidrug resistant strains have 9 emerged. Candida auris, a multidrug resistant fungus, has shown a global increase in recent years. 10 Importantly, some of these infections are resistant to almost all current antifungal agents [4]. In New 11 Delhi, it was reported that 15 COVID-19 patients had secondary candidiasis in the intensive care 12 unit (ICU), two-thirds of which were caused by C. auris, and the mortality rate was up to 60 % [5]. 13 Currently, the first-line antifungal agents for invasive fungal infections are voriconazole, 14 itraconazole, amphotericin B, and echinocandins [6]. However, due to the existence of toxicity and 15 drug-resistant strains, the present antifungal options have become more restricted. A variety of 16 approaches have been employed to conduct antifungal therapies, such as the synthesis of new 17 substances, the use of extracts from organisms, the development of old drugs to change the use or 18 form of fungal disease, and an association between known antifungal drugs and non-antifungal 19 agents [7]. Drug repurposing is an established strategy for the treatment of invasive fungal infections 20 due to the excellent antifungal activity of these drugs. Several agents have recently been confirmed 21 to serve as antifungal candidates in the treatment of mycoses. The purpose of this review is to present 22 a series of known drugs that have been investigated for their application in the treatment of fungal 23 infections. Firstly, the strategies, mechanisms, and challenges of current antifungal drugs are 24 described. Secondly, the extensive application and antifungal mechanisms of drugs with antifungal 25 activity that had been used in the clinic to treat non-mycotic infections are highlighted. 26

Current antifungal drugs used in the clinic 27
Since the first active antimycotic griseofulvin was recognized in 1939, a multitude of antifungal 28 agents have been used clinically. The three main types currently used in the clinic are polyenes, 29 azoles, and echinocandins. In fungi, ergosterol, located in the cell membrane, regulates membrane 30 structure permeability, mobility, and substance transportation by making direct linkages with the 31 phospholipid membrane [8]. The representative polyene drug is amphotericin B, which can bind to 32 ergosterol from lipid bilayers and form large and extramembranous aggregates [8]. These 33 extramembranous aggregates lead to the formation of transmembraneal pores, which can leak 34 cellular components. This results in the death of pathogenic fungi [8]. As the "gold standard" for 35 combating invasive fungal infections for decades, amphotericin B has a broad spectrum of 36 antifungal activity against yeasts and molds [9]. For instance, an investigation of 78 Candida spp. 37 clinical strains, showed that all examined free-living cells, were susceptible to amphotericin B [10]. 38 When amphotericin B was combined with caspofungin and voriconazole in Aspergillus species, the fractional inhibitory concentration (FIC) index was only 0.10-0.22 [11]. However, it has limited 40 clinical applications due to toxicity, which includes nephrotoxicity and infusion-related reactions 41 such as chest pain, dyspnea, hypoxia, flushing, and urticaria [12]. To resolve this problem, lipid 42 formations of amphotericin B, including liposomal amphotericin B (LAmB), AmpB lipid complex 43 (ABLC), and AmpB colloid dispersion (ABCD) were developed [9]. Toxicity was greatly reduced 44 using these formulations; however, the results were disappointing due to their low permeability at 45 therapeutic concentration [13]. 46 Due to the safety and wide availability, the azoles (including fluconazole, itraconazole, 47 voriconazole, respectively, and increases the permeability of the cell wall and cell membrane of 118 showed that the antifungal applications of antibiotics interfere with the homeostasis of symbiotic 142 bacteria and fungi in the body. Moreover, dysbiosis of microbiota is responsible for the occurrence 143 of many other diseases in humans such as cardiovascular, cancer, allergy, and the microbiota also 144 affect the human immune system and the synthesis of nutrients [58,59]. In addition, the 145 pharmacokinetics of antibiotics with antifungal activity in vivo also require further investigation. 146

Statins 191
Statins are firstly known as lipid-lowering and cholesterol-lowering drugs as they inhibit  CoA reductase (an essential enzyme in cholesterol biosynthesis) [114], and are classified according 193 to their hydrophobicity into hydrophilic statins (pravastatin and rosuvastatin) and lipophilic statins 194 (atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, and simvastatin). It has been confirmed that statins exert a broad spectrum of anti-fungal effects on Candida spp., Aspergillus 196 spp., and Zygomycetes [115]. Table 3 shows a brief summary of the statins with antifungal ability. 197 The antifungal mechanism of statins is focused primarily on the biofilm. For example, the changes 198 in the main components of the biofilm or genes associated with fungal biofilm formation following 199 monotherapy or/and synergism of antifungal agents have been verified. Inexplicably, antifungal 200 activity findings are inconsistent. For instance, pravastatin has synergistic effects with fluconazole 201 by inhibiting farnesol production against C. albicans [116], and in contrast, no synergy was found 202 between pravastatin and fluconazole in vitro in another investigation [117]. In addition, a study even 203 reported that pravastatin did not inhibit the growth of Candida spp. [118]. The reason for this may 204 be due to differences in fungi strains, and different methodology. These contradictory findings 205 require clarification in further investigations. 206

Antiarrhythmic drugs 207
Antiarrhythmic drugs, which are used in the prevention and treatment of tachycardia, bradycardia, 208 or arrhythmia, include sodium channel antagonists, β -receptor blockers, potassium channel 209 blockers (PCRs), and calcium channel blockers (CCBs). Trails have shown that amiodarone, PCRs, 210 and CCBs exhibit favorable antifungal activity when administered alone or combined with 211 conventional antifungals. CCBs, as the name suggests, prevent calcium ions entering cells and 212 maintain metabolic processes. Verapamil (verapamil hydrochloride), a phenylalkylamine CCB, 213 mainly combats C. albicans by affecting hyphal development, adhesion, gastrointestinal 214 colonization or increasing strain susceptibility to oxidative stress [131,132]. In addition, verapamil 215 enhances the antifungal activity of tunicamycin or fluconazole against C. albicans during biofilm 216 formation and pre-formed biofilms [133]. In A. fumigatus, the drug efflux pump was blocked and 217 ergosterol content was decreased following treatment with verapamil and itraconazole 218 simultaneously [134]. Other CCBs, such as diltiazem, nicardipine, and nifedipine, have shown 219 antifungal activity against C. albicans, C. glabrata, Ascomycetous and Mucoralean fungi, either 220 alone or in combination with antifungals [135][136][137]. Amiodarone is known to block potassium, 221 sodium and calcium channels, and is commonly used to treat and prevent tachyarrhythmia [138-222 140]. In pathogens, amiodarone mainly disrupted calcium homeostasis to elicit high levels of 223 cytoplasmic calcium, leading to cell death in Cryptococcus spp., Aspergillus spp., Fusarium 224 oxysporum, C. albicans, C. tropicalis, and Saccharomyces cerevisiae [141][142][143][144][145][146][147]. In addition, 225 amiodarone displayed potent fungicidal effects at low dose combined with fluconazole and 226 miconazole [148]. Hence, calcium channels may be potential targets in the therapy of fungal-related 227 infections. 228

Antipsychotic drugs 229
Antipsychotic drugs include benzamides, butyrophenones, dibenzoxazepine, phenothiazines, and 230 thioxanthene. Phenothiazines are the first generation of antipsychotics, and are mainly used to treat 231 schizophrenia and mania. In addition, phenothiazines possess multiple effects such as altering the 232 metabolism of cyclic nucleotides, modifying the structure of membranes, binding to calmodulin and 233 they participate in many intracellular responses [149], which may explain the antifungal action of phenothiazines [150,151]. Chlorpromazine and trifluoperazine are representative phenothiazines. 235 Both block the central dopamine D2 receptor to improve symptoms in mentally ill patients. They 236 have excellent activity against Candida spp., C. neoformans either alone or in combination with 237 ketoconazole and amphotericin B [150][151][152][153][154]. Trifluoperazine also has fungicidal effects on C. 238 neoformans, especially on melanized cells [155]. In addition, chlorpromazine and trifluoperazine 239 have fungicidal effects on Zygomycetes when concentrations reach 25-200 μg/mL. Moreover, it has 240 synergistic effects with amphotericin B [156]. The minimum fungicidal concentration of 241 chlorpromazine and trifluoperazine in Aspergillus spp., Scedosporium, and Pseudallescheria ranged 242 between 10 and 64 μg/mL [157,158]. Flunarizine is a difluorinated derivative of piperazine as well 243 as a potent CCB. It has the same structure as phenothiazines. Flunarizine also exhibits broad-244 spectrum antifungal effects against Candida spp., Cryptococcus spp. and Zygosaccharomyces spp. 245 alone or jointly with ketoconazole in vitro probably by inhibiting calmodulin activity and increasing 246 the penetration of ketoconazole through cell walls [159]. 247 Compared to the traditional antifungal drugs, the main advantage of phenothiazines is that they 248 can cross the blood-brain barrier and improve bioavailability. Moreover, the levels achievable in the 249 brain with antipsychotic therapeutic doses range from 50 to 100 μg/mL; however, the range in 250 plasma is only between 0.5 and 1 μg/mL [158]. 251

Antidepressant drugs 252
There are currently many types of antidepressant drugs used in the clinic. These mainly include 253 monoamine oxidase inhibitors (e.g., phenelzine), tricyclic (e.g., amitriptyline and doxepin), 254 tetracyclic (e.g., maprotiline), and selective serotonin reuptake inhibitors (SSRIs) (e.g., fluoxetine 255 and paroxetine). Of these, SSRIs are first-line antidepressant drugs with low side-effects. They not 256 only have anti-depression activity, but also have anti-anxiety activity [160]. SSRIs effectively inhibit 257 the uptake of serotonin by neurons from synaptic spaces, which increase the availability of this 258 neurotransmitter in these spaces and improves the emotional states, and treats depressive mental 259 disorders [161]. Encouragingly, SSRIs have been shown to have positive antifungal activity in 260 various studies. An antifungal experiment showed that fluoxetine could kill some azole-resistant 261 Candida spp. strains in vitro with or without fluconazole. Moreover, they also improved the survival 262 rate of Galleria mellonella in vivo. The antifungal mechanism involves inhibition of extracellular 263 phospholipase activity by down-regulated SAP1-4 genes in resistant C. albicans [162,163]. The SAP 264 genes encode secreted aspartyl proteinases (SAP), which are key virulence factors and play an 265 important role in the growth, development, and pathogenicity of Candida spp. [164]. In addition, 266 fluoxetine exhibited synergistic effects against C. albicans biofilms and relieved oral candidiasis in 267 infected mice when combined with caspofungin [165]. Sertraline, another type of SSRI, also showed 268 positive antifungal activity alone or in combination with antifungal agents. It can reduce the fungal 269 burden, improve survival rate and impair tissue damage in mice and G. mellonella [166,167]. Similar results showed that sertraline had fungistatic or fungicidal effects in Candida spp., 271 Coccidioides immitis, C. neoformans, Trichosporon asahii, and A. fumigatus [166][167][168][169][170][171]. 272

Non-steroidal anti-inflammatory drugs (NSAIDs)
NSAIDs, act mainly by inhibiting the activity of cyclooxygenase to reduce the production of 274 prostaglandins (PGs), thus have antipyretic, analgesic, anti-inflammatory, and other functions [172]. 275 NSAIDs, especially aspirin, etodolac, diclofenac, celecoxib, nimesulide, ibuprofen, meloxicam, 276 ketoprofen, tenoxicam, and ketorolac exhibited favorable anti-C. albicans effects by inhibiting the 277 synthesis of fungal PGs, which play an important role in biofilm development, adhesion, and 278 morphogenesis in C. albicans [173,174]. In addition, most of these drugs had synergistic or additive 279 (ketorolac) activity with fluconazole against C. albicans [174]. Subsequently, the antifungal 280 mechanisms of aspirin and ibuprofen have become clearer. They can, by activating the high-281 osmolarity glycerol pathway, induce the accumulation of reactive oxygen species (ROS), and then 282 simultaneously damage the integrity of cell membranes leading to the death of Cryptococcus cells 283 [175]. In addition, aspirin and ibuprofen combined with fluconazole, caspofungin, and amphotericin 284 B have effects on fungi [175,176]. Ibuprofen also has anti-Sporothrix activity singly (median MIC 285 of 256 μg/mL) or in combination with antifungal agents including amphotericin B, itraconazole, and 286 terbinafine [177]. Diclofenac sodium can down-regulate the expression of Ef-1 gene, which is 287 involved in cellular RNA transport, cell cycle, and apoptosis [178], thus resulting in a reduction in 288 the formation of A. fumigatus filaments [179]. 289

Conclusion 290
In this review, we summarized the antifungal effects of a number of non-antifungal agents. In 291 addition, some antitumor agents such as miltefosine [180], tamoxifen [181], methotrexate, [182] 292 and antiepileptic drugs [183] have also been reported to have antifungal effects. The use of drug 293 repurposing strategies in the discovery of novel antifungal agents is a revelation in the identification 294 of new antifungal drugs through structural readjustment. With regard to their related antifungal 295 targets, there are still many antifungal mechanisms of the above-mentioned drugs which are unclear 296 (Fig. 1). To reveal the precise targets, further investigations should be performed using transcriptome 297 analysis and molecular techniques, which will lay the foundation for the development of novel 298 antifungal drugs for example using target design. In addition, these antifungal experiments have 299 only focused on either in vitro studies or animal model experiments. There is not enough clinical 300 evidence to prove their practical use in the clinic. Moreover, many factors, such as changes in 301 medium composition will perhaps lead to different or completely opposite results. In vivo studies, 302 differences between animal models or homogeneous animal models and differences in 303 pharmacokinetic and pharmacodynamic parameters of compounds in these models, and the effects 304 of host-derived serum and, cellular factors should be clarified. Hence, it is essential to use systematic 305 and standard research approaches, as well as collect more clinical data to evaluate the antifungal 306 effectiveness of these agents.  were mentioned in the corresponding references; +, the drug has antifungal effect, but no specific data in the corresponding references.