Emerging New Targets for the Treatment of Resistant Fungal Infections

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Perspective

Emerging New Targets for the Treatment of Resistant Fungal Infections Na Liu, Jie Tu, Guoqiang Dong, Yan Wang, and Chunquan Sheng J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 2, 2018

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Journal of Medicinal Chemistry

Emerging New Targets for the Treatment of Resistant Fungal Infections

Na Liu†, Jie Tu†, Guoqiang Dong, Yan Wang, Chunquan Sheng* School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China

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Abstract

With the increasing morbidity and mortality of invasive fungal infections and the emergence of severe antifungal drug resistance, new drug targets and novel antifungal agents are urgently needed. Recently, better understanding of fungal pathogenesis has contributed to the rapid emergence of potential antifungal drug targets. This perspective aims to provide a comprehensive review of new antifungal targets and of medicinal chemistry efforts toward inhibitor discovery. Particular focus will be placed on the druggability of the targets and their potential to treat resistant fungal infections. Innovative strategies for the next generation of antifungal therapy, such as virulence factors, protein-protein interactions, and immune response-based proteins, will also be highlighted.

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1. Introduction Invasive fungal infections (IFIs) represent a growing threat with high morbidity and mortality.1, 2 IFIs are estimated to be responsible for approximately 1.5 to 2 million deaths every year.1, 2 Over the past few decades, the incidence of IFIs has significantly increased with the rising number of immunocompromised patients, mainly due to the widespread application of central venous catheters, broad use of antibiotics, increased frequency of bone marrow and organ transplants, increased use of antitumor agents, and prolonged use of corticosteroids.3 Patients with AIDS (acquired immune deficiency syndrome), transplant recipients, and patients undergoing cancer chemotherapy or cared for in intensive care units (ICU) are highly vulnerable populations for IFIs. Despite the large number of immunocompromised hosts at great risk of IFIs, mortality rates continue to be high, i.e., generally in the range from 20% to 40%, depending on the clinical treatment and the infecting fungal species. More than 90% of the life-threatening IFIs are caused by the Candida, Cryptococcus, and Aspergillus species. The Candida species, particularly Candida albicans (C. albicans), are the most common cause of IFIs and are ranked as the fourth most common cause of nosocomial bloodstream infections in modern hospitals with approximately 40% mortality.4, 5 Cryptococcus neoformans (C. neoformans) is an opportunistic pathogen that causes meningitis globally, particularly in HIV-infected patients.6, 7 C. neoformans kills approximately 600,000 people per year with up to a 60% mortality rate in developing countries.8 Aspergillus fumigatus (A. fumigatus) is the major cause of invasive aspergillosis in immunocompromised patients, which has 3



crude mortality rates ranging from 30% to 95%.9 Even when such infections are treated with antifungal agents, the mortality remains at approximately 50%.1 Despite the high incidence and mortality of IFIs, treatment options are rather limited. Only three classes of antifungals (polyenes, azoles, and echinocandins) are clinically available to treat IFIs. In addition to them, 5-fluorocytosine is typically used as adjunctive therapy. Substantial clinical failures were observed with these antifungal agents because of their limited clinical efficacy, narrow antifungal spectrum, unfavorable pharmacokinetic profiles, significant side effects and drug-drug interactions. More importantly, increasing drug resistance has been observed, which has further reduced the clinical success. Thus, the limited arsenal of effective antifungal agents highlights the urgent need to develop a new generation of therapeutic agents. Unlike bacteria, eukaryotic fungal cells are similar to mammalian cells. Thus, identifying fungal-specific targets and developing selective drugs are highly challenging. There are two major strategies for novel antifungal drug discovery and development: (1) optimization and improvement of existing drugs and (2) identification of new antifungal targets and design of effective inhibitors. Currently, most antifungal candidates in clinical development are structural analogues of known drugs.10 However, these follow-on drugs acting on the same targets can hardly solve the resistance problem. In contrast, the identification and validation of new antifungal targets represents a good opportunity to tackle resistant fungal infections. In recent years, better understanding of the biology of fungal pathogens and fungal-host 4


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interactions has led to important progress in the identification of new antifungal targets and the development of new inhibitors. This review will provide a comprehensive review of emerging new antifungal targets and the medicinal chemistry efforts to discover and optimize inhibitors of these targets. In particular, their potential to treat resistant fungal infections will be highlighted. 2. Traditional Antifungal Targets, Mechanism of Resistance and New Inhibitors 2.1 Ergosterol in the Fungal Cell Membrane Amphotericin B (AmB, 1), a polyene antibiotic, is still used as the “gold standard” for the treatment of severe IFIs. AmB binds to ergosterol in fungal plasmatic membranes and forms barrel-stave channels,11 leading to cell death by altered permeability and leakage of vital cytoplasmic components.12 However, AmB has serious nephrotoxicity and many other side effects, which have limited its broader application. Interestingly, AmB can evade drug resistance because of its unique mode of action.13 AmB is unaffected by most resistance mechanisms, such as target mutations and efflux pumps. Consequently, resistance to AmB rarely appears in the clinic. Improvements in the therapeutic index of AmB are focused on the development of new formulations14 and design of less toxic AmB derivatives.15 The lipid formulations of AmB that have been marketed, such as noncovalent lipid complexes (ABLC), liposomal forms (AmBisome)16 and colloidal dispersions (Amphocil), have shown reduced renal toxicity.17 Additionally, less toxic and more effective AmB derivatives have been discovered (Figure 1).18 Davis et al. designed a series of AmB urea derivatives (2, 3) that can selectively bind to fungal ergosterol over human cholesterol.18 This binding 5



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selectivity led to improved antifungal potency and reduced toxicity. The MHC (minimum hemolytic concentration) value of both compounds was greater than 500 µM against human red blood cells, whereas AmB is significantly more toxic (MHC = 8.6 µM). Compared to AmB, both compounds 2 and 3 were substantially more effective in a mouse model of disseminated candidiasis (reducing the fungal burden by 1.2 log units and 3 log units, respectively) and were proven to be nontoxic and resistance evasive.18 OH

OH

O HO

O

OH OH

OH

OH OH O

R H

O

H O

HO H2N

OH

O R=

COOH

N H

O N H

CH3

2

1 (amphotericin B)

N H

N H

NH2

3

Figure 1. Chemical structures of AmB and its urea derivatives. 2.2 Lanosterol 14α-Demethylase Lanosterol 14α-demethylase (Erg11 or CYP51) is the primary target of triazole antifungal agents (Figure 2, 4-8). The broad use of triazole antifungal agents in the prophylaxis and treatment of IFIs and the prolonged therapeutic courses has led to severe drug resistance.19 The molecular mechanism involved in triazole resistance mainly includes the overexpression of the ERG11 gene, alterations in CYP51 protein, activation of membrane-associated efflux pumps and development of fungal 6


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biofilms.20 Among these pathways, mutations in CYP51 predominantly contributed to triazole resistance.21 Fungal CYP51s are membrane-bound proteins, and determining their structures is challenging. Recently, crystal structures of Saccharomyces cerevisiae CYP51 complexed with various triazole antifungal agents have been solved, offering insight into the resistance mechanisms (Figure 3).22-25 For example, the Y140F/H mutation (tyrosine 140 to phenylalanine or histidine) could disrupt a water-mediated hydrogen bonding network between fluconazole and CYP51 and thus reduce susceptibility to fluconazole.23, 24 Very recently, the crystal structure of CYP51 from C. albicans was reported, and the functions of several resistant mutations, such as Y132H, Y132F, K143R, G307S, and S405F, were further clarified.26 Notably, the structures of CYP51s from pathogenic fungi are still rare. Our previous work indicated that molecular modeling studies of fungal CYP51s provided a complementary tool to elucidate the resistance mechanism.27-30 Advancements in structural biology studies of CYP51s will offer better understanding of the resistance mechanism and guide the design of new inhibitors to reduce the risk of resistance.

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Figure 2. Chemical structures of triazole antifungal agents.

Figure 3. Binding mode of fluconazole (A, PDB code: 4WMZ) and itraconazole (B, PDB code: 4ZDY) with Saccharomyces cerevisiae CYP51.

The development of new CYP51 inhibitors is still an active area in current 8


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antifungal drug research. Isavuconazole (6) was approved in 2015 for treatment of invasive aspergillosis and mucormycosis.31 Albaconazole (9)32 and VT-1161 (10),33 (Figure 4) which are under clinical evaluations, showed potent activities against clinically resistant fungal strains.34,

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Another strategy to treat triazole-related

resistance is the design of novel non-azole CYP51 inhibitors.36 Our group discovered a series of new CYP51 inhibitors by structure-based drug design (SBDD).37-39 Among them, compound 11 (Figure 4) showed excellent inhibitory activity against four fluconazole-resistant C. albicans strains (MIC range: 0.0625 µg/mL ~ 0.125 µg/mL).37

Figure 4. Chemical structures of novel azole and non-azole CYP51 inhibitors with potent activity against resistant fungal strains.

2.3 β -(1,3)-D-Glucan Synthase Echinocandins such as caspofungin (12), micafungin (13) and anidulafungin (14) act by specific and non-competitive inhibition of β-(1,3)-D-glucan synthase (GS), which is an important enzyme in the synthesis of the essential component of the fungal cell wall.40 GS inhibitors have the advantage of selective toxicity because the cell wall is totally absent in human cells. However, resistance to echinocandins has also developed, particularly in Candida spp.41 The resistance mechanisms of 9



echinocandins mainly involve amino acid substitutions and naturally occurring polymorphisms in hot-spot regions of FKS subunits of GS, cellular stress responses and increased chitin synthesis.42

Figure 5. Chemical structures of echinocandins and small-molecule GS inhibitors.

To overcome the limitations of echinocandins, orally active small-molecule GS 10


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inhibitors have been examined. Because knowledge of GS structures and inhibitor-binding mechanism has been rather limited to date, new GS inhibitors have been mainly discovered through high-throughput screening (HTS) of compound libraries from natural products or synthetic molecules.43 Onishi et al. discovered the first non-lipopeptide GS inhibitor: the acidic terpenoid enfumafungin (15).44 Further optimization of enfumafungin led to the discovery of SCY-078 (MK-3118, 16)45 as a phase 2 clinical candidate for intravenous and oral treatment of IFIs.46-48 Compound 16 showed excellent broad-spectrum activity against various clinical isolates of Candida spp.49 and Aspergillus spp.,50 including those with resistance to azoles or echinocandins.45, 46 Additionally, pyridazinone (17) and piperazine propanol (18) GS inhibitors have been reported.51-55 However, further preclinical or clinical development of these compounds has not been reported. 3. New Targets in the Fungal Cell Wall The fungal cell wall is a unique structure without a counterpart in mammalian cells; thus, it provides ideal drug targets for the development of selective antifungal agents.56 The structure of the fungal cell wall is complex, mainly containing glucans, chitins and mannoproteins.57 The proportion of fungal cell wall components varies among different fungal species. The great success of GS inhibitors has inspired the discovery and validation of new targets involved in the biosynthesis of the fungal cell wall. 3.1 β -1,6-Glucan Synthase Similar to β-(1,3)-D-glucan, β-1,6-glucan is another important component of the 11



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fungal cell wall.58 Pyridobenzimidazole derivative D75-4590 (19) was identified as the first inhibitor of β-1,6-glucan synthesis by targeting Kre6p protein.59 Compound 19 was effective against various Candida spp., including fluconazole-resistant strains (MIC range: 1 µg/mL ~16 µg/mL). However, it was inactive against C. neoformans and Aspergillus species. Subsequent studies of compound 19 were focused on improving the antifungal activity and physicochemical properties (Figure 6). After optimization of C1-C4 substitutions and the pyridobenzimidazole scaffold

60, 61

, the

resulting compound D21-6076 (20) showed excellent activity against C. glabrata (MIC = 0.125 µg/mL) and suitable physicochemical properties.60, 62 Moreover, it was orally active and could effectively prolong the survival of mice infected by C. glabrata at a dose of 3.3 or 10 mg/kg.63 However, further development of compound 20 has not been reported, possibly because of its narrow spectrum and limited efficacy against C. albicans.

Figure 6. Chemical structures and antifungal activity of β-1,6-glucan inhibitors.

3.2 Chitin Synthase Chitin, the β-(1→4)-linked polymer of N-acetylglucosamine, is an essential 12


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component in the fungal cell wall. Chitin synthase is responsible for the biosynthesis of chitin, and its inhibitors can induce fungal cell death. Polyoxin D (21) and nikkomycin Z (22) are representative fungal chitin synthase inhibitors (Figure 7).64 Generally, nikkomycins have better antifungal activity and a broader antifungal spectrum than polyoxins.65 Compound 22 has been evaluated in clinical trials, but its further development was terminated because of limited antifungal potency. Additionally, structural optimization of compound 22 did not yield more promising compounds. The detailed structure-activity relationship (SAR) analysis is described in our review.66

Figure 7. Chemical structures of chitin synthase inhibitors.

3.3 Glycosylphosphatidylinositol-anchored proteins Glycosylphosphatidylinositols (GPIs) are a family of complex glycolipids that covalently attach to the protein C-terminus as a unique post-translational modification in eukaryotic cells. GPIs share a common backbone consisting of ethanolamine phosphate (EtNP), three mannoses (Mans), one non-N-acetylated glucosamine (GlcN), and inositol phospholipid (Ins).67 GPIs function as cell surface anchors for a number of cell wall proteins in yeast via an amide bond between the free amino group of EtNP and C-terminal carboxyl group of the anchored protein (Figure 8A).68 GPIs and 13



GPI-anchored proteins (GPI-APs) play a vital role in diverse biological processes, including adherence, virulence, and cell wall biogenesis.69 In fungi, GPI-APs belong to the family of cell wall mannoproteins and are essential for the adhesion to human epithelium and maintenance of cell wall homeostasis.70 Moreover, GPI biosynthesis is necessary for fungal growth. Thus, key enzymes involved in specific steps of GPI precursor biosynthesis can serve as promising antifungal targets. The GPI biosynthetic pathway in fungi is different from that in mammalian cells, providing a basis to discover selective inhibitors. In particular, two GPI biosynthetic steps, Gwt1 (GPI-anchored wall protein transfer 1)-dependent acylation of inositol and Mcd4 (morphogenesis checkpoint dependent)-mediated EtNP addition to Man1 of the GPI core, have been used to discover novel antifungal agents (Figure 8B).

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Figure 8. Targets in and inhibitors of GPI biosynthesis. (A) General structure of GPI-APs and possible modifications. EtNP is linked to the C-terminus of the protein, and Ins, which carries the lipophilic residue, interacts with lipid bilayer membranes. (B) Predicted process and inhibitors of GPI precursor biosynthesis. The mammalian ortholog of Gwt1 and Mcd4 is PIG-W (phosphatidylinositol glycan anchor biosynthesis class W) and PIG-N (phosphatidylinositol glycan anchor biosynthesis 15



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class N), respectively. Gwt1 and Mcd4 inhibitors have been identified.

Gwt1, a kind of acyltransferase, is involved in inositol acylation at an early step in the GPI biosynthetic pathway (Figure 8B). To date, two classes of Gwt1 inhibitors (phenoxyacetanilides and aminopyridines) have been identified.71, 72 Gepinacin (23) was identified by HTS and was shown to inhibit the growth of C. albicans, C. glabrata and A. terreus. 72 In addition, the compound was shown to inhibit the growth of azole-resistant strains of C. albicans72 and to have little toxicity to mammalian cells. However, the in vivo antifungal activity of compound 23 has not been reported, and more medicinal chemistry optimization studies and biological evaluations are still required to characterize its therapeutic potential. In contrast, the SAR and antifungal activity of aminopyridine Gwt1 inhibitors 24-26 have been well characterized. Initial hit 24 was identified via a yeast cell-based screening.71 Compound 24 was moderately active against C. albicans (MIC = 1.56 µg/mL) and had no activity against A. fumigatus.73 After scaffold hopping and side chain optimization (Figure 9, 25-27)73, E1210 (27) was identified to possess excellent antifungal activity with a very broad spectrum.

74-76

In addition to Candida spp., compound 27 was also highly active

against Aspergillus, Cladosporium, Phialophora, Fusarium, and Scedosporium species, among others. Importantly, compound 27 also showed good efficacy in the treatment of azole-resistant and echinocandin-resistant C. albicans strains.77,

78

Compound 27 was orally active, and its in vivo antifungal potency was proven in various models, including disseminated candidiasis, oropharyngeal candidiasis, 16


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pulmonary aspergillosis, and disseminated fusariosis.77 In particular, compound 27 showed good protective effects in an infection model of azole-resistant C. albicans.77, 78

At a dose of 5 mg/kg, compound 27 achieved a survival rate of 100% for the mice

at day 14 (ED50 = 1.3 mg/kg), whereas the survival rate of the group treated with voriconazole at the same dose was 25%.77, 78 The results highlighted the potential of compound 27 to treat resistant IFIs. Compound 27 also had suitable pharmacokinetic and toxicological profiles.77 The elimination half-life and oral bioavailability were 2.2 h and 57.5% in mice, respectively. It was well tolerated in rats when given orally for 1 week at doses up to 300 mg/kg. Compound 24 is currently under evaluation as a preclinical candidate.79

Figure 9. Structural optimization of compound 27.

Mcd4 is an ethanolamine phosphotransferase in the GPI biosynthetic pathway. 17



Recently, the potential of Mcd4 as a new antifungal target was validated using a chemical genomics-based strategy.80 Two terpenoid lactone ring-based natural products, M743 (28) and M720 (29), were identified as Mcd4 inhibitors (Figure 8B).80 Both of them displayed potent activity against a variety of fungal pathogens, including C. albicans, C. parapsilosis, C. glabrata, C. krusei, C. lusitaniae and A. fumigatus, with MIC values ranging from 0.25 µg/mL to 1 µg/mL. Compound 29 was more stable in mouse plasma than compound 28, and its in vivo antifungal potency was validated in a C. albicans infection model.80 After administration of compound 29 by intraperitoneal (IP) injection, fungal burden was significantly reduced in a dose-dependent manner (12.5 mg/kg to 50 mg/kg). Moreover, the toxicity of compound 29 was low (LD50 > 300 mg/kg) when administered IP.80 Interestingly, strong synergistic effects were observed by combining Gwt1 and Mcd4 inhibitors with FICI (fractional inhibitory concentration index) values in the range of 0.25−0.313.80 The results revealed the advantages of simultaneously targeting different enzymes involved in GPI biosynthesis. Despite the proof-of-concept study, several problems of Mcd4 inhibitors must be addressed. First, only one class of Mcd4 inhibitor has been reported. Identifying new structurally distinct inhibitors is thus highly desirable. Second, medicinal chemistry optimization of compound 29 is important to understand the SAR and discover more potent derivatives. Particularly, the selectivity of Mcd4 inhibitors for fungal cells over human cells needs to be further improved because compounds 28 and 29 showed only relatively narrow therapeutic indices (∼10-fold). Third, the antifungal efficacy of Mcd4 inhibitors in treating 18


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resistant infections remains to be evaluated. 3.4 Biosynthesis of UDP–GlcNAc UDP (uridine diphosphate)–GlcNAc (N-acetylglucosamine) functions as the sugar donor in the biosynthesis of cell wall chitin, mannoproteins and GPI-APs.81,

82

Starting from fructose 6-phosphate (Fru-6P), UDP–GlcNAc is synthesized by four successive reactions (Figure 10A).83 Among the enzymes catalyzing these reactions, AGM1

(N-acetylglucosamine-phosphate

mutase)

and

UAP

(UDP-N-acetylglucosamine pyrophosphorylase) have been investigated as potential antifungal

targets

in

A.

fumigatus.84,

85

AGM1

is

a

member

of

the

α-D-phosphohexomutase superfamily that catalyzes the intramolecular phosphoryl transfer on GlcNAc-6P (N-acetylglucosamine-6-phosphate) to form GlcNAc-1P (N-acetylglucosamine-1-phosphate). AGM1 was proven to be essential for cell viability and cell wall synthesis in A. fumigatus. Inhibitors of A. fumigatus AGM1 were identified by HTS (Figure 10A, 30-32).84 However, none of them showed antifungal activity, possibly due to their poor solubility, limited cell penetration or low efficacy.84 UAP1 catalyzes the final enzyme in eukaryotic UDP–GlcNAc biosynthesis, converting UTP and (GlcNAc-1P) to UDP–GlcNAc. UAP1 is essential for survival; cell wall ultrastructure, integrity and synthesis; and morphogenesis in A. fumigatus.85 However, no inhibitors of UAP1 have been reported to date. The crystal structures of C. albicans and A. fumigatus AGM1 (Figure 10B)84,

86

and A. fumigatus UAP1

(Figure 10C)85 have been solved, providing the basis for SBDD of novel inhibitors. Notably, both enzymes also exist in eukaryotes, and identifying selective inhibitors is 19



of key importance. Fungal and human AGM1 possess differences near the substrate-binding site, which offers an opportunity to discover selective inhibitors of fungal AGM1 over the human ortholog.

Figure 10. Potential targets in the biosynthesis of UDP–GlcNAc. (A) Biosynthetic pathway of UDP–GlcNAc and chemical structures of AGM1 inhibitors; (B) Crystal structure of A. fumigatus AGM1 (PDB code: 2DKC); (C) Crystal structure of A. fumigatus UAP1 (PDB code: 1JV1).

4. New Targets in the Fungal Cell Membrane The fungal cell membrane mainly comprises sterols, glycerophospholipids and sphingolipids, which are important for cellular functions, pathogenesis and signal transduction pathways.87 Targeting ergosterol biosynthesis in the fungal cell 20


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membrane is a successful approach to develop effective antifungal agents.87 Triazoles have been widely used as the first-line antifungal therapy. Moreover, topical allylamine antifungal agents (terbinafine and naftifine) act by inhibiting squalene epoxidase, another important enzyme in ergosterol biosynthesis. Inspired by these successes, researchers have made numerous efforts toward identifying new antifungal targets from proteins involved in sterol, glycerophospholipid and sphingolipid biosynthesis (Figure 11).87 However, successful examples are rather limited. Most reported inhibitors targeting these biosynthetic enzymes lack antifungal potency and selectivity.87,

88

Notably, these potential targets have counterparts in human cells,

making it highly challenging to achieve selective inhibition. Thus, more experimental evidence is required to evaluate the druggability of these proteins.

Figure 11. Potential targets in the fungal cell membrane. 21



Upc2, a member of the conserved transcription factors in fungi, contributes to regulating the ergosterol biosynthetic pathway.89 Upc2 can induce biosynthetic gene expression of ergosterol in response to triazole antifungal drug treatment and thus is associated with drug resistance.90 As a critical regulator of the antifungal drug response, Upc2 transcription factor can serve as an excellent target for antifungal drug discovery. In particular, inhibition of Upc2 would eliminate the most downstream event required for resistance, thereby showing the potential of this approach to treat resistant infections. However, developing small molecules targeting transcription factors is highly challenging. Gallo-Ebert et al. developed HTS assays to screen small-molecule Upc2 inhibitors.91 Three compounds (Figure 12, 33-35) reduced Upc2 activity without inhibiting DNA binding. These compounds inhibited the growth of S. cerevisiae and C. glabrata strains at low micromolar concentrations. However, they were inactive against C. albicans cell growth. Mechanistic studies indicated that compounds 33-35 restricted Upc2 DNA binding by attenuating the signaling pathway(s) for regulation of Upc2 function rather than by directly interacting with Upc2.

Figure 12. Chemical structures of Upc2 transcription factor inhibitors.

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5. New Targets in the Biosynthesis of Fungal Proteins, DNA and Other Essential Substances 5.1 N-Myristoyltransferase Myristoyl-CoA: protein N-myristoyltransferase (NMT) catalyzes the transfer of the myristoyl group (a C14 fatty acid) from myristoyl-CoA to the N-terminal glycine of a number of proteins. N-myristoylation is important for the viability and survival of fungi, parasites, humans, etc.92 NMT is extensively investigated as a therapeutic target in cancer and parasite and fungal infections.93, 94 The availability of crystal structures of C. albicans NMT (CaNMT) facilitates the structure-based design of selective inhibitors (Figure 13). Benzofuran derivative RO-09-4879 (36, IC50 = 5.7 nM)95 and benzothiazole derivative FTR1335 (37, IC50 = 0.49 nM)96 are highly active CaNMT inhibitors with good selectivity over human NMT. Both of them were fungicidal and showed excellent antifungal activity against Candida spp.95, 96 In particular, they were effective against fluconazole-resistant clinical isolates.95, 96 Compound 36 showed in vivo antifungal activity in a rat model of systemic candidiasis (EC50 = 7.1 mg/kg), but it was less effective than fluconazole.95 In vivo data of compound 37 have not been reported. Our group performed structural optimization studies of NMT inhibitors, and several derivatives showed a broader antifungal spectrum and improved in vivo efficacy.97-99 More recently, NMT is proven to be a cell wall target in A. fumigatus.100 However, a highly potent A. fumigatus NMT inhibitor, DDD86481 (38, IC50 = 12 nM), showed poor fungicidal activity against A. fumigatus (MIC = 925 µM).100 Several limitations remain to be overcome before NMT inhibitors can be developed as novel

23



antifungal agents. First, more knowledge of the functions of NMT in pathogenic fungi is required. Second, known inhibitors should be optimized with a focus on pharmacokinetic (PK) profiles. Third, structurally diverse NMT inhibitors still need to be discovered.

Figure 13. Chemical structures (A), antifungal activity and binding mode (B) of NMT inhibitors (PDB Code: 1IYL).

5.2 Aminoacyl-tRNA Synthetases Aminoacyl-transfer RNA synthetases (AaRSs) catalyze the attachment of the correct amino acid to its corresponding tRNA during the translation stage.101 AaRSs are essential enzymes for protein biosynthesis and have been widely investigated as antimicrobial drug targets.102 In the field of antifungal drug discovery, leucyl-tRNA 24


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synthetase (LeuRS) and isoleucyl-tRNA synthetase (IleRS) have been validated as promising antifungal targets. Tavaborole (39, AN2690, Figure 14A), a specific LeuRS inhibitor (S. cerevisiae LeuRS, IC50 = 2.1 µM), was marketed in 2014 for the topical treatment of onychomycosis.103 Compound 39 contains an unusual benzoxaborole scaffold, and the X-ray crystal structure of the LeuRS-inhibitor complex indicated that the boron atom in the oxaborole ring could covalently bind to the 3’-terminal adenosine (Figure 14B).104 However, the half-life of compound 39 was too short for it to be developed as a systemic antifungal agent, and it is therefore currently used to treat tropical fungal infections. Icofungipen (40), an IleRS inhibitor, is a synthetic derivative of the naturally occurring β-amino acid cispentacin (41).105, 106 The SAR of compound 40 is very steep, and minor modifications led to a significant decrease in or loss of the antifungal activity.107 Compound 40 has weak inhibitory activity against IleRS (Ki = 1 mM), but protein biosynthesis in yeasts is very sensitive to the inhibition of IleRS activity.105 Thus, compound 40 showed excellent in vitro and in vivo antifungal activity against both fluconazole-susceptible and fluconazole-resistant C. albicans infections.106, 108 Moreover, compound 40 had favorable PK profiles (T1/2 = 7 h in man and 100% oral bioavailability in rats, dogs and rabbits).109 Icofungipen has been evaluated in phase II clinical trials for the treatment of Candida infections. However, no progress has been reported to date. Although fungal AaRSs have been validated as promising antifungal targets, the discovery of new inhibitors has been dormant in recent years. Progress in the structural biology of fungal AaRSs110 will promote the structure-based design of 25



Page 26 of 100

new inhibitors.

Figure 14. AaRSs as potential antifungal targets. (A) Chemical structures of AaRS inhibitors; (B) Binding mode of a LeuRS inhibitor.

5.3 Fungal Amino Acids Biosynthesis Fungal microorganisms can synthesize all proteinogenic amino acids. Several enzymes catalyzing particular steps of amino acid biosynthesis are absent from mammalian cells; thus, these fungal-specific enzymes are potential antifungal targets.111 Unique enzymes involved in the fungal biosynthetic pathways of human-essential

amino

acids

and

their

inhibitors

have

been

extensively

investigated.111 Although a number of inhibitors were identified, only a small portion of them showed potent in vitro antifungal activities. Several potential validated targets 26


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and their inhibitors and antifungal activities are summarized in Table 1. In contrast, successful examples of enzymes and inhibitors of human-non-essential amino acids biosynthesis in fungi are rather limited. Poor correlation between the enzyme inhibition and antifungal activity and lack of antifungal potency (particularly in vivo potency) are the bottlenecks in the development of antifungal agents targeting amino acid biosynthetic enzymes. Most inhibitors are analogues of the amino acid substrates, which are too hydrophilic to cross the fungal cell membrane. In addition, inhibition of the biosynthesis of a particular amino acid can be compensated by exogenous supply or other by-pass mechanisms, which will abolish the antifungal effect. Nevertheless, further validation of these fungal-specific target candidates is worthwhile.

Table 1. Potential targets in and inhibitors of the biosynthetic pathway of fungal amino acids. Targets

Structures and activity of inhibitors

Homoserine

Refs

112

dehydrogenase (Hom6p)

C. kefyr MIC = 6.25 – 12.5 µg/mL H3C

OH

N

Homoserine

N

kinase

N

CF3

(Thr1p) 43

C. neoformans MIC80 = 32 – 64 µg/mL

27


113


Threonine

synthase

Page 28 of 100

114, 115

(Thr4p) C. albicans MIC = 0.35 µg/mL

Homoserine transacetylase

113

(Met2p)

C. albicans MIC = 64 µg/mL

Sulfite reductase (Met4,

116-118

Met10) A. fumigatus IC80 = 0.72 µg/mL

Homoisocitrate

119

dehydrogenase (Lys12p)

C. tropicalis MIC = 1 mg/mL

120

Homoaconitase (Lys4P)

C. albicans MIC > 512 µg/mL

28


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Acetohydroxyacid

121

synthase (Ilv2p)

C. albicans MIC90 = 0.72 µg/mL−1

Ketol-acid

122

reductoisomerase (Ilv5p)

Good in vivo fungicidal activity against F. oxysporum at a concentration of 500 µg/mL

NAD-dependent 123

glutamate dehydrogenase (Gdh2P)

Inhibits germ tube formation during the yeast-to-mycelia transition of C. albicans

Acetohydroxyacid synthase (AHAS) or acetolactate synthase (ALS) is a fungal-specific enzyme that catalyzes the first step in the biosynthesis of branched-chain amino acids.111, 124 Due to the absence of a human counterpart, AHAS inhibitors can possess selective antifungal activity. Triazolopyrimidine-sulfonamide (52) and sulfonylurea (53-54) AHAS inhibitors have been identified (Figure 15).125, 126

Compound 52 is an S. cerevisiae AHAS inhibitor that showed broad-spectrum

antifungal activity without significant cytotoxicity.108 Sulfonylurea AHAS inhibitors were derived from herbicides. Compound 53, the initial screening hit (AHAS Ki = 7 29



nM), showed potent activity against C. albicans (MIC50 = 2 µM).126 Further structural optimization identified iodine analogue 54 with improved AHAS inhibitory activity (Ki = 3.8 nM) and antifungal activity (MIC50 = 0.6 µM). However, its inhibitory activity against resistant fungal isolates and its in vivo antifungal potency have not been reported.

Figure 15. Chemical structures and antifungal activity of AHAS inhibitors.

5.4 Dihydrofolate Reductase Targeting DNA synthesis is an effective strategy in antifungal drug development. For example, 5-fluorocytosine is widely used as adjunctive therapy for IFIs. However, the selectivity or toxicity is an important issue that should to be considered in drug design. Dihydrofolate reductase (DHFR) is a traditional antitumor target that plays an important role in thymidine synthesis. Structural biology studies revealed that the differences in the active site of DHFR between human and Candida species could be used to design selective inhibitors.127-129 2,4-Diaminopyrimidine propargyl derivatives were identified as potent Candida DHFR inhibitors with good selectivity over human DHFR (Figure 16).127-129 X-ray crystal structure revealed that the hydrogen bonding 30


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and hydrophobic interactions between the pyrimidine ring and C. albicans DHFR (i.e., Glu32, Ile9, Phe36, Met33, and Ile121) were conserved.130 Adding hydrophobic substituents to the para position of the distal C-ring could enhance the interactions with Phe66 in C. albicans and decrease the interactions with Asn64 in human DHFR. Thus, a series of new para-linked compounds was designed, most of which showed improved potency and selectivity.130 For example, compound 55 was highly active against C. albicans (IC50 = 49 nM) and C. glabrata (IC50 = 27 nM) DHFR with 13and 23-fold greater selectivity over human DHFR (IC50 = 625 nM), respectively. Moreover, it showed good antifungal activity against both C. albicans (MIC = 0.39 µg/mL) and C. glabrata (MIC = 0.2 µg/mL). However, the antifungal activity of DHFR inhibitors against drug resistant isolates and their in vivo potency remain to be further evaluated. Additionally, the problem of the poor correlation between DHFR inhibitory activity and antifungal activity remains to be addressed by more medicinal chemistry efforts. 131

Figure 16. DHFR as a potential antifungal target. (A) Chemical structures and antifungal activity of fungal DHFR inhibitors; (B) the binding mode of a 31



2,4-diaminopyrimidine propargyl inhibitor with C. albicans DHFR (PDB code: 4HOF).

5.5 Purine Metabolism The purine metabolic pathway is responsible for providing the cell with a ready supply of adenosine triphosphate (ATP) and guanosine triphosphate (GTP). Purine metabolism is particularly important in C. neoformans, and the potential of critical enzymes in this biosynthetic pathway as novel antifungal targets has been investigated.132,

133

Inosine monophosphate (IMP) dehydrogenase (IMPDH) is the

rate-limiting enzyme that catalyzes the first committed step of de novo GTP biosynthesis. IMPDH is critical for the growth and virulence of C. neoformans.133 Mycophenolic acid (56), a non-selective inhibitor of C. neoformans IMPDH (Figure 17A), was able to inhibit the growth of the fungi at a concentration of 5 µg/mL. Moreover, it showed potent protective effects in a nematode model of cryptococcosis infection.133 Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the ATP biosynthetic pathway that catalyzes the formation of adenylosuccinate (s-AMP) from IMP and aspartate. Loss of function of AdSS in C. neoformans resulted in adenine auxotrophy and complete loss of virulence in a murine model.132 Moreover, AdSS is not an essential enzyme for mature human erythrocytes. However, potent inhibitors of C. neoformans AdSS have not been developed, which has limited target validation. Fortunately, the crystal structures of C. neoformans IMPDH (Figure 17A) and AdSS (Figure 17B) have been solved, revealing significant structural differences between 32


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the fungal and human enzymes.132, 133 These structures could facilitate the rational design of fungal-specific inhibitors and the investigation of their potential as novel antifungal therapeutics.

Figure 17. Potential antifungal targets in purine metabolism. (A) Crystal structure (PDB code: 4AF0) and an inhibitor of C. neoformans IMPDH; (B) Crystal structure of C. neoformans AdSS (PDB code: 5I34).

5.6 Flavin-dependent Monooxygenase Siderophore A Flavin-dependent monooxygenase siderophore A (SidA) catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores (Figure 18A), which are essential for extracellular iron scavenging in fungi.134 SidA plays an important role in the pathogenesis of A. fumigatus and is considered an attractive antifungal drug target.135 Sobrado’s group performed an HTS study and discovered celastrol (59) (Figure 18B) as a non-competitive inhibitor of SidA (IC50 = 11 µM, KD = 1.4 µM).136 33



Compound 59 is a natural quinone methide that showed inhibitory activity against A. fumigatus (MIC = 2 µM). Importantly, the crystal structure of A. fumigatus SidA has been solved (Figure 18C), which paves the way for the rational design of more specific inhibitors.137

Figure 18. SidA as a potential antifungal target. (A) SidA catalyzes the oxygen- and NADPH-dependent hydroxylation of L-ornithine (57) to N5-L-hydroxy-L-ornithine (58); (B) Chemical structure of sidA inhibitor 59; (C) Crystal structure of A. fumigatus sidA (PDB code: 4B63). 5.7 Trehalose Pathway Trehalose, which is also known as α-D-glucopyranosyl-(1→1)-D-glucopyranoside (61), is a non-reducing disaccharide. Compound 61 can be synthesized in fungi, bacteria, plants and invertebrates but has not been identified in mammalian hosts. In most fungi, compound 61 is synthesized by a two-step enzymatic reaction catalyzed 34


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by rehalose-6-phosphate synthase (Tps1), and trehalose-6-phosphate phosphatase (Tps2) (Figure 19A). During infections, compound 61 is essential for the cell stress response and survival of fungi.138 Moreover, targeting the trehalose pathway poses little risk of developing drug resistance.139 Thus, Tps1 and Tps2 can serve as potential targets in the search for novel antifungal targets with little toxicity to the host. Currently, reports of Tps1 and Tps2 inhibitors are rare. Brennan’s group developed HTS assays for the screening of Tps1 and Tps2 inhibitors and identified an initial hit (closantel, 62).139 Compound 62 (Figure 19B) showed marginal in vitro activity against C. neoformans and C. gattii (MIC < 1 mg/mL), but it was inactive in a murine cryptococcosis model.139 Very recently, the same group solved the crystal structures of fungal Tps1 and Tps2, which will pave the way for structure-based design of novel inhibitors (Figure 19C). 139, 140

35



Figure 19．．Potential antifungal targets in the trehalose pathway. (A) Biosynthetic pathway of trehalose; (B) Chemical structure of Tps inhibitor 62; (C) Crystal structure of the C. albicans Tps2 (PDB code: 5DXF).

5.8 Phosphopantetheinyl Transferases Phosphopantetheinyl transferases (PPTases) (PptB in A. fumigatus) are important enzymes involved in mitochondrial fatty acid synthesis. Recent studies indicated that PPTases in A. fumigatus and C. albicans were essential for their viability.141, 142 Thus, PPTases have been proposed as potential antifungal targets. Currently, no inhibitor of fungal PPTases has been reported. Recently, a fluorescence polarization assay amenable to HTS was developed142, which will accelerate the process of inhibitor discovery and target validation.

6. Virulence Factors Traditional antifungal agents act by inhibiting the growth of or killing the fungal cells, which exerts high selective pressure and results in the emergence of drug resistance. In contrast, targeting virulence represents a promising alternative for the development of novel antifungal agents with the potential to reduce, eliminate or even reverse the selective pressures and thus overcome drug resistance.143 Virulence factors are molecules produced by pathogens that promote disease or damage the host. The encouraging potential of anti-virulence agents mainly includes decreased evolutionary pressure for the development of resistance, low cytotoxicity, rapid target inactivation and synergistic effects with antifungal agents.144 Notably, there are also several limitations of virulence factor inhibitors, such as narrow spectrum, limited efficacy 36


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(often used in combination therapy), and therapeutic delay. Thus, anti-virulence agents should be carefully evaluated regarding whether they can be used alone or in combination with current antifungal agents and whether they can be used as preventative agents or therapeutic agents. Currently, research on fungal virulence is mainly focused on C. albicans, which has promoted better understanding of pathogenic mechanisms and identification of potential novel antifungal targets. For example, C. albicans metabolic pathways, signal transduction pathways, invasion-related processes, and transcription factors have been validated to be essential not only for fungal growth but also for fungal virulence.145 Selected examples of virulence factor targets and inhibitors are summarized in Table 2.

Table 2. Virulence factors as potential antifungal targets Targets

Inhibitors

Antifungal potency

Refs

Glyoxylate cycle

No inhibitory effects on C. albicans growth in glucose; potent inhibitory

ICL

activity

against

C.

albicans grown in acetate (MIC = 12.5 µg/mL).

IC50 = 7.62 µM HOG pathway

37


146


Page 38 of 100

No in vitro antifungal

Hog1

activity; in vivo inhibition

147

of Hog1 kinase activity.

IC50 = 7.4 nM

TOR signaling pathway

Potent in vitro antifungal activity

against

C.

albicans, C. neoformans, A.

TORC1

fumigatus,

M.

148

circinelloides and several pathogenic

Penicillium

species.

Hydrolytic enzymes Potent in vivo antifungal activity

in

experimental

Sap2

model

an of

149

vaginal candidiasis caused by a fluconazole-resistant

74% inhibition at 20 µM

strain.

Potent in vitro antifungal PLB

activity

against

C.

albicans

and

C.

150

neoformans (MIC range: 0.5 ~ 10 µM) active at 2.5 µM HOG, high-osmolarity glycerol; TOR, target of rapamycin; TORC1, TOR complex 1; ICL, isocitrate

38


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lyase; Sap2, secreted aspartyl proteinase 2; PLB, Phospholipase B.

6.1 Filamentation and Biofilm Formation Filamentation and biofilm formation are important reasons for the emergence of severe resistance to antifungal agents.151 Thus, targeting these pathogenic processes with drug-like molecules represents a promising strategy to overcome drug resistance.152 Biofilms are highly structured microbial communities attached to or associated with a surface.152 The formation of biofilms is a kind of survival mechanism of fungi. The cells within biofilms have different features from those of planktonic populations.153 Consequently, fungal biofilms are associated with high-level resistance to most antifungal agents.154 Filamentation consists of the morphology transition among yeast, pseudohyphae, and hyphae.155 The pathogenic processes of fungal infection mainly include the adhesion of fungi to surfaces, morphology transition between yeast and hyphal and tissue invasion.156,

157

Filamentation and biofilm formation have been considered major virulence factors.158 Currently, a number of filamentation and biofilm inhibitors have been identified. The medicinal chemistry of fungal biofilm inhibitors is detailed in our recent review.159 Herein, selected examples that show the potential of treating resistant infections are highlighted (Figure 20). The anti-leishmaniasis agent miltefosine (68), an alkyl phospholipid, showed broad-spectrum antifungal activity against Candida spp., Cryptococcus spp., Aspergillus spp., etc.160 Interestingly, it was proven to inhibit not only the growth of fluconazole-resistant C. albicans isolates (MIC range: 0.5 ~ 2 µg/mL) but also the 39



biofilm formation (90% inhibition at 2 to 4 µg/mL) and mature biofilms (>90% inhibition at 8 to 32 µg/mL) of these isolates.161, 162 Moreover, repeated exposure of C. albicans to compound 68 could not induce drug resistance. Compound 68 also showed potent in vivo antifungal potency in a murine model of oropharyngeal candidiasis (topical treatment, 50 mg/kg).162 However, its efficacy in treating azole-resistant and biofilm-associated systemic candidiasis remains to be investigated further. 4-(Dimethylamino)styrylpyrylium derivative SM21 (69) is a yeast-to-hypha transition inhibitor identified by HTS.163 Compound 69 showed potent antifungal, anti-biofilm and anti-virulence activities. It had excellent antifungal activity against Candida and other pathogenic fungi (MIC range: 0.2 ~ 6.25 µg/mL), including resistant Candida spp. (MIC range: 0.5 ~ 1 µg/mL). Compound 66 could significantly prevent candidal adhesion and biofilm formation on denture acrylic surfaces and inhibit mature Candida biofilms. Similar to compound 68, compound 69 showed good in vivo antifungal potency in an oral candidiasis mouse model.163 Organoselenium compound 70 was reported to be a promising antifungal lead compound due to its triple antifungal, anti-biofilm and normal cell protective effects. 164, 165

It inhibited C. albicans cell growth and biofilm formation at similar

concentrations (1 ~ 2 µM).165 Moreover, compound 70 could significantly inhibit the transition from the budding yeast form to the filamentous form.165 Interestingly, compound 70 could protect animal cells from oxidative damage, highlighting its potential ability to treat biofilm-related resistant fungal infections without affecting 40


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host cell viability.164, 165

Figure 20. Chemical structures of fungal biofilms inhibitors.

Our group identified β-carboline derivative 71 as possessing both antifungal and anti-biofilm effects.166 Its antifungal activity (C. albicans MIC = 2 µg/mL, C. neoformans, MIC = 1 µg/mL) was comparable to that of fluconazole.166 In addition, compound

71

showed

excellent

fungicidal

activities

against

both

fluconazole-sensitive and fluconazole-resistant C. albicans strains and had synergistic effects with fluconazole (FICI = 0.252) for the treatment of resistant C. albicans infections.166 Further studies indicated that compound 71 also inhibited C. albicans biofilm formation and hyphal growth in a dose-dependent manner.166 Compound 71 has a low risk of causing drug-drug interactions due to its weak inhibition of human cytochrome P450 (CYP) enzymes.166 Our group also discovered a series of biofilm inhibitors derived from natural 41



products.167-169 Tetrandrine (72), a bis-benzylisoquinoline alkaloid, was observed to show synergistic antifungal effects with ketoconazole against resistant C. albicans isolates, although its antifungal activity against both fluconazole-susceptible and fluconazole-resistant C. albicans strains was weak (MIC80 = 64 µg/mL).170 Our group further confirmed that compound 72 was a potent biofilm inhibitor that significantly inhibits C. albicans biofilm formation in a dose-dependent manner.168. Compound 72 could also inhibit the C. albicans yeast-to-hypha morphological transition by down-regulation of the Ras1p-cAMP-PKA pathway.168 The in vivo antifungal potency of compound 72 was validated in a C. albicans-infected C. elegans model.168 However, it was inactive in a C. albicans-infected mouse model, and further structural optimization is required. 6.2 Calcium-calcineurin Signaling Pathway Calcium is a secondary messenger in eukaryotic cells and regulates various cellular functions. The fungal calcium-calcineurin signaling network plays an important role in the survival of fungi171, 172 and the emergence of fungal resistance.173, 174 Thus, targeting the key components in the fungal calcium-calcineurin signaling network provides an effective strategy to develop novel antifungal agents and overcome drug resistance (Figures 21 and 22). Recently, several components that are important in the regulation of Ca2+ homeostasis, such as calcium channels, calcineurin and calmodulin, have been investigated as potential antifungal targets.175 6.2.1 Calcium Channels The Cch1-Mid1 protein complex is the main channel for external calcium absorption 42


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and is associated with drug resistance in C. albicans. Calcium channel blockers (CCBs) such as verapamil (73) can inhibit Cch1-Mid1 and decrease the Ca2+ concentration. Compound 73 showed a moderate inhibitory effect on C. albicans hyphal development.176 Moreover, it had synergistic effects with fluconazole on inhibiting biofilm formation and pre-formed biofilms with FICI values of 0.19 and 0.25, respectively.177 Recently, four CCBs, namely, amlodipine (74), nifedipine (75), benidipine (76) and flunarizine (77), were found to significantly increase the sensitivity of resistant C. albicans strains to fluconazole (FICI range: 0.017 ~ 0.035).178 Mechanistic studies indicated that the synergism between compound 74 and fluconazole was caused by down-regulation of the genes CNA1, CNB1 and YVC1. CNA1 and CNB1 encode calcineurin, whereas YVC1 encodes calcium channel protein in the vacuole membrane. Notably, there are no structural homologs of YVC1 in mammalian cells. Thus, YVC1 can serve as a potential target for the development of selective antifungal agents.

43



Figure 21. Potential targets in and representative inhibitors of the calcium-calcineurin signaling pathway.

44


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6.2.2 Calmodulin The calcium-binding protein calmodulin (CaM) is responsible for the transduction of Ca2+ to its effector proteins.179 The Ca2+-calmodulin complex specifically binds to calcineurin (CN), which is activated to regulate the stress response. Most of the reported calmodulin inhibitors were discovered by drug reposition approaches.174 The calmodulin inhibitors fluphenazine (78) and calmidazolium (79) are inactive by themselves but show synergistic effects with azole antifungal agents.174,

180

More

recently, the estrogen receptor antagonists toremifene (80) and tamoxifen (81) were identified as C. neoformans calmodulin inhibitors.180 This calmodulin inhibitory activity was directly related to their triphenylethylene scaffold. Compounds 80 and 81 could inhibit the growth of C. neoformans and synergize with fluconazole, both in vitro and in vivo.180

45



Page 46 of 100

NO2 NO2 O

Cl O

O

O O N H

O

O O

O

O

O

O

N H

N H

NH2

74 (amlodipine)

N

O

75 (nifedipine)

76 (benidipine) N Cl

F Cl

Cl

N N

O

O

N

Cl

Cl

Cl

N Cl Cl

F

77 (flunarizine)

79 (calmidazolium)

O

80 (toremifene)

N HO O N

N

81 (tamoxifen)

N

O O N H

O

O

N

O O

O

H N

N

O O

H N

N O

N

H N

O

O

COOH

82 (cyclosporine A)

85 (plakortide F acid)

Figure 22. Chemical structures of inhibitors of the calcium-calcineurin signaling pathway. 6.2.3 Calcineurin CN is a protein phosphatase that consists of catalytic subunit A (CnA) and regulatory subunit B (CnB). CN is responsible for maintaining calcium homeostasis and contributes to mediating fungal resistance.175 The immunosuppressants cyclosporine (82) and tacrolimus (83, FK506) are potent CN inhibitors.181 In fungal cells, compounds 82 and 83 first bind to their receptors, cyclophilin (CyP) and FK506 46


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binding protein (FKBP), respectively, to form a protein-drug complex, which further interacts with the CnA/CnB interface and inhibits CN activity. Compound 82 (MIC = 0.39 µg/mL) and 83 (MIC = ~ 0.01 µg/mL) showed good antifungal activity against C. neoformans.182, 183 Moreover, they showed synergistic effects with fluconazole both in vitro and in vivo to immunosuppressants

as

treat C.

albicans infection.184-186 However,

antifungal

agents

is

paradoxical

using

because

immunocompromised patients are highly vulnerable to IFIs. Fortunately, several analogues of compounds 82 and 83 were found to be non-immunosuppressive while retaining their antifungal activity.

126, 127

Thus, medicinal chemistry efforts are

required to optimize compounds 82 and 83 and discover new types of CN inhibitors. 6.2.4 Other Targets in the Calcium-Calcineurin Signaling Pathway The fungal calcium secretory system, which is important in maintaining normal cytosol Ca2+ concentrations, has also been investigated as an antifungal target. The antiarrhythmic drug amiodarone (84) acts on the channel proteins in the calcium secretory system (Figure 22) and showed potent fungicidal activity against Candida, Cryptococcus, and Aspergillus species.187,

188

Plakortide F acid (PFA, 85), a

marine-derived polyketide endoperoxide, exerted good antifungal activity against C. albicans (MIC = 0.08 µg/mL), C. neoformans (MIC = 2.5 µg/mL), and A. fumigatus (MIC = 5.0 µg/mL) by perturbing Ca2+ homeostasis.189 Mechanistic studies revealed that compound 85 might target Ca2+ transporters, such as pumps (Pmr1 and Pmc1) and channels (Cch1 and Mid1).189 RTA2, a phospholipid translocase, plays an important role in the emergence of calcineurin-mediated azole resistance.173, 190 RTA2 47



is a potential new antifungal drug target because there is no homologs of RTA2 in mammalian or bacteria cells. However, RTA2 inhibitors have not been reported to date. 6.3 Heat Shock Protein 90-Calcineurin Pathway Heat shock protein 90 (Hsp90), an important molecular chaperone involved in the fungal stress response, is essential for fungal survival.191 Hsp90 controls the activity of its client proteins, such as CN and lysine deacetylases (KDAC), in stress responses and repair mechanisms of the cell wall (Figure 23). Hsp90 have been reported to be associated with drug resistance to azoles, echinocandins and polyenes.192 Hsp90 can promote the development of drug resistance by stabilizing signal transducers and stimulating the phenotypic effects of resistant mutations.193 Moreover, Hsp90 also regulates the morphogenesis and virulence of fungi.194 Thus, Hsp90 has been explored as a novel antifungal target for the treatment of invasive candidiasis and aspergillosis.195 The Hsp90 inhibitor geldanamycin (86) and its derivatives (87, 88) showed modest in vitro antifungal activity.195-197 Interestingly, Hsp90 inhibitors were able to potentiate the effect of azoles and echinocandins against various resistant clinical isolates, suggesting their potential as adjunctive therapy to treat resistant infections. 195, 196, 198 Although targeting Hsp90 has emerged as a promising approach to impair the evolution of drug resistance and improve the efficacy of antifungal agents in treating resistant pathogens, the development of fungal-selective Hsp90 inhibitors is challenging. Hsp90 is highly conserved in eukaryotes, and Hsp90 inhibitors are currently under development as antitumor agents, indicating their potential to cause 48


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significant side effects. Studies have indicated that the use of compound 86 and its derivatives in murine models of invasive candidiasis or aspergillosis failed to demonstrate significant therapeutic benefit because of their toxicity.199 Fortunately, the protein sequence, ATPase activity, and equilibrium of conformational states are different between the fungal and human orthologs and thus can be utilized to identify fungal-specific Hsp90 inhibitors.200 Alternatively, identification of new fungal Hsp90 chaperone network components that regulate drug resistance and share low similarity to the hosts provides another strategy to inhibit the Hsp90 chaperone machinery and discover novel antifungal agents.

Figure 23. Potential targets in and representative inhibitors of the Hsp90-CN pathway. 49



Page 50 of 100

6.3 Lysine deacetylases Hsp90 function and its interaction with its client proteins and co-chaperones are mediated

by

post-translational

modifications.

KDACs,

particularly

histone

deacetylases (HDACs), are important regulators of Hsp90 function and fungal drug resistance (Figure 23). Thus, indirect inhibition of the Hsp90-calcineurin axis by KDAC (HDAC) inhibitors represents a promising approach to combat the emerging resistance of fungal pathogens. Moreover, the difference between fungal and human KDACs is far larger than that for Hsp90, enabling the development of fungi-selective inhibitors.201 HDACs remove acetyl groups from histones, and fungal HDACs are mainly classified into three types: Class I (Rpd31, Rpd32, Hos1, and Hos2), Class II (Hda1 and Hos3) and Class III (Sir2 and Hst proteins).202 In addition to them, Set3 is a kind of fungal-specific HDAC with a sequence identity of less than 20% with human or other C. albicans HDACs.203 HDAC inhibition blocks the emergence and maintenance of Hsp90-dependent resistance, virulence and biofilm formation in C. albicans, 201, 203 C. neoformans,204 and A. fumigatus205, 206. Similar to Hsp90 inhibitors, the non-selective HDAC inhibitors trichostatin A (89) and vorinostat (90, SAHA) showed modest in vitro antifungal activity. However, they can significantly enhance the potency of azoles and echinocandins against various fungal pathogens, including resistant isolates.207-209 MGCD290 (structure not disclosed; may be an analogue of compound 91) is a fungal-specific Hos2 inhibitor 50


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with broad-spectrum antifungal activity against Candida spp. (MIC range: 0.5 ~16 µg/mL), Aspergillus spp. (MIC range: 8.0~ > 32 µg/mL) and C. neoformans (MIC range: 0.5 ~ 4 µg/mL).210 It also showed synergistic effects with the echinocandins and azoles both in vitro and in vivo for treating resistant infections.211 Its combinational use with fluconazole has been tested in phase 2 clinical studies to treat severe vulvo-vaginal candidiasis.203 However, the therapeutic outcome was not significantly improved compared with that of treatment with fluconazole alone. Thus, further clinical evaluations or structural optimizations are required. Notably, no enzymatic data have been reported regarding purified fungal HDACs, which has limited the development of fungal-specific inhibitors. Additionally, structural information on fungal HDACs will facilitate the discovery of selective inhibitors, which will have high clinical value to improve therapeutic efficacy and reduce side effects. 7. Protein-Protein Interaction The Pdr1–Gal11A interaction is involved in a key multidrug resistance pathway in C. glabrata, which is clinically important and intrinsically resistant to azoles.212 Arthanari’s group performed an HTS study to identify small-molecule C. glabrata Pdr1–Gal11A inhibitors.213 The lead compound iKIX1 (92, Figure 24) bound to the C. glabrata Gal11A KIX domain with Kd and Ki values of 0.32 µM and 18 µM, respectively.212 NMR and molecular docking studies revealed that compound 92 interacted with the KIX domain (a three-helix bundle) through a hydrogen bonding network and hydrophobic interactions (Figure 24). Compound 92 was able to restore

51



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azole sensitivity in azole-resistant C. glabrata strains with gain-of-function PDR1 point

mutations

(PDR1L280F).

The

efficacy

of

compound

92-fluconazole

combinational therapy in treating resistant disseminated C. glabrata infections was validated by in vivo models.212 Co-treatment with fluconazole and compound 92 significantly reduced the fungal burdens in the kidney and spleen in the mouse models. Moreover, compound 92 showed low toxicity to human HepG2 cells (IC50 ≈ 100 µM) and had favorable PK profiles. Notably, the SAR information of compound 92 is limited, and current data indicate that its SAR was narrow and that tiny modifications led to loss of activity. Thus, diverse inhibitors are highly desirable to further validate the druggability of the novel target.

Figure 24. Inhibition of the Gal11A KIX domain by iKIX1.

8. Novel Immune Response-based Targets Recently, novel immune response-based antifungal therapies were investigated by determining how the host immune system fights fungal infections. Recognition of fungal pathogens relies on the surface pattern-recognition receptors, such as the C-type lectin receptors (CLRs), to initiate the innate immune response against 52


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invading pathogens.214 E3 ubiquitin ligase casitas B lymphoma-b (CBLB) controls polyubiquitination and proteasomal degradation of CLRs (dectin-1 and dectin-2) and their downstream kinase (spleen tyrosine kinase, SYK) and inhibits innate immune responses. In 2016, two complementary studies clarified the function of CBLB as a negative regulator of fungal recognition during systemic C. albicans infection and highlighted its potential as a novel antifungal target.215, 216 CBLB deficiency could protect mice from lethal systemic C. albicans infection. Moreover, pharmacological inhibition of CBLB by systemically delivering CBLB-specific small interfering RNA or injecting a cell-permeable CBLB inhibitory peptide was demonstrated to enhance antifungal immunity, reduce kidney fungal burden and improve the survival rate.215, 216

However, specific inhibition of CBLB ubiquitin ligase activity by small molecules

still remains a formidable challenge. In 2017, c-Jun N-terminal kinase 1 (JNK1) was proven to negatively regulate the host antifungal innate immune response and serve as a therapeutic target in fungal infection.217 In C. albicans infection models, the JNK1 inhibitors SP600125 (93, Figure 25) and JNK-IN-8 (94) showed potent protective effects and significantly increased the survival rate.217 Due to the effectiveness of JNK inhibition in enhancing antifungal immunity, JNK inhibitors provide a new strategy for novel antifungal drug discovery.

Figure 25. Chemical structures of JNK1 inhibitors. 53



9. Chemogenomic Profiling to Identify Targets of Antifungal Molecules All the clinically available antifungal agents were originally discovered by antifungal activity screening prior to knowing their targets. Phenotypic screening is an effective approach to identify starting points for antifungal development.218 When antifungal lead compounds are available, identification and validation of their molecular targets are important to facilitate the subsequent lead optimization and drug development process. The chemogenomic profiling methods,219, 220 i.e., haploinsufficiency profiling (HIP) and homozygous profiling (HOP), have been widely applied to identify the targets of antifungal compounds.221 HIP/HOP assays systematically screen large collections of genetically defined mutant strains for their sensitivity to antifungal compounds and identify the primary target and the buffering cellular pathway.222 In the HIP assay, one copy of each gene is deleted per strain, and the strain library is screened for the sensitivity to a given compound. If the compound shows increased sensitivity to a heterozygous deletion, the corresponding protein may be the primary target. As a complementary assay, HOP can determine secondary targets or compensating pathways in the absence of direct targets by testing the drug sensitivity to deletion strains for nonessential genes. The advantage of the HIP/HOP assay is that prior knowledge of the target is not required and that only those targets that affect the fitness of fungi will be identified. However, it should be noted that a number of potential targets may be identified from the assay; thus, further validation studies are necessary to identify the direct binding target. Successful examples of target identification using HIP/HOP assays are listed in Table 3. In addition to HIP/HOP, affinity chromatography techniques have also been used to identify antifungal 54


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targets.223 Recently, new strategies for target identification and validation of bioactive molecules have been developed rapidly, which may facilitate the discovery of more antifungal targets.224-226 Table 3. Selected examples of antifungal target identification using HIP/HOP assays Compounds

Targets

Geranylgeranyltransferase I (GGTase I)

GGTase I

Genes GLC7, MET4, RFC2, YAE1, and PRP18

Endoplasmic reticulum-associated protein degradation components

Refs

227

227

228

229

HO

Endoplasmic reticulum integrity 99

55


230


Cl

Page 56 of 100

Cl

N S

N

CYP51

231

AHAS

125

Mia40-Erv1 pathway

232

100

Lipid transfer protein Sec14p

233

S HN

N NH

Fatty acid desaturase

234

Microtubules

235

104

56


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HO O

H OH

Erg26p

H HO O

H OH

236

O 106

10. Perspectives With the increasing morbidity and mortality of IFIs and the emergence of severe antifungal drug resistance, new drug targets and novel antifungal agents are urgently needed. Recently, better understanding of fungal pathogenic processes has contributed to the emergence of new antifungal drug targets. Ideally, an antifungal target without a human counterpart is highly promising for new drug development. Inspired by the success of echinocandin GS inhibitors, researchers have extensively investigated potential targets involved in the biosynthesis of the fungal cell wall, such as β-1,6-glucan synthase, chitin synthase, and the GPI biosynthetic pathway, and several small-molecule inhibitors have shown potent antifungal activity and anti-resistance profiles. Due to the complexity of the fungal cell wall components, structural information about essential proteins is still lacking, which has limited the efficiency of drug development. Progress in fungal structural biology can promote rational drug design and accelerate the drug discovery process. Conversely, fungal cells share high similarity with mammalian cells, and a number of targets involved in the cell membrane and protein (or DNA) biosynthesis are not fungal specific. Nevertheless, selectivity can be achieved by targeting unique binding site in fungal proteins. For example, Oliver et al. recently reported a selective inhibitor of A. fumigatus 57



dihydroorotate dehydrogenase that showed excellent in vivo efficacy in an azole-resistant murine pulmonary model of aspergillosis.237 In contrast to compounds that act on traditional targets to exert fungistatic or fungicidal activities, virulence factors provide a novel type of high value target. Anti-virulence agents have low risk to develop drug resistance and low host toxicity because they are generally inactive in vitro. However, developing virulence factor inhibitors represents a difficult task. First, knowledge of fungal virulence is still limited, although various virulence factors have been identified. Second, most of the reported anti-virulence agents have been identified from known compounds. Their further development is hampered by their non-selectivity and the lack of subsequent medicinal chemistry studies. Third, potential clinical applications of virulence factor inhibitors are unexploited. Whether anti-virulence agents can be used as a single therapy or a combinational therapy remains to be validated by systematic evaluations. Recently, new types of drug targets (such as protein-protein interactions and epigenetic targets) and novel therapeutic strategies (such as cancer immunotherapy) have emerged rapidly and achieved great success. These innovative ideas can also be used to develop a new generation of antifungal agents. Recently, proof-of-concept studies of Pdr1–Gal11A interaction, E3 ubiquitin ligase CBLB, and kinase JNK1 opened a new window for antifungal drug development. Thus, diverse inhibitors are highly desirable to further validate these novel targets. The genomes of clinically important fungal pathogens, such as C. albicans, C. neoformans and A. fumigatus, provide a good opportunity to identify novel antifungal 58


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drug targets.238, 239 Recently, genome-scale analysis of C. albicans and C. neoformans identified essential genes for antifungal drug target prioritization.240, 241 In particular, essential genes that have no mammalian homologs (such as ARO1, ARO7, ARO2 and RIB1) would be ideal for the development of selective antifungal drugs with broad-spectrum activity and low toxicity. Conversely, a number antifungal lead compounds with unknown antifungal mechanisms and target have been reported.66, 218 Thus, chemogenomic profiling approaches are powerful tools to identify drug targets and assist in antifungal lead optimization. It should be noted that there is a long road from target identification to clinical application. Importantly, the druggability of an antifungal target depends on whether it can bind drug-like compounds, and target validation by medicinal chemistry approaches is necessary. Although a number of potential antifungal targets have been identified, more effort should be focused on the discovery, evaluation and optimization of drug-like inhibitors. Several of them, such as GPIs, AaRSs and HDACs, have shown promising features for antifungal drug development. With better understanding of fungal pathogenic mechanisms and increased medicinal chemistry efforts, a new generation of antifungal drugs will become reality in the near future.

AUTHOR INFORMATION

Corresponding Authors * (C. S.) Phone / Fax: +86 21 81871205. E-mail: [email protected]. Author Contributions

59



†

N. L. and J. T. contributed equally to this perspective.

Notes The authors declare no competing financial interest.

Biographies Na Liu received her bachelor’s degree in pharmacy (2006) and master’s degree in medicinal chemistry (2013) from Second Military Medical University. Since 2016, she has been an associate professor in the Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University. Her current research is focused on antifungal drugs and anti-HCV drugs. Jie Tu received his bachelor’s degree in pharmacy and joined the Department of Medicinal Chemistry as a graduate-student (2015) at the School of Pharmacy, Second Military Medical University. His current research is focused on antifungal agents. Guoqiang Dong received his bachelor’s degree in pharmacy (2008) and PhD in medicinal chemistry (2013) from Second Military Medical University. Since 2013, he has been a lecturer in the Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University. His current research is focused on the modification and scaffold hopping of natural products and multi-targeting antitumor drugs. Yan Wang received her bachelor’s degree in pharmacy (2001) and PhD in Pharmacology (2006) from Second Military Medical University. Dr. Wang was appointed to the faculty of the Department of Pharmacology, School of Pharmacy, in 60


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2006, and presently, she is an associate professor and the vice director of the New Drug Research and Development Center in the School of Pharmacy. Her research interests include antifungal drug discovery and strategies against infectious diseases. Chunquan Sheng received his bachelor’s degree in pharmacy (2000) and PhD in medicinal chemistry (2005) from Second Military Medical University. In 2005, Dr. Sheng was appointed to the faculty of the Department of Medicinal Chemistry, School of Pharmacy, where he is currently a Professor and the Director of the Department of Medicinal Chemistry. His research interests include antifungal and antitumor drug discovery.

ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (Grants 81725020 and 81573283 to C. S.) and the Science and Technology Commission of Shanghai Municipality (Grant 17XD1404700 to C. S.).

ABBREVIATIONS USED

IFIs, invasive fungal infections; AIDS, acquired immune deficiency syndrome; ICU, intensive care units; AmB, amphotericin B; CYP51, lanosterol 14α-demethylase; GS, β-(1,3)-D-glucan

synthase;

glycosylphosphatidylinositols;

SAR,

structure-activity

PIG-W,

relationship;

phosphatidylinositol

glycan

GPIs, anchor

biosynthesis class W; PIG-N, phosphatidylinositol glycan anchor biosynthesis class N; EtNP, ethanolamine phosphate; Man, mannose; GlcN, non-N-acetylated glucosamine; Ins, inositol phospholipid; GPI-APs, GPI-anchored proteins; Gwt1, GPI-anchored wall protein transfer 1; Mcd4, morphogenesis checkpoint dependent; IP, 61



Page 62 of 100

intraperitoneal; FICI, fractional inhibitory concentration index; UDP, uridine diphosphate; GlcNAc, N-acetylglucosamine; Fru-6P, fructose 6-phosphate; AGM1, N-acetylglucosamine-phosphate

mutase;

pyrophosphorylase;

N-acetylglucosamine-6-phosphate;

GlcNAc-6P,

UAP,

UDP-N-acetylglucosamine GlcNAc-1P,

N-acetylglucosamine-1-phosphate; SBDD, structure-based drug design; NMT, N-myristoyltransferase; CaNMT, C. albicans NMT; AaRS, aminoacyl-transfer tRNA synthetase; LeuRS, leucyl-tRNA synthetase; IleRS, isoleucyl-tRNA synthetase; AHAS, acetohydroxyacid synthase; ALS, acetolactate synthase; DHFR, dihydrofolate reductase; ATP, adenosine triphosphate; GTP, guanosine triphosphate; IMP, inosine monophosphate;

IMPDH,

inosine

monophosphate

dehydrogenase;

AdSS,

adenylosuccinate synthetase; s-AMP, adenylosuccinate; SidA, siderophore A; Tps1, rehalose-6-phosphate synthase; Tps2, trehalose-6-phosphate phosphatase; PPTases, phosphopantetheinyl transferases; HOG, high-osmolarity glycerol; TOR, target of rapamycin; ICL, isocitrate lyase; Sap, secreted aspartyl proteinase; PLB, phospholipase B; CCBs, calcium channel blockers; CaM, calcium-binding protein calmodulin; CN, calcineurin; CyP, cyclophilin; FKBP, FK506 binding protein; PFA, plakortide F acid; Hsp90, heat shock protein 90; KDAC, lysine deacetylase; HDAC, histone deacetylase; CLR, C-type lectin receptor; CBLB, casitas B lymphoma-b; SYK, spleen tyrosine kinase; JNK1, c-Jun N-terminal kinase 1; HIP, haploinsufficiency profiling; HOP, homozygous profiling.

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