Review on fungal enzyme inhibitors – potential drug targets to manage human fungal infections

Abstract

Invasive fungal infections caused by opportunistic fungal pathogens have been emerging as a global problem of great concern as they are associated with increased morbidity and mortality. Despite this, there are very limited drugs of choice to treat fungal infections. The continuous usage of these drugs is associated with resistance development and thus this is another area of concern. Fungal enzymes represent one of the most important and potential targets for drug development, as they are essential for their growth and establishment in the host. In this review, we have discussed the well established and currently available enzyme inhibitors as therapeutic choices to treat fungal infections as well as those enzyme inhibitors that have been identified as suitable drug candidates to manage fungal infections. Thus, the study of fungal biosynthetic enzymes and their inhibitors could potentially show a promising way of drug development for emerging and re-emerging fungal infections of humans.

---

Introduction

Invasive fungal infections has increased dramatically over the past two decades, and are associated with an increase in morbidity and mortality. More specially, immunocompromised populations, like allogenic and haematopoietic stem cell and organ transplant patients, patients undergoing cancer therapy, HIV positive individuals, diabetics, patients under prolonged ICU stay, preterm babies, malnutrition etc., are highly susceptible to such opportunistic and invasive fungal infections.^1,2 The major etiological agents causing invasive fungal infections are Candida spp, Aspergillus spp and Cryptococcus spp.^3,4 Infections caused by these pathogens often result in life threatening systemic illnesses such as candidiasis, aspergillosis, and cryptococcus meningitis. These pathogens are acquired through inhalation of spores (aspergillosis, cryptococcosis), by nosocomial transmission and penetration into mucosa by normal flora (candidiasis).⁵

Invasive candidiasis caused by Candida albicans is the most prevalent infectious species among the 20 different species of Candida.^6,7 The increased use of medical devices such as central venous catheters has lead to the emergence of Candida spp as an important nosocomial pathogen.⁸ Although C. albicans is most common, non-albicans species namely, C. glabrata, C. tropicalis, C. parapsilosis, C. guilliermondii, C. krusei, C. lusitaniae and C. kefyr are also increasing recently.⁹ Cryptococcus neoformans is an emerging invasive fungal infection in immunocompromised patients. Cryptococcus meningitis and meningo-encephalitis are becoming quite common in patients with AIDS, and these have emerged as a leading cause of morbidity and mortality in HIV positive and negative patients.¹⁰ Recently, the Center for Disease Control estimated approximately one million new cases of cryptococcal meningitis each year, resulting in the death of 625 000 people worldwide.^11,12 Similarly, invasive aspergillosis has also become increasingly common in recent days, resulting in mortality ranging from 40–80%.^13,14 The predominant causative agent is Aspergillus fumigatus which also finds concurrence in a recent literature study reported from 1985 to 2013.^15–20

Biofilms of these invasive pathogens are difficult to manage with conventional antifungal agents. Hence, these infections are not eradicated and are emerging as life-threatening infections. Though the spectrum of fungal infections has increased recently, the choice of antifungal agents to treat invasive fungal infections is limited, owing to their toxicity. Thus, the objective of the present review is to explore the potential applications of enzyme inhibitors for the management of invasive fungal infections.

Potential application of enzyme inhibitors in management of fungal infections

β-(1,3)-Glucan synthase inhibitors

The fungal cell wall has a unique structure that is absent in human cells and hence it is identified as a potential target for development of antifungal agents.²¹ Though the cell wall composition of fungi varies with species, the major component of cell walls of many fungi is β-(1,3)-glucan, which are comprises 30–60% of the fungal cell wall. 2β-(1,3)-Glucan provides structural integrity and osmotic stability to the fungus.^22,23 Hence, the biosynthesis of β-(1,3)-glucan by glucan synthase is identified as a crucial drug target. When β-(1,3)-glucan synthase is inhibited, it eventually weakens the cell wall, giving rise to osmotic lysis.²⁴

Members of the β-(1,3)-glucan synthase inhibitors include echinocandins, enfumafungin and papulacandins. Echinocandins are synthetically modified naturally produced lipopeptides and are the first line of drug choice for invasive candidiasis, particularly for patients who are critically ill and those with previous exposure to triazole.²⁵ Their target is to comprise a GTP-binding and Rho protein that regulates the synthesis of β-(1,3)-glucan synthase and a FKS catalytic subunit, encoded by the FKS gene.²⁶ Echinocandins have potent antifungal activity against most common yeasts and molds with a broad spectrum activity against Candida sp. Unfortunately echinocandins are not effective against C. neoformans, as they have typically less β-(1-3) linked glucan.^27,28 However, the in vitro combination of caspofungin and amphotericin B has been shown to have synergism against C. neoformans.²⁹

Caspofungin is the first approved semisynthetic echinocandin product followed by micafungin, and anidulafungin (Fig. 1). Though the mechanisms of action are similar, the spectrum of antifungal activity varies. For instance anidulafungin is effective against a wide range of azole or polyene resistant Candida sp and Aspergillus sp.^30–32 Hence, these antifungal agents are truly life saving drugs. The frequency of echinocandin resistance remains low with Candida sp, as it facilitates escape through the formation of characteristic FKS hot-spot mutations.^33,34 However, enchinocandin resistance has been observed in C. glabrata, thus this poses a serious clinical challenge in immunocompromised patients. Even though β-(1,3)-D-glucan synthase inhibitors are a significant alternative to ergosterol-binding antimycotic agents, poor oral bioavailabilty is their main limitation.^35,36 Due to their large chemical structure echinocandins are available only as intravenous formulations.³⁶ To overcome this particular drawback, the semi-synthetic modification of a natural product, triterpene glycoside enfumafungin and its derivatives, is in progress. One such example is SCY-078, a novel oral glucan synthase inhibitor, developed and investigated for its pharmacodynamic property with a suggestion to promote SCY-078 as a promising oral option to treat Candida infections.³⁷ Papulacandins are another set of molecules belonging to the β-(1,3)-glucan synthase inhibitors, but are not currently being used therapeutically due to their narrow antifungal spectral activity and lower activity in vivo than in vitro.³⁸

Fig. 1 Inhibitors of β-(1,3)-glucan synthase.

Inhibitors of chitin synthesis

Chitin and chitosan are unique polysaccharides found in all fungal pathogens. Inhibition of chitin synthesis is another attractive target for antifungal therapies.^39,40 The well known chitin synthase inhibitors are polyoxins and nikkomycins (Fig. 2). They exhibits similar structural features, having a nucleoside skeleton and one or two peptidyl moieties. They act as competitive analogs of UDP-N-acetylglucosamine, a substrate for chitin synthesis.³⁹ Although polyoxins exhibit effective in vitro activity, they did not produce satisfactory results against systemic candidiasis in mice,^41–44 owing to their poor transport across membranes.⁴⁵ However, in combination with flucanozole, polyoxins exhibit a wide spectrum antifungal activity against flucanozole resistant C. neoformans, C. albicans and non-albicans Candida sp.⁴⁶ Moreover, the activity of nikkomycin has been well demonstrated against the medically important fungi Coccidioidis immitis and Blastomyces dermatitidis, though clinical trials are still pending.^21,47

Fig. 2 Inhibitors of chitin synthesis.

Inhibitors of cytochrome P450

Cytochromes of fungi contribute to cellular respiration, ATP generation and also to the biosynthesis of several cellular components. Lanosterol 14α-demethylase (CYP51) is a cytochrome P450 that co-catalyzes 14α-demethylation of lanosterol, an essential step in the biosynthesis of sterols.^48,49 Ergosterol serves as a regulator of membrane fluidity and provides membrane integrity in fungal cells. Azole drugs target the heme protein that co-catalyzes cytochrome P450-14α-demethylation of lanosterol. Inhibition of 14α-demethylase results in the depletion of ergosterol and accumulation of 14-methylsterols, which in turn leads to membrane dysfunction.⁵⁰ However in humans, CYP51 mediates conversion of lanosterol to meiosis-activating sterols, which are the intermediates in the biosynthesis of cholesterol. However, the selectivity and safety of the azoles are attributed to their greater affinity for fungal CYP51 than mammalian, especially when azoles are present at therapeutic concentrations.^51–53

Azole antifungal drugs have been in use for more than two decades. Azoles are classified into two different classes, namely the imidazole and triazoles. Clotrimazole, ketoconazole, and micanozole are categorized under imidazoles and the triazole agents are fluconazole, itraconazole, voriconazole, isavuconazole and posaconazole⁵⁴ (Fig. 3). The imidazole derivatives are not very selective, as they are found to inhibit several membrane bound enzymes and membrane lipid biosynthesis.⁵⁵ The triazoles demonstrate a broader antifungal spectrum, notably against many Candida spp and Aspergillus spp. Though posaconazole has a wide spectrum of activity, including against azole resistant Candida sp, the oral suspension has been shown to have some limitations due to low bioavailability attributed to elevated gastrointestinal pH and also meal status.^5,56 Nevertheless the development of delayed-release oral tablets and intravenous solution has proved advantageous in terms of maximum bioavailability of the drug. A recent addition to the azole class is isavuconazonium (Cresemba®). It is the sixth approved product of Qualified Infectious Disease Product (QIDP) to treat invasive aspergillosis and mucormycosis.⁵⁷ Moreover, it has superior characteristics including linear dose-dependent pharmacokinetics, intravenous and oral formulations, absence of nephrotoxic solubilizing agents and excellent oral bioavailability which is independent of meal status and gastric acidity. For more detailed information please refer to the recent discussion on pharmacokinetics of oral and intravenous posaconazole and isavuconazole formulations by Jukius, (2015).⁵⁶

Fig. 3 Inhibitors of cytochrome P450.

Inhibitors of amino acid biosynthetic pathways

Fungi obtain essential amino acids for their growth and development through biosynthetic pathways, whereas mammals acquire the same from their diet. The enzymes that catalyse amino acid biosynthesis pathway are identified as potential targets for antifungal drugs, as they are highly conserved in fungi.⁵⁸

The enzymes which are involved in amino acid biosynthetic pathways are unique to fungi and are not found in mammals, with the exception of the arginine biosynthetic pathway which is identical with the mammalian one.⁵⁸ Numerous studies have shown that fungal pathogens that have defects in a particular amino acid biosynthetic pathway show growth defects and have growth defects and reduced virulence factors.⁵⁸ In conjunction with this, various studies have been carried out to explore potential antifungal agents that are targeting specific enzymes involved in amino acids biosynthetic pathways. High throughput screening approaches were used in some studies to screen small molecule enzyme inhibitors of major amino acid biosynthesis pathways. These include inhibitors of homoserine dehydrogenase, homoserine kinase and homoserine transacetylase. For example, the natural product ebelactone A, and its synthetic derivatives, are inhibitors of bacterial homoserine transacetylase and appear to be promising antifungal agents.^59,60 Azoxybacillin, another natural product from B. cereus, exhibits antagonistic activity against Aspergillus sp, however poor bioavailability was observed in animal models.⁶¹ Similarly, 6-carbamoyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4-carboxylic acid (CTCQC), a homoserine transacetylase enzyme inhibitor, demonstrated poor antifungal activity.⁶² Several compounds with similar enzyme inhibitory activity were demonstrated to be effective in vitro but were not always potent under in vivo conditions. Though the in vitro antifungal action was very specific, under in vivo conditions, the fungal cells acquire the required amino acids from the hosts blood, thus rendering the therapy ineffective.^60,63 However, the availability of methionine and tryptophan is much lower in mammalian serum, and is not sufficient for the fungi to make use of for its growth and development. Hence, inhibitors of methionine and tryptophan biosynthetic enzymes seem to be the most promising candidates.^64,65 The readers are directed to a recent review on this topic.⁶⁰ It is clear that such studies on amino acid synthesis inhibitors could be saddled with Pan Assay Interference Compounds (PAINS) and other promiscuous compounds that could prove to be a pitfall in trials of such compounds as potential antifungal therapies.⁶⁶ Thus, any inhibitor reported should be thoroughly evaluated for their mechanism of action. The details of inhibitors of amino acid synthesis pathway that are identified as potential drug targets are summarized in Table 1.^60,67

Table 1 Inhibitors of aminoacid synthesis pathway as potential drug targets. Sources: ref. 60–67

S. no.	Amino acid family	Amino acids	Biosynthesis pathway	Effect on mutant strain	Target pathogen	Potential drug target	Inhibitory compound
1	Aspartate family	L-Threonine, L-isoleucine, and L-methionine	Aspartate pathway	Mutant strain not viable in minimal media	Bacteria and fungi	Threonine biosynthesis (role as a intermediate of isoleucine synthesis aspartate pathway)
			Aspartate aminotransferase (Aat1p)				Potential antifungals compound not reported
			Aspartate kinase (Hom3p)	Mutant strain depends on temperature and nitrogen source and it will reduce virulence	S. cerevisiae, C. neoformans		Derivatives of 7-chloro-4([1,3,4]thiadiazol-2-yl sulfonyl)-quinoline
			Homoserine dehydrogenase (Hom6p)	Due to homoserine accumulation thr1 and thr4 mutant specific phenotypes were suppressed	C. albicans		(S)-2-Amino-4-oxo-5-hydroxypentanoic acid (RI-331 compound produce by Streptomyces)
				Mutant strains are more sensitive compared to wild strain
			Asparagine synthetase (Asn1p)				Potential antifungals compound not reported
			Aspartate semialdehyde dehydrogenase (Hom2p)				Potential antifungals compound not reported
1a	Threonine branch	Threonine	Homoserine kinase (Thr1p)	Mutant strain depends on temperature and nitrogen source	C. neoformans		Potential antifungals compound not reported
				Mutant strains are more sensitive for antifungal agents compared to wild	S. cerevisiae, C. albicans		Potential antifungals compound not reported
				Reduction in virulance	C. neoformans, C. albicans		Potential antifungals compound not reported
				No in vivo survival	S. cerevisiae		Potential antifungals compound not reported
			Threonine synthase (Thr4p)	No in vivo survival	S. cerevisiae		L-(Z)-2-Amino-5-phosphono-3-pentenoic acid
1b	Methionine	Methionine	Homoserine transacetylase (Met2p)	Lack of sexual development, reduce fungal virulance (Fusarium graminearum)	C. albicans, C. neoformans, Fusarium graminearum	Homoserine transacetylase	6-Carbamoyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4-carboxylic acid (CTCQC)
							Ebelactone A
				In vitro effective, active against mycelial fungi	Aspergillus sp, Absidia sp., Microsporum sp., Trichophyton sp.		Azoxybacillin
			Met3	In vivo (mouse model) melanin production decreased; in vitro slow growth in minimal media supplemented with methionine (C. neoformans)	C. neoformans		Potential antifungals compound not reported
			Methionine synthase (Met6p)	Decrease in virulance (C. albicans, C. neoformans); hypersensitive to antifungal drugs (C. neoformans); homocysteine accumulation (C. neoformans)	C. albicans; C. neoformans		Fluconazole; the calcineurin inhibitor tacrolimus FK506
			Acetylhomoserine aminocarboxypropyltransferasde (Met15p)		C. albicans		Potential antifungals compound not reported
			Cystathionine-synthase (Str2p)		Aspergillus sp	Not potential antifungal target	Pyrimethanil (SC-0858)
			Cystathionine-lyase (Str3p)		Aspergillus sp, C. albicans	Not potential antifungal target	3,3,3-Trifluoro-N-(2-methylphenyl)-2-(trifluromethyl)propanamide
			Asparagine synthase (Asn1p)	Inhibit pathogenicity (Magnaporthe grisea)	Magnaporthe grisea		Potential antifungals compound not reported
2	Lysine biosynthesis	Lysine	α-Aminoadipate pathway	Attenuated virulence	Aspergillus sp, C. albicans	Homoisocitrate dehydrogenase	Thiahomoisocitrate
				Moderate inhibition	Aspergillus nidulans		(R)-Homocitrate and (2R,3S)-homoisocitrate
					Aspergillus nidulans		(R)-(2-p-Carboxybenzyl)malate trimethyl ester
					Aspergillus nidulans, C. albicans		(2R,3S)-3-(p-Carboxybenzyl)malate trimethyl ester
					Aspergillus nidulans		(2R,3S)-3-(m-Carboxybenzyl)malate trimethyl ester
					Aspergillus sp, C. albicans, C. neoformans		(2R,3S)-2-Fluoro-3-allylsuccinate
					Aspergillus sp, C. albicans, C. neoformans		(1R,2S)-1-Fluorobutane-1,2,4-tricarboxylate
					Aspergillus sp, C. albicans, C. neoformans		Methyl esters
					C. albicans		trans-Homoaconitate
					C. albicans		trans-1,2-Epoxypropane-1,2,3-carboxylate
						Saccharopine dehydrogenase	No information
3	Branched-chain amino acids biosynthesis	Leucine, valine, and isoleucine	Acetohydroxyacid synthase (Ilv2p)	Inhibit growth	C. albicans	Acetohydroxyacid synthase	Sulfonylureas derivatives (a: R = CH2CH3, X = I; b: R = CH2CH3, X = Br; c: R = CH3, X = I; d: R = CH3, X = Cl)
					C. albicans		Ethoxysulfuron
					C. albicans		Chlorimuron ethyl
					S. cerevisiae, C. albicans, A. fumigatus, R. oryzae, and C. neoformans		Triazolo-pyrimidine-sulfonamides derivatives
			Ketol-acid reductoisomerase Ilv5p	Inhibit growth	R. solanii, F. oxysporum, C. cassiicola, B. cinerea	Ketol-acid reductoisomerase	N-(5-Substituted-1,3,4-thiadiazol-2-yl)cyclopropanecarboxamides
4	Histidine biosynthesis	Histidine	Imidazole glycerol phosphate synthase His7p	Reduce virulance	Cryptococcus sp, Candida sp, and Ajellomyces sp		Potential antifungals compound not reported
			Histidinol dehydrogenase His4p				Potential antifungals compound not reported
5	The glutamate family	L-Glutamate, L-glutamine, L-proline, and L-arginine					Potential antifungals compound not reported
5a	L-Glutamate biosynthesis	L-Glutamate	NAD(P)+-dependent glutamate dehydrogenase Gdh2p		Aspergillus niger	Iminoglutarate	2-Methyleneglutarate
			NAD(P)+-dependent glutamate dehydrogenase Gdh3p				Isophthalate
							2,4-Pyridinedicarboxylate
					Candida albicans, Yarrowia lipolytica		1,2,3-Triazole-linked β-lactam-bile acid conjugates: B18 and B20
5b	L-Glutamine biosynthesis	L-Glutamine	Glutamine synthetase Gln1p				Potential antifungals compound not reported
5c	L-Proline biosynthesis	L-Proline	γ-Glutamyl kinase Pro1p		Antibacterial		Potential antifungals compound not reported
			Glutamate-5-semialdehyde dehydrogenase Pro2p		Antibacterial		Potential antifungals compound not reported
			Pyrroline-5-carboxylate reductase Pro3p	Inhibit pathogenicity	Magnaporthe grisea		Potential antifungals compound not reported
5d	Arginine biosynthesis	L-Arginine	ARG5,6-acetylglutamate kinase and acetylglutamyl-phosphate reductase	Inhibit growth	Candida albicans		Potential antifungals compound not reported
			Arg4p argininosuccinate lyase	Inhibit growth and pathogenicity	Fusarium oxysporum, C. albicans and A. nidulans		Potential antifungals compound not reported
6	Serine biosynthesis	L-Serine	de novo cysteine biosynthesis pathway		Antifungal		Potential antifungals compound not reported
7	Cysteine biosynthesis	L-Cysteine	Cystathionine γ-lyase Cys3p		Antifungal		2-Amino-2-pentynoic acid (propargylglycine)

---

Glucosamine-6-phosphate synthase inhibitor (GlcN-6-P)

GlcN-6-P is a crucial enzyme for the biosynthesis of N-acetyl-D-glucosamine, the major component of chitin and mannoproteins of fungi.⁶⁸ The major role of GlcN-6-P is that it catalyzes the transfer of an amino group from L-glutamine to D-fructose-6-phosphate with the formation of D-glucosamine-6-phosphate. The resultant D-glucosamine-6-phosphate is the substrate for the synthesis of macromolecules containing chitin and mannoproteins. Hence antifungal activity with GlcN-6-P inhibitors is not surprising.³ However, the target is not very specific to fungi, as it is well known that GlcN-6-P synthase is also required for the biosynthesis of glycoproteins, glycolipids and proteoglycan in humans.⁶⁹ Some studies have demonstrated that treatment with a GlcN-6-P synthase inhibitor as a short term course was not dangerous, probably due to the rapid turnover of the mammalian GFAT gene.⁷⁰ The well known specific GlcN-6-P synthase inhibitors belong to two different structural groups: L-glutamine analogues and analogues of the transition state of the reaction in the C-terminal sugar isomerising domain. The best example for glutamine analogues are N-3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP),^65,67,71 which demonstrates specific inhibitory action on GlcN-6-P synthase, and not on any other enzymes that use glutamine as the nitrogen source. Nevertheless, FMDP shows no in vivo antifungal activity as it has very poor diffusion into the cell. Several approaches have been adopted to increase the antifungal activity of FMDP, one such successful approach is the incorporation of FMDP into short peptides that aid its entry of FMDP via peptide permeases.^72–74 In order to overcome FMDP peptide resistance strains of C. albicans, the FMDP molecules were modified to improve lipophilic activity by introducing four to seven carbon atoms into the alkyl chains. Unfortunately this modification reduced the antifungal activity.⁷⁵ Recently, three novel inhibitors of GlcN-6-P synthase, namely the primary amide of FMDP, N-methyl and N,N-dimethylamide of FMDP were reported. The primary amide of FMDP was shown to have higher anticandidial activity.⁷⁶ Zong (2014) reported a series of novel glycosylthiadiazole derivatives and evaluated for their inhibitory action against GlcN-6-P synthase and their SAR based design revealed that “compounds with two electron-withdrawing substituents in the benzene ring have better fungicidal activities than those with two electron-donating substituents”.⁷⁷ Thus compounds with the protecting groups in the sugar ring have less inhibitory activities against GlcN-6-P synthase. A recent addition to the GlcN-6-P synthase inhibitors is a novel acetamide derivative, 2-chloro-N-(2-phenylthiazol-5-yl) which is found to be effective against A. niger and C. albicans.⁷⁸

Inhibitors of carbonic anhydratase

Carbonic anhydratases (CA) are metalloenzymes that enhance the hydration of CO₂ to generate bicarbonate, which are subsequently used in cellular metabolism.⁷⁹ The five distinct divisions of CA are α-, β-, γ-, δ- and ζ. The α-class of CA are found in mammals, prokaryotes, plants and fungi. Whereas the β-class has been identified only in plants, bacteria and fungi, γ-CAs are predominantly found in archaea.^80,81 They are also involved in other functions like gluconeogenesis, fatty acid biosynthesis, ureagenesis, tumorigenesis and the growth of various pathogens.⁶⁷ C. neoformans, has two potential CA homologues (Can1 and Can2). Can2 is involved in both survival and proliferation in the environment. In addition, it is involved in the induction of capsule synthesis in C. neoformans through activation of adenylyl cyclase by bicarbonate.⁸² Transcriptional analysis of C. neoformans reveals that CA-dependent genes are involved in fatty acid biosynthesis, organization of the polysaccharide capsule, sexual reproduction, environmental stress response and oxidative stress response.⁸³ Similarly β-CAs are involved in the pathogenesis of Candida spp. The polymorphic nature of Candida spp is one of the important virulence factors facilitated by CO₂.⁸⁴ The two important proteins that are involved in the Candida CO₂ sensing pathway are adenylyl cyclase and CA (Nce 103).⁸⁵ Muhlschlegel’s and Heitman’s groups demonstrated the essentiality of β-CAs in the pathogenesis of C. albicans in niches where the available CO₂ is limited and also in immunocompromised patients, where the CO₂ concentration is about ∼5%. Many studies support the presence of elevated CO₂, and yeast-mycelial shift in Candida sp, and capsule induction in C. neoformans. Thus, CAs are attractive targets for the development of antifungal agents^80,86–88 against Aspergillus sp and C. neoformans.

Sulfonamides are widely used classical carbonic anhydratase inhibitors. They are used as diuretics and also against glaucoma, cancer drugs and obesity.^89,90 The presence of RSO₂NH₂ group in their structure makes these drugs the most ideal of all human α-CA (Fig. 4). Hence, a wide range of sulfonamides derivatives has begun to be investigated as anti-infectives. Many such compounds including methazolamide, ethoxzolamide, brinzolamide, benzolamide displayed Can2 inhibitory activity; methazolamide, the oldest drug is found to have major side effects like electrolyte imbalance and development of acidotic state.⁹¹ Acetazolamide appears to be the best inhibitor of both Can2 and Nce103.^85,92 However, acetazolamide, benzolamide, dichlorophenamide were shown to have low potency and poor selectivity against fungi. Hence, the search for a new class of β-CA inhibitors with high specificity is currently being investigated by many research groups. Dithiocarbamates were reported as a new class of inhibitors, with a similar mechanism of action as that of the sulphonamides by binding to the metal ion at the enzyme active site. Recently the Supuran lab investigated a new series of Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide as β-carbonic anhydrase inhibitors.⁸⁸ The inhibitory activity was similar, but slightly better compared to acetazolamide and bromosulfanilamide. Their study demonstrated that Schiff base sulfonamides are highly selective in the inhibition of β-CAs over α-CAs, suggesting that these new derivatives might have different modes of action compared to clinically used sulfonamides. A recent study has shown that acetazolmide, sulfamates, sulfamides, as well as some 4-substituted ureido-benzene-sulfonamides, possess inhibitory activity against the C. glabrata enzyme, CgNce103.⁹³ Clinical strains of C. glabrata are highly resistant to the classical antifungal agents. CA inhibitors thus could be an interesting and efficient agent to control clinically important fungal pathogens.

Fig. 4 Inhibitors of carbonic anhydratase.

A schematic representation of the overview of inhibitors of fungal biosynthetic enzymes as drug targets is shown in Fig. 5.

Fig. 5 Overview of inhibitors of fungal biosynthetic enzymes as drug targets.

Drug resistance mechanisms

Development of secondary resistance and clinical resistances in fungi are of great concern in the management of invasive fungal infections.⁹⁴ In addition, other factors such as reduced drug bioavailability, patient’s underlying disease condition, and immune status, might also play a significant role in drug resistance.⁹⁵

Some of the possible mechanisms contributing to fungal drug resistance include gene mutation, up-regulation of multi-drug efflux pumps, target site modification, development of bypass pathways and more importantly biofilm formation. The consequences of drug resistance are elevated minimum inhibitory drug concentrations and poorer clinical outcomes.⁹⁶ In this section, we have provided a brief discussion on resistance mechanisms against antifungal agents that significantly target fungal biosynthetic enzymes. Drug resistance mechanisms of sulfonamides and its derivatives are not reported in fungi, hence azole and echinocandins resistances alone are discussed in this section.

Azole resistance

Resistance to azole antifungal agents were a rare event until the 1980s, however, it has become an increasing problem recently. Azole drug resistance mechanisms are well studied in C. albicans, and are broadly categorized as (i) overexpression of membrane efflux pumps membrane proteins; (ii) mutation of target site; (iii) development of bypass pathway.⁹⁷

Induction or overexpression of multi-drug pumps actively expel drugs out of the cell. This is mediated by multidrug resistance transporters namely ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS), which are encoded by CDR and MDR genes in C. albicans respectively.⁹⁸ Activation of membrane efflux pump proteins leads to insufficient drug concentration at the target site. The up-regulation of CDR genes has been shown to confer resistance to almost all azoles, whereas induction of MDR encoded efflux pumps is selective for fluconazole resistance.⁹⁸

Another azole resistance mechanism involves the mutation or up-regulation of the target enzyme lanosterol 14-α-sterol demethylase, which is encoded by ERG11 gene and thus prevents or reduces the binding of azoles to the target site. However, ERG11 mutations are reported in both azole resistant and susceptible isolates, suggesting the unrelatedness of flucanozole resistance in C. albicans. However, a recent study reports the most important role of ERG11 mutations, especially G487T and or T916C in fluconazole resistance. During their study with 14 flucanozole resistant isolates, five of the isolates were shown to have no up-regulation of mRNA, and transcription of MDR1, ERG11, or FLU1 and CDR1 & CDR2 was also not detected. This result suggests the importance of mutations G487T and or T916C in the ERG11 gene in conferring fluconazole resistance.^98–101 The tertiary structure of the enzyme was altered due to the mutation effect and consequently this altered the affinity of the enzyme for binding with azole. Even though many distinct almost 160 amino acid substitutions have been reported only a very few have been confirmed to cause fluconazole resistance.¹⁰⁰ On the other hand, overexpression of 14α-demethylase has also been reported as an azole resistance mechanism. This particular resistance mechanism is observed in C. glabrata alone, suggesting its limited role in clinical resistance.

Development of a bypass pathway is a less common resistance mechanism that is found to produce an alteration to the sterol biosynthetic pathway. The mechanism involves the mutation of the ERG3 gene, which encodes 5,6-desaturase (essential for ergosterol biosynthesis), that prevents the formation of 14α-methyl-3,6-diol from 14α-methylfecosterol, which in turn replaces the ergosterol in the cell membrane and blocks the effect of azole on the ergosterol biosynthetic pathway. However, this mechanism does not necessarily affect filamentation and virulence.¹⁰⁰

Resistance to azole compounds was uncommon until the 1980s except for flucanozole. The widespread use of itraconazole and fluconazole is thought to have been the major cause of azole resistance. The overall drug resistance in Candida sp to fluconazole and voriconazole is considered to be around 3–6%. Also, cross-resistance among Candida sp to multiple antifungal agents is generally uncommon, however it has been demonstrated for many Candida sp including C. glabrata, C. albicans, C. tropicalis and C. parapsilosis.⁹⁵ Resistance mechanisms to azole drugs are also well demonstrated among Aspergillus sp. Triazole resistance in clinical isolates of A. fumigatus has increased up to 6% in the United Kingdom and Netherlands.^100,101

Echinocandin resistance

β(1,3)-Glucan is a key component of the fungal cell wall which is non-competitively inhibited by the echinocandins. Echinocandins destroy fungi by inducing defects in cell wall formation.¹⁰² Echinocandins are the first choice of drug to treat invasive Candida and Aspergillus infections, with low toxicity as their main advantage. The development of echinocandin resistance is reported to be comparatively less,²⁹ however echinocandin resistance was observed after prolonged therapy. Echinocandin resistance is associated with mutation in target site namely, the FKS gene,¹⁰³ particularly in a short conserved region, known as “hot-spot” region of the gene, that encodes the FKS subunit of β(1,3)-glucan synthase.^95,104 In C. albicans mutation takes place in Fks1p at amino acid positions 641 to 649 (hot spot 1) and 1345 to 1365 (hot spot 2).⁹⁵ Other than C. albicans, hot spot mutation were reported in FKS1 for C. glabrata, C. dubliniensis, C. krusei and C. tropicalis.^95,105 Mutations in these hot-spot regions elevate MIC, produce cross resistance among the echinocandins and exhibit reduced β(1,3)-glucan synthase inhibition.^95,105,106 Balashov (2006) observed major changes in the serine 645 position, which was replaced with proline, tyrosine or phenylalanine. All echanocandin resistant isolates showed either a heterozygous (62% exhibits a characteristic S645P substitution) or a homozygous (93% showed changes at Ser645) mutation in FKS1 such that mutant enzyme sensitivity decreases by 1000 fold.^102,107 In C. glabrata also resistance occurs in homologous regions of FKS1 and FKS2. The prominent resistance is conferred by nonsense mutations in either FKS1 or FKS2. Echinocandin resistance in C. glabrata were found to be increased from 2–3% to >13%, with a parallel increase in azole resistance.¹⁰⁸ Elevated MIC and clinical failures were widely observed in non C. albicans species.^26,33,34 However, the echinocandin resistance mechanism in A. fumigatus is still not clear.¹⁰⁹

Adaptive stress response is another potential echinocandin resistance mechanism. The highly dynamic nature of the fungal cell wall allows it to synthesize cell wall components even if any specific cell wall biosynthetic pathway is inhibited.^95,110 Echinocandins induce a set of genes including HOG1, CEK1 and PKC MAP kinase cascade which is involved in the alteration of protein kinase C/Ca²⁺/calcineurin/Crz1, and high osmolarity glycerol, which results in compensatory increases in chitin synthesis.^95,111–113 An increase in chitin synthesis protects against destabilizing environmental stresses created by echinocandin exposure.^108–111 However, echinocandin resistance is uncommon unless other conditions like repeated therapy are undertaken among immunosuppressed patients.²⁶ Moreover, echinocandins and chitin synthase inhibitors like calcineurin inhibitors, FK506 and cyclosporin A have been shown to demonstrate synergism, highlighting the potential for combination therapies with enhanced antifungal activity against A. fumigatus and C. neoformans.¹¹⁵ In addition, few in vitro studies have observed the supra MIC concentration of caspofungin in C. albicans strains. This is known as the paradoxical effect, wherein it synthesizes more chitin at low concentrations of caspofungin.^95,116–118 The details of the therapeutic applications of antifungal agents are discussed in Table 2.

Table 2 General overview about antifungal agents and their target sites, organisms, route and side effects^a

Target site	Drug	Target pathogen	Drug resistance	Drug administration routes	Side effects	FDA approved	References
a — no reports.
β-1,3-Glucan synthase inhibitor	Echinocandins
	Caspofungin	Candida glabrata, Aspergillus sp., Saccharomyces boulardii	Candida albicans, Aspergillus fumigatus	Intravenous	Rash, swelling, and nausea (rare)	2001	117–122
	Anidulafungin	Candida glabrata, Candida albicans, Aspergillis sp., Candida parapsilosis, Coolia tropicalis	Rarely observed in Candida and Aspergillus	Intravenous	Fever; facial erythema; juvenile toxicity (in rat)	2006	117, 123 and 124
	Micafungin	Aspergillis sp., Candida sp.	Rear observed in Cnadida and Aspergillus	Intravenous	—	2005	117, 123 and 125
	Enfumafungin
	SCY-078 (MK-3118)	Candida albicans, C. glabrata, C. parapsilosis	—	Oral	Invasive	2015	36
Inhibitors of cytochrome P450	Imidazole family
	Ketoconazole	Broad spectrum antifungal agent	Cross-resistance	Oral, cream	Hepatotoxicity	2007	126 and 127
	Clotrimazole	Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus	Candida guilliermondi	Oral, cream	Erythema, stinging, blistering, peeling, edema, pruritus, urticaria, burning, and general irritation of the skin, and cramps	2009	128
	Miconazole	Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus	C. albicans	Ointment and cream	General irritation of the skin	2009	128 and 129
Inhibitors of cytochrome P450	Triazole family
	Itraconazole	Cryptococcus neoformans, Candida spp	Aspergillus fumigatus	Oral	No major side effects observed	2001	114, 130 and 131
	Posaconazole	Cryptococcus neoformans, Candida glabrata, Candida tropicalis	Aspergillus fumigatus	Oral, intravenous	No major side effects observed	2006	132 and 133
	Fluconazole	Cryptococcus neoformans, Candida spp	Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus	Oral, intravenous, topical	Overdose include hallucinations and paranoid behavior	1990	131 and 134
	Voriconazole	Aspergillus fumigatus, Cryptococcus neoformans	Cross-resistance	Oral, intravenous	Mydriasis, titubation (loss of balance while moving), depressed behavior, prostration, partially closed eyes, and dyspnea	2003	135 and 136
	Isavuconazole	Candida, Aspergillus, Cryptococcus	Cross-resistance	Oral, intravenous	Should be avoided in breastfeeding women	2014	137
Inhibitors of carbonic anhydratase	Sulfonamides	Aspergillis sp., Candida sp.	Saccharomyces cerevisiae	Topical	Itching, burning, skin rash, redness, swelling and may cause cancer of the thyroid gland	1973	138 and 139
	Methazolamide	Aspergillis sp., Candida sp.	—	Oral	Electrolyte imbalance, development of an acidotic state	1959	138 and 140
	Brinzolamide	Fusarium sp., Aspergillis sp., Candida sp.	—	Ophthalmic	—	1998	141 and 142
	Acetazolamide	Aspergillis sp., Candida sp.	—	Oral, intravenous	—	1953	138 and 140
	Ethoxzolamide	Aspergillis sp., Candida sp.	—	—	—	Withdrawn	138
Inhibitors of chitin synthesis	Polyoxin B	Cryptococcus neoformans, Candida albicans, Aspergillus flavus, Saccharomyces cerevisiae, Blumeria graminis f. sp.	Botrytis cinerea, Candida albicans	In vitro only and agricultural fungicides	—	In progress	143
	Nikkomycin Z	Candida albicans, other Candida spp., Aspergillus spp., Cryptococcus neoformans, Coccidioides immitis	—	In vitro only and agricultural fungicides	—	In progress	144

---

Biofilm formation

Biofilm formation is another most important contributing factor in drug resistance. It is an emerging global problem of great concern. The three main drug resistance mechanisms proposed in biofilms are

I. Incomplete or slow penetration of antimicrobials due to exopolysachharide polymers.

II. Resistant phenotype, known as persister cells characterized by a slower growth rate and greater resistance to antimicrobials.

III. An altered environment that may affect the antibiotic action, such that fungal biofilm persists even when appropriate antifungal treatment is given.¹⁴⁶

Candida biofilms are the leading cause for intravascular catheter-related infections. It makes the treatment of catheter related-blood stream infections difficult as it resists the action of clinically important drugs, including amphotericine B and azole.^111,146 A recent study suggests that the combinations of miconazole with artesunate or other artemisinins could be an effective therapeutic strategy to treat C. albicans biofilm-related infections.¹⁴⁷ Also, the concept of using antibiofilm agent has identified as a promising method to treat biofilm related infections. However, no specific drugs are available today for the treatment of any biofilm-based microbial infection. Studies have shown that Aspergillus biofilms exhibit antifungal tolerance by an increase in efflux pump activity and the extrapolysachharide matrix which confer resistance to itraconazole and, to some extent, to caspofungin.² C. neoformans biofilms are becoming common due to the increased use of medical devices such as ventriculoatrial shunt catheters, cardiac valves, and prosthetic joints. Exo polysachharide matrix and melanin confers the resistance to drugs. However, the knowledge of mechanism of biofilm formation by Cryptococcus is still in infancy and there are very limited reports on the evaluation of antifungals on C. neoformans biofilms. An in vitro study reveals that amphotericin B and caspofungin are effective against the mature biofilms of C. neoformans; however the efficacy profiles are not similar for melanized Cryptococcus biofilms¹⁴⁸ correlating the production of melanin with drug resistance in C. neoformans. Summing up, the development of drug resistance in fungi is slow and the frequency is relatively low, with the exception of certain Candida spp. But there is a possibility of an increase in drug resistance in other fungi groups as well.

Conclusion

It is not surprising that enzymes are prominent drug targets, as they are well known for their biological activities in fungi. The well known antifungal agents like echinocandins and azole drugs are the potential inhibitors of β-1,3-glucan synthase and lanosterol 14-α-demethylase respectively. The availability of limited antifungal drugs has made azole drugs vital for therapeutic use for more than two decades. The increased use of triazoles has led to the emergence of drug-resistant Candida sp and Aspergillus sp. Hence, exploring antifungal agents with new drug targets is a long term goal for many research groups. Enzymes are essential for all life, thus selective inhibition of critical enzymes are the major focus for many researchers to combat infectious diseases. Inhibitors of carbonic anhydratase, GlcN-6-P, and amino acid synthesis are identified as commanding drug targets. However CA inhibitors are shown to have low potency and selectivity against fungi. Hence, the search for a new class of β-CA inhibitors with high specificity in antifungal action is currently being investigated by many research groups. GlcN-6-P inhibitors are as well studied by many researchers as they cause no lethal effects in humans. Similarly amino acid synthesis pathways are also exploited as suitable drug targets. Enzymes are considered as the potential drug targets not only because of their significant function, but also due to their ability to be inhibited by small molecules. Summing up, the exploration of new targets and new enzyme inhibitors is certainly a promising avenue of drug development for emerging and re-emerging fungal infections of humans.

Acknowledgements

We thank the Science and Engineering Research Board (SERB), Department of Science and Technology, New Delhi, for support under the Fast Track Young Scientist Scheme (No: SB/FT/LS-249/2012) to JR and (SERB/F/1266/2012-13) to TR. We also sincerely thank the SASTRA University and its management for providing us the infrastructure needed to carry out our research work.

References

1. J. Riddell and E. K. Shuman, Neuroimaging Clin., 2012, 22, 543–556.
2. P. Badiee and Z. Hashemizadeh, Indian J. Med. Res., 2014, 139, 195–204.
3. M. Wojciechowski, S. Milewski, J. Mazerski and E. Borowski, Acta Biochim. Pol., 2005, 52, 647–653.
4. E. Paramythiotou, F. Frantzeskaki, A. Flevari, A. Armaganidis and G. Dimopoulos, Molecules, 2014, 19, 1085–1119.
5. P. Badiee and Z. Hashemizadeh, Indian J. Med. Res., 2014, 139, 195–204.
6. C. D. C. Atlanta, Centres Dis. Control Prev., 2013, pp. 1–113.
7. A. A. Cleveland, L. H. Harrison, M. M. Farley, R. Hollick, B. Stein, T. M. Chiller, S. R. Lockhart and B. J. Park, PLoS One, 2015, 10, 2008–2013.
8. J. S. Kim, P. Holtom and C. Vigen, Am. J. Infect. Control, 2011, 39, 640–646.
9. V. Krcmery and A. J. Barnes, J. Hosp. Infect., 2002, 50, 243–260.
10. C. K. Muzoora, T. Kabanda, G. Ortu, J. Ssentamu, P. Hearn, J. Mwesigye, N. Longley, J. N. Jarvis, S. Jaffar and T. S. Harrison, J. Infect., 2012, 64, 76–81.
11. P. G. Pappas, Trans. Am. Clin. Climatol. Assoc., 2013, 124, 61–79.
12. S. S. Rathore, T. Raman and J. Ramakrishnan, Front. Microbiol., 2016, 7, 1–16.
13. S. B. Ascher, P. Brian Smith, K. Watt, D. K. Benjamin, M. Cohen-wolkowiez, R. H. Clark, D. K. Benjamin Jr and C. Moran, Pediatr. Infect. Dis. J., 2012, 31, 439–443.
14. J. María Aguado, L. Vázquez, M. Fernández-Ruiz, T. Villaescusa, I. Ruiz-Camps, P. Barba, J. T. Silva, M. Batlle, C. Solano, D. Gallardo and I. Heras, Clin. Infect. Dis. Adv., 2014, 1–15.
15. F. Barchiesi, L. F. Di Francesco, P. Compagnucci, D. Arzeni, A. Giacometti and G. Scalise, J. Antimicrob. Chemother., 1998, 41, 59–65.
16. T. Ogawa, K. Matsumoto, K. Tsujimoto, N. Hishiya, Y. Yamada, K. Uno, K. Kasahara, K. Maeda, K. Nario, K. Mikasa and K. Morita, J. Infect. Chemother., 2015, 21, 134–137.
17. G. P. Bodey, M. Mardani, H. A. Hanna, M. Boktour, J. Abbas, E. Girgawy, R. Y. Hachem, D. P. Kontoyiannis and I. I. Raad, Am. J. Med., 2002, 112, 380–385.
18. J. P. Donnelly, Clin. Infect. Dis., 2006, 42, 487–489.
19. D. L. Horn and M. I. Morris, Eur. J. Clin. Microbiol. Infect. Dis., 2010, 29, 223–229.
20. J. W. Baddley, D. R. Andes, K. A. Marr, D. P. Kontoyiannis, B. D. Alexander, C. A. Kauffman, R. A. Oster, E. J. Anaissie, T. J. Walsh, M. G. Schuster, J. R. Wingard, T. F. Patterson, J. I. Ito, O. D. Williams, T. Chiller and P. G. Pappas, Clin. Infect. Dis., 2010, 50, 1559–1567.
21. B. I. Aderiye and O. A. Oluwole, J. Agric. Biol. Sci., 2015, 1, 206–216.
22. F. M. Klis, P. De Groot and K. Hellingwerf, Med. Mycol., 2001, 39, 1–8.
23. F. M. Klis, P. Mol, K. Hellingwerf and S. Brul, FEMS Microbiol. Rev., 2002, 26, 239–256.
24. S. S. Walker, Y. Xu, I. Triantafyllou, M. F. Waldman, C. Mendrick, N. Brown, P. Mann, A. Chau, R. Patel, N. Bauman, C. Norris, B. Antonacci, M. Gurnani, A. Cacciapuoti, P. M. McNicholas, S. Wainhaus, R. J. Herr, R. Kuang, R. G. Aslanian, P. C. Ting and T. A. Black, Antimicrob. Agents Chemother., 2011, 55, 5099–5106.
25. E. Levesque, S. El Anbassi, E. Sitterle, F. Foulet, J. C. Merle and F. Botterel, J. Clin. Microbiol., 2015, 53, 771–776.
26. D. S. Perlin, Ann. N. Y. Acad. Sci., 2015, 1354, 1–11.
27. F. Meunler, C. Lambert and P. Van Der Auwera, J. Antimicrob. Chemother., 1989, 24, 325–331.
28. M. Pfaller, J. Riley and T. Koerner, Eur. J. Clin. Microbiol. Infect. Dis., 1989, 8, 1067–1070.
29. K. Bartizal, C. J. Gill, G. K. Abruzzo, A. M. Flattery, L. Kong, P. M. Scott, J. G. Smith, C. E. Leighton, A. Bouffard, J. F. Dropinski, K. E. N. Bartizal, C. J. Gill, G. K. Abruzzo, A. M. Y. M. Flattery, L. I. Kong, P. M. Scott, J. G. Smith, C. E. Leighton, A. Bouffard, J. F. Dropinski and J. Balkovec, Antimicrob. Agents Chemother., 1997, 0991, 2326–2332.
30. F. Carta, A. Innocenti, R. A. Hall, F. A. Muhlschlegel, A. Scozzafava and C. T. Supuran, Bioorg. Med. Chem. Lett., 2011, 21, 2521–2526.
31. S. P. Bachmann, T. F. Patterson and J. L. Lopez-Ribot, J. Clin. Microbiol., 2002, 40, 2228–2230.
32. K. Niimi, K. Maki, F. Ikeda, A. R. Holmes, E. Lamping, M. Niimi, B. C. Monk and R. D. Cannon, Antimicrob. Agents Chemother., 2006, 50, 1148–1155.
33. G. Garcia-Effron, S. Lee, S. Park, J. D. Cleary and D. S. Perlin, Antimicrob. Agents Chemother., 2009, 53, 3690–3699.
34. G. Garcia-Effron, S. Park and D. S. Perlin, Antimicrob. Agents Chemother., 2009, 53, 112–122.
35. H. Morikawa, M. Tomishima, N. Kayakiri, T. Araki, D. Barrett, S. Akamatsu, S. Matsumoto, S. Uchida, T. Nakai, S. Takeda and K. Maki, Bioorg. Med. Chem. Lett., 2014, 24, 1172–1175.
36. J. Onishi, M. Meinz, J. Thompson, J. Curotto, S. Dreikorn, M. Rosenbach, C. Douglas, G. Abruzzo, A. Flattery, L. Kong, A. Cabello, F. Vicente, F. Pelaez, M. T. Diez, I. Martin, G. Bills, R. Giacobbe, A. Dombrowski, R. Schwartz, S. Morris, G. Harris, A. Tsipouras, K. Wilson and M. B. Kurtz, Antimicrob. Agents Chemother., 2000, 44, 368–377.
37. A. J. Lepak, K. Marchillo and D. R. Andes, Antimicrob. Agents Chemother., 2015, 59, 1265–1272.
38. I. M. Martins, J. C. G. Cortes, J. Munoz, M. B. Moreno, M. Ramos, J. A. Clemente-Ramos, A. Duran and J. C. Ribas, J. Biol. Chem., 2011, 286, 3484–3496.
39. C. A. Munro, S. Selvaggini, I. De Bruijn, L. Walker, M. D. Lenardon, B. Gerssen, S. Milne, A. J. P. Brown and N. A. R. Gow, Mol. Microbiol., 2007, 63, 1399–1413.
40. N. H. Georgopapadakou and J. S. Tkacz, Trends Microbiol., 1995, 3, 98–104.
41. R. Surarit, P. K. Gopal and M. G. Shepherd, J. Gen. Microbiol., 1988, 134, 1723–1730.
42. J. A. Shaw, P. C. Mol, B. Bowers, S. J. Silverman, M. H. Valdivieso, A. Durdn and E. Cabib, J. Cell Biol., 1991, 114, 111–123.
43. R. Kollár, B. B. Reinhold, E. Petra, H. J. C. Yeh, G. Ashwell, J. C. Kapteyn, F. M. Klis and E. Cabib, J. Biol. Chem., 1997, 272, 17762–17775.
44. R. Kollár, E. Petráková, G. Ashwell, P. W. Robbins and E. Cabib, J. Biol. Chem., 1995, 270, 1170–1178.
45. A. Ray and S. Anand, Indian J. Chest Dis., 2000, 42, 357–366.
46. R. K. Li and M. G. Rinaldi, Antimicrob. Agents Chemother., 1999, 43, 1401–1405.
47. M. Hori, J. Eguchi, K. Kakiki and T. Misato, J. Antibiot., 1974, XXVII, 260–266.
48. P. D. M. H. Liver, Y. Sekigawa, M. Fukuhara, Y. Sonoda and Y. Sato, Lipids, 1995, 30, 1067–1073.
49. S. V. Sambasivarao, Biochim. Biophys. Acta, 2013, 18, 1199–1216.
50. A. G. Warrilow, J. E. Parker, D. E. Kelly and S. L. Kelly, Antimicrob. Agents Chemother., 2013, 57, 1352–1360.
51. M. Glynn, W. Jo, K. Minowa, H. Sanada, H. Nejishima, H. Matsuuchi, H. Okamura, R. Pillai and L. Mutter, Reprod. Toxicol., 2015, 52, 18–25.
52. D. J. Sheehan, C. A. Hitchcock and M. Carol, Clin. Microbiol. Rev., 1999, 12, 40–79.
53. Y. Koltin and C. A. Hitchcock, Curr. Opin. Chem. Biol., 1997, 1, 176–182.
54. E. Mellado, M. Cuenca-Estrella and J. L. Rodriguez-Tudela, Enfermedades Infecciosas Microbiología Clínica, 2002, 20, 523–529.
55. H. C. Neu and T. D. Gootz, Medical Microbiology, 4th edn, 1996, Ch. 11.
56. J. Li, C. Nguyen and J. Garcia-Diaz, J. Fungi, 2015, 1, 345–366.
57. P. L. McCormack, Drugs, 2015, 75, 817–822.
58. J. M. Kingsbury, Z. Yang, T. M. Ganous, G. M. Cox and J. H. McCusker, Microbiology, 2004, 150, 1547–1558.
59. G. De Pascale, E. J. Griffiths, T. Shakya, I. Nazi and G. D. Wright, ChemBioChem, 2011, 12, 1179–1182.
60. K. Jastrzebowska and I. Gabriel, Amino Acids, 2015, 47, 227–249.
61. Y. Aoki, M. Kondoh, M. Nakamura, T. Fujii, T. Yamazaki, H. Shimada and M. Arisawa, J. Antibiot., 1994, 47, 909–916.
62. I. Nazi, A. Scott, A. Sham, L. Rossi, P. R. Williamson, J. W. Kronstad and G. D. Wright, Antimicrob. Agents Chemother., 2007, 51, 1731–1736.
63. J. Loeffler and D. A. Stevens, Clin. Infect. Dis., 2003, 36, 31–41.
64. J. D. S. Fernandes, K. Martho, V. Tofik, M. A. Vallim and R. C. Pascon, PLoS One, 2015, 10, 1–22.
65. K. J. Motil, A. R. Opekãoen, C. M. Montandon, H. K. Berthold, T. A. Davis, P. D. Klein and P. J. Reeds, J. Nutr., 1994, 124, 41–51.
66. M. Pouliot and S. Jeanmart, J. Med. Chem., 2016, 59, 497–503.
67. C. T. Supuran, Nat. Rev. Drug Discovery, 2008, 7, 168–181.
68. S. Milewski, Biochim. Biophys. Acta, 2002, 1597, 173–192.
69. G. L. McKnight, S. L. Mudri, S. L. Mathewes, R. R. Traxinger, S. Marshall, P. O. Sheppard and P. J. O’Hara, J. Biol. Chem., 1992, 267, 25208–25212.
70. E. Borowski, Farm., 2000, 55, 206–208.
71. R. Jedrzejczak, D. Zgodka, S. Milewski and E. Borowski, Bioorg. Med. Chem., 2001, 9, 931–938.
72. D. Zgódka, S. Milewski and E. Borowski, Microbiology, 2001, 147, 1955–1959.
73. S. Milewski, F. Mignini, L. Micossi and E. Borowski, Med. Mycol., 2008, 36, 177–180.
74. J. Nowak-Jary and R. Andruszkiewicz, Pol. J. Microbiol., 2009, 58, 295–299.
75. T. Tanida, F. Rao, T. Hamada, E. Ueta and T. Osaki, Infect. Immun., 2001, 69, 3883–3890.
76. D. Pawlak, M. Stolarska, M. Wojciechowski and R. Andruszkiewicz, Eur. J. Med. Chem., 2015, 90, 577–582.
77. G. Zong, H. Zhao, R. Jiang, J. Zhang, X. Liang, B. Li, Y. Shi and D. Wang, Molecules, 2014, 19, 7832–7849.
78. S. Bawa, R. Ali, S. Kumar and O. Afzal, Drug Dev. Ther., 2015, 6, 79.
79. C. T. Supuran, J. Enzyme Inhib. Med. Chem., 2015, 27, 759–772.
80. F. Carta, C. Temperini, A. Innocenti, A. Scozzafava, K. Kaila and C. T. Supuran, J. Med. Chem., 2010, 53, 5511–5522.
81. S. Elleuche and S. Pöggeler, Microbiology, 2010, 156, 23–29.
82. J. M. Tobal and M. E. D. S. F. Balieiro, J. Med. Microbiol., 2013, 63, 15–27.
83. Y. S. Bahn, G. M. Cox, J. R. Perfect and J. Heitman, Curr. Biol., 2005, 15, 2013–2020.
84. F. L. Mayer, D. Wilson and B. Hube, Virulence, 2013, 4, 119–128.
85. R. A. Hall, L. de Sordi, D. M. MacCallum, H. Topal, R. Eaton, J. W. Bloor, G. K. Robinson, L. R. Levin, J. Buck, Y. Wang, N. A. R. Gow, C. Steegborn and F. A. Muhlschlegel, PLoS Pathog., 2010, 6, 1–11.
86. A. Innocenti, J. Winum, R. A. Hall, F. A. Mühlschlegel, A. Scozzafava and C. T. Supuran, Bioorg. Med. Chem. Lett., 2009, 19, 2642–2645.
87. C. Schlicker, R. A. Hall, D. Vullo, S. Middelhaufe, M. Gertz, C. T. Supuran, F. A. Mühlschlegel and C. Steegborn, J. Mol. Biol., 2009, 385, 1207–1220.
88. M. P. Toraskar, ACS Med. Chem. Lett., 2014, 5, 8–11.
89. O. Guzel, A. Maresca, A. Scozzafava, A. Salman, A. T. Balaban and C. T. Supuran, Bioorg. Med. Chem. Lett., 2009, 19, 2931–2934.
90. M. I. Hassan, B. Shajee, A. Waheed, F. Ahmad and W. S. Sly, Bioorg. Med. Chem., 2013, 21, 1570–1582.
91. C. T. Supuran, Future Med. Chem., 2016, 8, 311–324.
92. A. Innocenti, R. A. Hall, C. Schlicker, A. Scozzafava, C. Steegborn, F. A. Mühlschlegel and C. T. Supuran, Bioorg. Med. Chem., 2009, 17, 4503–4509.
93. D. Vullo, W. Leewattanapasuk, F. A. Mühlschlegel, A. Mastrolorenzo, C. Capasso and C. T. Supuran, Bioorg. Med. Chem. Lett., 2013, 23, 2647–2652.
94. Z. A. Kanafani and J. R. Perfect, Clin. Infect. Dis., 2008, 46, 120–128.
95. M. Sanguinetti, B. Posteraro and C. Lass-Florl, Mycoses, 2015, 58, 2–13.
96. K. K. Lee, D. M. MacCallum, M. D. Jacobsen, L. A. Walker, F. C. Odds, N. A. R. Gow and C. A. Munro, Antimicrob. Agents Chemother., 2012, 56, 208–217.
97. F. A. Drobniewski, Y. Balabanova, L. Heifets and G. Cangelosi, Antimicrob. Drug Resist., 2009, 901–915.
98. L. A. Vale-Silva, A. T. Coste, F. Ischer, J. E. Parker, S. L. Kelly, E. Pinto and D. Sanglard, Antimicrob. Agents Chemother., 2012, 56, 1960–1968.
99. R. S. Shapiro, N. Robbins and L. E. Cowen, Microbiol. Mol. Biol. Rev., 2011, 75, 213–267.
100. M.-J. Xiang, J.-Y. Liu, P.-H. Ni, S. Wang, C. Shi, B. Wei, Y.-X. Ni and H.-L. Ge, FEMS Yeast Res., 2013, 13, 386–393.
101. B. D. Esquivel, A. R. Smith, M. Zavrel and T. C. White, Antimicrob. Agents Chemother., 2015, 59, 3390–3398.
102. M. A. Pfaller, Am. J. Med., 2012, 125, S3–S13.
103. S. V. Balashov, S. Park and D. S. Perlin, Antimicrob. Agents Chemother., 2006, 50, 2058–2063.
104. N. D. Beyda, R. E. Lewis and K. W. Garey, Ann. Pharmacother., 2012, 46, 1086–1096.
105. G. Garcia-Effron, D. P. Kontoyiannis, R. E. Lewis and D. S. Perlin, Antimicrob. Agents Chemother., 2008, 52, 4181–4183.
106. S. Park, R. Kelly, J. N. Kahn, J. Robles, M. J. Hsu, E. Register, W. Li, V. Vyas, H. Fan, G. Abruzzo, A. Flattery, C. Gill, G. Chrebet, S. A. Parent, M. Kurtz, H. Teppler, C. M. Douglas and D. S. Perlin, Antimicrob. Agents Chemother., 2005, 49, 3264–3273.
107. D. S. Perlin, BMC Public Health, 2009, 10, 121–130.
108. D. S. Perlin, Drugs, 2014, 74, 1573–1585.
109. S. J. Howard and M. C. Arendrup, Med. Mycol., 2011, 49(1), S90–S95.
110. D. S. Perlin, Ann. N. Y. Acad. Sci., 2015, 1354, 1–11.
111. L. A. Walker, N. A. R. Gow and C. A. Munro, Fungal Genet. Biol., 2010, 47, 117–126.
112. L. A. Walker, C. A. Munro, I. De Bruijn, M. D. Lenardon, A. McKinnon and N. A. R. Gow, PLoS Pathog., 2008, 4, 1–12.
113. N. A. R. Gow, M. G. Netea, C. A. Munro, G. Ferwerda, S. Bates, H. M. Mora-Montes, L. Walker, T. Jansen, L. Jacobs, V. Tsoni, G. D. Brown, F. C. Odds, J. W. M. Van der Meer, A. J. P. Brown and B. J. Kullberg, J. Infect. Dis., 2007, 196, 1565–1571.
114. L. Xiao, V. Madison, A. S. Chau, D. Loebenberg, R. E. Palermo and P. M. McNicholas, Antimicrob. Agents Chemother., 2004, 48, 568–574.
115. M. Del Poeta, M. C. Cruz, M. E. Cardenas, J. R. Perfect and J. Heitman, Antimicrob. Agents Chemother., 2000, 44, 739–746.
116. N. P. Wiederhold, D. P. Kontoyiannis, R. A. Prince and R. E. Lewis, Antimicrob. Agents Chemother., 2005, 49, 5146–5148.
117. D. a. Stevens, M. Espiritu and R. Parmar, Antimicrob. Agents Chemother., 2004, 48, 3407–3411.
118. R. K. Shields, M. H. Nguyen, C. Du, E. Press, S. Cheng and C. J. Clancy, Antimicrob. Agents Chemother., 2011, 55, 2641–2647.
119. G. Eschenauer, D. D. Depestel and P. L. Carver, Ther. Clin. Risk Manage., 2007, 3, 71–97.
120. R. Herbrecht, J. Maertens, L. Baila, M. Aoun, W. Heinz, R. Martino, S. Schwartz, A. J. Ullmann, L. Meert, M. Paesmans, O. Marchetti, H. Akan, L. Ameye, M. Shivaprakash and C. Viscoli, Bone Marrow Transplant., 2010, 45, 1227–1233.
121. N. Lolis, D. Veldekis, H. Moraitou, S. Kanavaki, A. Velegraki, C. Triandafyllidis, C. Tasioudis, A. Pefanis and I. Pneumatikos, Crit. Care, 2008, 12, 414.
122. E. Spreghini, F. Orlando, M. Sanguinetti, B. Posteraro, D. Giannini, E. Manso and F. Barchiesia, Antimicrob. Agents Chemother., 2012, 56, 1215–1222.
123. U. S. F. and D. Administration, 2001.
124. N. D. Grover, Indian J. Pharmacol., 2010, 42, 422.
125. U. S. F. and D. Administration, 2006.
126. U. S. F. and D. Administration, 2005.
127. C. Costa, C. Pires, T. R. Cabrito, A. Renaudin, M. Ohno, H. Chibana, I. Sá-Correia and M. C. Teixeira, Antimicrob. Agents Chemother., 2013, 57, 3159–3167.
128. M. D. Johnson and J. R. Perfect, Curr. Fungal Infect. Rep., 2010, 4, 87–95.
129. R. Ross, M. Lee and A. Onderdonk, Obstet. Gynecol., 1995, 86, 925–930.
130. M. Levy, I. Polacheck, Y. Barenholz and S. Benita, Pharm. Res., 1995, 12, 223–230.
131. D. Denning and K. Venkateswarlu, Antimicrob. Agents Chemother., 1997, 41, 1364–1368.
132. M. Martin, J. Antimicrob. Chemother., 1999, 44, 429–437.
133. V. Flores, Curr. HIV Res., 2012, 10, 620–623.
134. S. Kuipers, R. J. M. Brüggemann, R. G. L. de Sévaux, J. P. F. A. Heesakkers, W. J. G. Melchers, J. W. Mouton and P. E. Verweij, Antimicrob. Agents, 2011, 55, 3564–3566.
135. M. S. Saag, R. J. Graybill, R. A. Larsen, P. G. Pappas, J. R. Perfect, W. G. Powderly, J. D. Sobel and W. E. Dismukes, Clin. Infect. Dis., 2000, 30, 710–718.
136. K. Ogaki, K. Noda, J. Fukae, T. Furuya, T. Hirayama, K. Fujishima, N. Hattori and Y. Okuma, Brain Nerve, 2010, 62, 1337–1340.
137. N. Greer, Proc. (Bayl. Univ. Med. Cent)., 2003, 16, 241.
138. D. Falci and A. Pasqualotto, Infect. Drug Resist., 2013, 6, 163.
139. A. Innocenti, R. A. Hall, C. Schlicker, A. Scozzafava, C. Steegborn, F. A. Mühlschlegel and C. T. Supuran, Bioorg. Med. Chem., 2009, 17, 4503–4509.
140. Z. H. Chohan, Mahmood-Ul-Hassan, K. M. Khan and C. T. Supuran, J. Enzyme Inhib. Med. Chem., 2005, 20, 183–188.
141. C. Brugnara, L. de Franceschi and S. L. Alper, J. Clin. Invest., 1993, 92, 520–526.
142. C. T. Supuran, J. Y. Winum and B. Wang, Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications, John Wiley & Sons, 2009.
143. G. Gece, Corros. Sci., 2011, 53, 3873–3898.
144. K. Isono, K. Asahi and S. Suzuki, J. Am. Chem. Soc., 1969, 91, 7490–7505.
145. R. Li and M. Rinaldi, Antimicrob. Agents Chemother., 1999, 43, 1401–1405.
146. A. Chatzimoschou, A. Katragkou, M. Simitsopoulou, C. Antachopoulos, E. Georgiadou, T. J. Walsh and E. Roilides, Antimicrob. Agents Chemother., 2011, 55, 1968–1974.
147. K. De Cremer, E. Lanckacker, T. L. Cools, M. Bax, K. De Brucker, P. Cos, B. P. A. Cammue and K. Thevissen, Antimicrob. Agents Chemother., 2015, 59, 421–426.
148. Z. Bin Liao, X. Z. ZhangGuan, Z. Y. Zhu, X. W. Yao, Y. Yang, Y. Y. Jiang and Y. Y. Cao, Int. J. Antimicrob. Agents, 2014, 46, 45–52.

---