Antifungal Drugs and Associated Problems
The current arsenal of antifungal compounds used for the treatment of aspergillosis consists of three classes of antifungal agents, two of them targeting ergosterol, the functional fungal analogue of cholesterol and the major component specific to fungal membranes, and a third class that targets the synthesis of β1,3 glucan, the major component of the fungal cell wall (
610–614) (
Fig. 20). Amphotericin B (AMB), a polyene class of antifungal drug, irreversibly binds to ergosterol, resulting in fungal cell death. Binding of AMB to sterols causes membrane leakage, the proposed mechanism leading to cell death (
615). Detailed structural and biophysical studies have demonstrated that polyenes act like an ergosterol sponge, forming large, extramembranous aggregates that extract the essential membrane-lipid ergosterol from the plasma membrane. It also has been proposed that AMB has an oxidative killing mechanism of action (
616). A main advantage of AMB as an anti-
A. fumigatus drug is the absence of resistance emergence, due in part to its cidal activity (
617). Less toxic, lipid-associated drugs have been formulated. Amphotericin B lipid complex (ABLC) consists of amphotericin B in a complex with two lipids:
l-α-dimyristoyl phosphatidylcholine and
l-α-dimyristoyl phosphatidylglycerol (
618). The large molecular size of the compound results in rapid uptake of the drug by macrophages. The other formulation is liposomal amphotericin B (L-AmB), which is composed of a small, unilamellar vesicle consisting of hydrogenated soy phosphatidylcholine, cholesterol, and distearoyl phosphatidylglycerol (
619). These compounds have comparable activity, are far less toxic, and exhibit pharmacokinetics equal to that of the mother compound.
The synthesis of ergosterol begins with acetyl-coenzyme A (CoA) and involves 20 steps that have been subjected to multiple reviews (
620,
621). The antimycotic azoles have a five-membered nitrogen-containing heterocyclic moiety, which binds to an iron atom in the heme group located in the active site of the lanosterol 14-α-demethylase encoded by
CYP51A and
CYP51B. Consequently, azoles block the demethylation of lanosterol, resulting in ergosterol depletion and the accumulation of a toxic sterol produced by Erg3. This toxic sterol exerts a severe membrane stress on the cell. The fungicidal activity is the result of a compensatory cell wall stress pathway following ergosterol depletion that results in carbohydrate patch formation with subsequent penetration of plasma membrane and fungal killing (
622). Because of favorable bioavailability, pharmacokinetics, and lack of host toxicity, triazoles (primarily voriconazole and posaconazole) have become the primary choice of treatment for IA (
25). The azoles have a drawback, since they also inhibit cytochrome P450 (CYP 450) enzymes that are responsible for the metabolism of various chemicals, including numerous other drugs. In addition, they serve as substrates of the CYP 450 enzymes; therefore, drugs that inhibit or induce the activity of these enzymes also can lead to clinically significant changes in azole concentrations. To overcome the problem of drug-drug interactions associated with the azoles, attempts have been undertaken to replace the triazole metal-binding group with a tetrazole.
The echinocandins are the only other class of antifungals to reach the clinic in decades. Three echinocandins are currently available for clinical use: caspofungin, micafungin, and anidulafungin. These compounds are cyclic hexapeptides that act as noncompetitive inhibitors of β1,3 glucan synthase, a key enzyme for cell wall synthesis. The safety profile of these compounds is impressive and attributable to a fungus-specific target that is not conserved in mammals. However, a precise biochemical mode of action of echinocandins and their membrane target(s) remain elusive. Moreover, these drugs, which are active at 0.5 μg/ml, show a paradoxical effect
in vitro; higher concentrations (>4 μg/ml) result in incomplete growth inhibition against
A. fumigatus. This paradoxical growth also seems to exist
in vivo (
623–625). The slow-growth phenotype resulting from high-dose exposure is characterized by hyperbranching, occasional lysis of hyphal apical compartments, translocation of the β1,3 glucan synthase to vacuoles, disappearance of cell wall β1,3 glucan, and compensatory increase of cell wall chitin. After approximately 2 to 3 days, paradoxically growing hyphae emerge from the β1,3 glucan-depleted and growth-inhibited microcolonies. These paradoxically growing hyphae are characterized by fast growth, normal morphology, renewed localization of the β1,3 glucan synthase to the hyphal tips, reconstitution of β1,3 glucan synthesis, and normalization of the cell wall chitin levels (
625). In addition to solubility problems of this drug at high concentration, the stability of the echinocandin concentration or the presence of a high concentration of serum (50%), which may play a role in this paradoxical effect (
626), the genetic and molecular mechanisms responsible for this phenomenon remain speculative. The MpkA-RlmA-CWI pathway has been shown to be involved in the control of paradoxical growth (
627). The echinocandin-induced increase in cell wall chitin is thought to at least partly mediate this paradoxical growth. Moreover, calcineurin also plays an important role in this paradoxical growth, since
cnaA and
crzA deletion mutants showed increased caspofungin susceptibility and no paradoxical growth (
628). The TF ZipD, responding to Ca
2+/calcineurin signaling, has been shown to be involved in paradoxical growth via regulation of chitin biosynthesis genes (
627). Inhibition of Hsp90 function (involved in
A. fumigatus calcineurin-dependent stress response to echinocandins), which results in increased caspofungin susceptibility, also abolishes paradoxical growth (
629). In spite of the association between paradoxical growth and many fungal proteins and pathways, paradoxical growth remains inadequately understood.
The CLSI (Clinical & Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) methods allow the determination of MICs and can identify strains resistant to drugs. To date, no resistance problems associated with echinocandins and amphotericin B have been identified in clinics. However, echinocandin-resistant mutants can be induced easily
in vitro (
630,
631), and hot spots for point mutations inducing the resistance to amphotericin were identified in
A. fumigatus after use of natamycin (
632–634) and are starting to be seen in laboratories (
635,
636). However, resistance to AMB is only anecdotal. In contrast, resistance to azoles has been reported with increased frequency (
538,
637,
638). Importantly, there was not an absolute association between
in vitro MIC and clinical response (
681).
Azole resistance in
A. fumigatus reflects an increase in drug use for prophylactic and long-term treatment regimens, and acquired azole resistance was reported in patients who received long-term treatment with azoles (
528,
538,
639–641). Although resistance to azoles has become an important concern, global surveillance studies reveal 3.2% of
A. fumigatus isolates are resistant to one or more azoles, with large discrepancies between regions, from 25% to less than 1% in the multicenter epidemiological SCARE study with 22 centers from 19 countries (
637,
642–644). The azole target is the 14-α sterol demethylase encoded by cyp51A and -B genes in
A. fumigatus (
645), and the main mechanisms of resistance involve mutations in the
CYP51A gene (
646–651). A common mechanism of azole resistance found in environmental and clinical isolates of
A. fumigatus now involves modification of
CYP51A and its promoter (TR34/Leu98His; TR46/Tyr121Phe/Thr289Ala). Complex regulation is expected, since TR34 was bound by both the sterol-regulatory binding protein SrbA and the CCAAT binding complex (
652). Other proven mechanisms of resistance in
A. fumigatus are Cyp51A amino acid substitutions at Gly54, Gly138, Met220, and Gly448. A 53-bp tandem repeat in the promoter region of
A. fumigatus has also been reported, as well as a triple mutation in the
CYP51A gene (
653,
654). Indeed, the heterogeneity of the
A. fumigatus population has been linked to azole resistance by genome sequencing, leading to 4 to 7 different clusters, depending on the methodology used (
655,
656).
The insertion of repeated sequences in the promoter region was speculated to be related to the extensive use of fungicides in agriculture (
657). Azoles are the most widely used class of agricultural fungicides, and it is unlikely that the pesticide industry will cease marketing fungicides; these chemicals provide protection to plants against phytopathogenic fungi and are a key tool in world food production (
658,
659). Moreover, the role of these azoles used in agriculture as the source for emergence of the azole-resistant strains has not been demonstrated conclusively (
660). For example, in agriculture, the worldwide emergence of azole-resistant phytopathogens occurred 2 to 6 years after commercial application of these fungicides. However, this was not the case with
A. fumigatus. Similarly, the cereal pathogen
A. flavus, a producer of foodborne mycotoxins that has been exposed yearly to fungicide applications, has no reported azole resistance. It was shown that no clear fitness has been associated with the induction of azole resistance (in contrast to plant pathogens) (
661). The mechanisms of acquiring resistance are also more complex than the integration of 34 or 46 repeated sequences in the
CYP51A promoter. Many resistant isolates do not have amino acid substitutions in Cyp51A (
662) but use alternative mechanisms. For example, the upregulation of ABC and MFS transporter genes (
663); the modification of HapE (
652); the involvement of mitochondrion metabolism, especially the mitochondrial complex 1 and the cytochrome
b5-CybE redox systems (
664,
665); the role of TF SrbA and AtrR, regulating
CYP51A (
666,
667); and
Cyp51B overexpression (
646) have been associated with azole resistance. Resistance to azoles is a fashionable topic (>600 references in PubMed), but does it deserve so much attention? Resistance to antifungals in
A. fumigatus has not been recognized as a substantial clinical problem in medical mycology (
668). However, case reports show that patients who are infected with azole-resistant strains have a higher mortality incidence than patients who are infected with susceptible strains (
638,
669,
670). It is necessary to take into account the emergence of these forms of resistance when one considers the overarching problem of multidrug-resistant bacteria. The paucity of fungus-specific targets is an additional problem because of the prevalence of cross-resistance to all drugs with a common target. Therefore, there is a need to identify additional antifungal drugs to overcome future challenges that will be posed by emerging antifungal resistance. A major concern is that there are few discovery programs devoted to developing new antifungal therapeutics, because pharmaceutical companies expect limited financial returns due to the costliness of clinical trials. Indeed, the major pharmaceutical companies selling antifungal drugs each have an azole and an echinocandin molecule and seem to be reluctant to invest money on the search for new lead compounds.
Regardless, many new drug targets have been identified or revisited, and some of them have led to the discovery of new antifungal drugs (
671,
672). Below is a nonlimitative list of putative drugs and targets followed by startup companies which may reach the clinics in the near future. Lysine deacetylases and acetyltransferases, which catalyze the removal or addition of acetyl groups from core histones, resulting in changes in chromatin structure and gene expression, have been selected recently (
673). Farnesyltranferase, which catalyzes posttranslational lipidation on the C terminus of many important signaling proteins, also has been identified as a promising target. Inhibitors of GPI anchor biosynthesis as well as of GPI-anchored proteins, essential in the construction of the cell wall and, accordingly, fungus specific (
162), have been spotted. An inhibitor of inositol acyltransferase (AX001) that blocks fungal glycosylphosphatidylinositol biosynthesis is currently being developed. Fungal pyrimidine biosynthesis also has been recognized as a target for a long time, and the compound F901318, which inhibits the oxidoreductase enzyme dihydroorotate dehydrogenase, was launched recently. Inhibitors of the glyoxylate cycle also look promising. The arylamide T-2307, which is structurally similar to aromatic diamidines, causes collapse of fungal mitochondrial membrane potential. The cyclic metallohexapeptide VL-2397 is structurally related to the siderophore ferrichrome; a rapid antifungal effect follows its transport into fungal cells via siderophore iron transporter 1, which is absent from mammalian cells. Additional inhibitors of β1,3 glucan synthase, such as the echinocandin rezafungin, exhibit a prolonged half-life, allowing weekly treatments, and rezafungin has completed phase II studies. A new molecule, different from echinocandin SCY-078, is available in oral and in intravenous formulations and has recently received fast-track FDA approval. Even though chitin synthesis is an evident target, no clinically relevant inhibitors of this enzyme have been identified. Nikkomycins and polyoxins are specific chitin synthase inhibitors of chitin synthases, and although they potently inhibit enzyme activity
in vitro, they are not efficiently taken up
in vivo and consequently often are not effective antifungals (
674).
Figure 20 represents the old and new molecules based on the inhibition of old ones and new, promising drug targets.