7.14 Fungi and Fungal Disease

7.
14 Fungi and Fungal Disease

P Dorr, Pfizer Global Research and Development, Sandwich, UK
& 2007 Elsevier Ltd. All Rights Reserved.
7.14.1 Medically Important Fungi and Major Fungal Diseases 420

7.14.1.1 General Classification and Structure of Medically Important Fungi 420
7.14.1.2 Human Pathogenic Fungi and Disease States 420
7.14.1.2.1 Superficial mycoses 420
7.14.1.2.2 Subcutaneous mycoses 420
7.14.1.2.3 Cutaneous mycoses 420
7.14.1.2.4 Dimorphic systemic mycosis 423
7.14.1.2.5 Opportunistic systemic mycoses 424
7.14.1.3 Emerging Fungal Diseases 425
7.14.2 Pathogenesis of Major Fungal Diseases 426
7.14.2.1 Candida albicans and Candida Species 426
7.14.2.2 Aspergillus 427
7.14.3 Host Defenses 427
7.14.4 Diagnosis of Fungal Infection 427
7.14.4.1 General 427
7.14.4.1.1 Microscopy and culture of pathogenic fungi 428
7.14.4.1.2 Diagnosis of fungal infection by metabolite detection 428
7.14.4.1.3 Diagnosis of fungal infection by gene sequence detection 429
7.14.5 Current Antifungal Drugs: Clinical Pharmacology and Treatment
Modalities 429
7.14.5.1 General 429
7.14.5.2 Superficial Mycoses 429
7.14.5.3 Systemic Mycoses 431
7.14.5.3.1 Amphotericin 432
7.14.5.3.2 Fluconazole 432
7.14.5.3.3 New antifungal drugs 432
7.14.6 Antifungal Drug Discovery and Development: Future Directions and
New Modes of Action 433
7.14.6.1 Antifungal Compounds in Clinical Development 433
7.14.6.1.1 Isoleucyl-tRNA synthetase inhibitors (ITRS) 433
7.14.6.1.2 Sphingolipid biosynthesis inhibitors – isophosphorylceremide synthase (IPCS) 434
7.14.6.2 New Antifungal Targets and Leads – Preclinical Discovery 434
7.14.6.2.1 N-Myristoyltransferase (NMT) 436
7.14.6.2.2 Ribonucleic acid (RNA) polymerase (Pol) III 436
7.14.6.2.3 Acetyl-CoA carboxylase (ACCase) 439
7.14.6.2.4 Fungal protein biosynthesis inhibitors 439
7.14.7 Further Reading 440
References 440
419
7.14.1 Medically Important Fungi and Major Fungal Diseases

7.14.1.1 General Classification and Structure of Medically Important Fungi
All living organisms are classified into five kingdoms, one of which is Fungi. This represents a diverse group of
eukaryotic microorganisms (usually with dimensions of 3–50 mm) that are heterotrophic (i.e., require elaborated organic
substrate for growth, such as human tissues in the case of pathogenic fungi), and are devoid of chlorophyll. In light of
their heterotrophic nature, all fungi exist either as saprophytes or parasites. It is the latter that will be discussed in
more detail in this chapter.
The basic classification of fungi is based on their appearance and method (or indeed absence) of sexual
reproduction. Their nomenclature is in accordance with the International Code of Botanical Nomenclature, in light of
their original identification as plants in Roman times. Most fungi, including fungal pathogens such as Aspergillus
fumigatus (Figure 1), exist in a vegetative phase as microscopic filaments or hyphae. Hyphae usually consist of tubular
cells that are surrounded by a rigid, chitin-containing cell wall. The hyphae extend by tip growth, and multiply by
branching, creating a fine network called a mycelium, which can be of sufficient biomass to be macroscopic. Consistent
with their eukaryotic nature, hyphae contain nuclei, mitochondria, ribosomes, Golgi, and membrane-bound vesicles
within a plasma membrane-bound cytoplasm. The subcellular structures are supported and organized by microtubules
and endoplasmic reticulum. Under certain conditions, septa develop in hyphae, which initiate the differentiation into
nonvegetative resting spore structures. The yeasts represent a large group of fungi that consist of separate round, oval,
or elongated cells that propagate by budding out similar cells from their surface. Many fungi, including the most
significant fungal pathogen Candida albicans, can exist in a yeast or mycelial form (Figure 1).
A comprehensive review of the structural biology and taxonomy of pathogenic fungi can be found in the recently
published textbook edited by San-Blas and Calderone.1
7.14.1.2 Human Pathogenic Fungi and Disease States

It is estimated that about 300 000 different species of fungi are currently in existence, of which about 600 are associated
with human disease, with about 20 that cause 499% of human fungal infections (mycoses). The vast majority of fungi
are therefore free-living organisms with no dependence on humans for survival. With few exceptions, fungal infections
originate from an exogenous source in the environment, and are acquired through inhalation, ingestion, or traumatic
implantation.
Fungal diseases are described in many ways, although the most practical is a subdivision into the mycoses groups
described below.
7.14.1.2.1 Superficial mycoses

Superficial (or cutaneous) mycoses are fungal diseases that are confined to the outer layers of the skin, nail, or hair,
(keratinized layers), rarely invading the deeper tissue or viscera, without inducing a cellular response from the host.
The fungi involved are called dermatophytes (listed in Table 1). Superficial fungal infections are very common, of
worldwide distribution, and are rarely serious or difficult to treat. These infections are often so innocuous that patients
are often unaware of their condition. All topical antifungal drugs such as clotrimazole (Canestan) or terbinafine
(Lamisil) are highly efficaceous and available where treatment is sought. The epidemiology and therapeutic options for
the treatment of these infections has been extensively reviewed by Brandt and Warnock.2
7.14.1.2.2 Subcutaneous mycoses

These are rare but chronic localized infections of the skin and subcutaneous tissue following the traumatic implantation
of the etiologic agent. The causative fungi are all soil saprophytes of regional epidemiology, whose ability to adapt to the
tissue environment and elicit disease is extremely variable (Table 2). Management options are limited, difficult, and
can vary according to pathogen.3 Surgical intervention in combination with systemic antifungal chemotherapy is often
required, with variable success.4 Amphotericin B has been generally used for the treatment of these infections, although
azole therapy is becoming more widespread as sensitivities to these drugs are being reported.5,6 Sporotrichosis and
chromoblastomycosis have been reported to respond to oral therapy with the azole itraconazole, and, more recently,
voriconazole and allylamines, sometimes in combination with amphotericin.6
7.14.1.2.3 Cutaneous mycoses

These extremely common and worldwide diseases are superficial fungal infections of the skin, hair, or nails. Although
no living tissue is invaded, a variety of pathological changes occur in the host because of the presence of the infectious
Fungi and Fungal Disease 421
Figure 1 Micro- and macroscopic images of pathogenic fungi. (a) Electron micrograph of Candida albicans in yeast form and
(b) mixed yeast–mycelium form from a urine sample (per acid stained). (c) Methenamine silver staining of clinical samples
highlights the hyphal growth of the filamentous pathogen Aspergillus fumigatus invading lung tissue. The spore-forming (conidial
head) structure of A. fumigatus is also highlighted (d) within invaded lung tissue, and (e) in more detail as an electron micrograph
following laboratory culture. Extensive invasion can lead to macroscopic emergence of Aspergillus biomass, as shown by
(f) visible growth on lung tissue. Colony presentation of (g) Aspergillus and (h) C. albicans on agar plates. (a–d; f–h: reproduced
by permission of Dr David Ellis, School of Molecular & Biomedical Science, University of Adelaide and the Kaminski digital image
library; e: reproduced by permission of Dr Jean-Paul Latge of the Pasteur Institute.)
Table 1 Diseases and causative organisms of superficial mycoses
Disease Causative organism
Pityriasis versicolor (Figure 1a) Malassezia furfur (a lipophilic yeast)
Tinea negra Exophiala werneckii
White piedra Trichosporon beigelii
Black piedra Piedraia hortae

Table 2 Subcutaneous mycoses. The causative pathogen is emboldened for the clinical case highlighted
Disease Causative organism Presentation
Sporotrichosis Sporothrix schenckii
Chromoblastomycosis Fonsecaea, Phialophora, Cladosporium
Phaeohyphomycosis Cladosporium, Exophiala, Wangiella,

Bipolaris, Exserohilum, Curvularia
Mycotic mycetoma Pseudallescheria, Madurella,

Acremonium, Exophiala, etc.
Subcutaneous zygomycosis (including Basidiobolus ranarum, Conidiobolus

mucoromycosis) coronatu, Rhizopus, Mucor, Rhizomucor
Rhinosporidiosis Rhinosporidium seeberi
Reproduced by permission of Dr David Ellis, School of Molecular & Biomedical Science, University of Adelaide.
agent and its metabolic products. The causative pathogen is usually a filamentous dermatophyte (Table 3), from which
Trychophyton (usually T. mentagrophytes or T. rubrum), Epidermophyton, and Microsporium species are by far the most
common pathogens, although superficial candidiasis is not uncommon.7 These pathogens infect various areas of the
body, from which the infection description is derived (e.g., Tinea capitis (scalp and/or hair), Tinea corporis (a dermatophyte
Table 3 Cutaneous mycoses. The causative pathogen is emboldened for the clinical case highlighted
Dermatophytosis. Dermatophytes (Microsporum,

Ringworm of the scalp, Trichophyton, Epidermophyton)
glabrous skin, and nails
Candidiasis of skin, mucous Candida albicans and related species

membranes and nails
Dermatomycosis Nondermatophyte moulds (Hendersonula

toruloidea, Scytalidium hyalium,
Scopulariopsis brevicaulis)
infection of the trunk, legs, and arms), T. pedis (feet), T. mannum (hands), T. unguium (nails)). The vast majority of
infections are mild, and readily diagnosed by microscopy and culture of samples, following symptoms of localized
irritation, lesions, and inflammation. These infections respond well to topical therapies such as azole and allylamine
drugs (in particular, terbinafine).8 In cases where the infection is showing signs of deeper and wider invasion into
peripheral tissues, oral systemic therapy is required, usually by itraconazole or terbinafine.9,10 Of these infections,
T. unguium or onychomycosis is the least well addressed in terms of therapy.8 Although generally a mild infection due to
dermatophytes and also Candida species, it can spread to case discomfort and localized disfigurement. Effective therapy
is restricted to long-term (3–6 month) oral terbinafine or itraconazole. Topical agents have very limited efficacy,8,10 but
would be preferred to limit systemic exposure of the oral agents, where liver tests are often required to monitor possible
side effects.
7.14.1.2.4 Dimorphic systemic mycosis

Dimorphic systemic mycoses are rare fungal infections of the body caused by dimorphic fungal pathogens that can
overcome the physiological and cellular defenses of the normal human host by changing their morphological form. They
are geographically restricted, and the primary site of infection is usually pulmonary, following the inhalation of conidia
(fungal spores from the associated pathogen). The severity of disease is extremely variable, sometimes inapparent (e.g.,
most cases of histoplasmosis), but can be extreme, leading to gross disfiguration and fatal outcomes7 (Table 4). Most
immunocompetent patients have a benign, self-limiting illness, and will recover without antifungal treatment.
However, in patients who require treatment, amphotericin B is currently the drug of choice, often with azole therapy
for maintenance, although the broad spectrum of voriconazole (Vfend) and recent demonstration of its successful
treatment of these infections offer a therapeutic option in the absence of the side effects associated with the
polyenes.11–13
Table 4 Dimorphic systemic mycoses
Histoplasmosis Histoplasma capsulatum
Coccidioidomycosis Coccidioides immitis
Blastomycosis Blastomyces dermatitidis
Paracoccidioidomycosis Paracoccidioides brasiliensis
7.14.1.2.5 Opportunistic systemic mycoses

These are fungal infections of the body that occur almost exclusively in debilitated patients whose normal defense
mechanisms are impaired. Of all the mycoses, these represent by far the greatest challenge in terms of unmet medical
need. The great increase in this patient population has led to a massive increase in the frequency of these diseases,
which are associated with high morbidity and mortality. The organisms involved (Table 5 and Figure 2) are
cosmopolitan fungi that have a very low inherent virulence. The increased incidence of these infections and the
diversity of fungi causing them has paralleled the decrease in host resistance due to the emergence of AIDS, more
aggressive cancer and post-transplantation chemotherapy, and the use of antibiotics, cytotoxins, immunosuppressives,
and corticosteroids.14–16 In light of these mycoses, in particular candidiasis and aspergillosis becoming more prevalent,
scientists and the pharmaceutical industry have focused considerable effort toward the discovery and development of
Table 5 Opportunistic systemic mycoses
Disease Causative organism
Candidiasis Candida albicans and other Candida spp.
Aspergillosis Aspergillus fumigatus
Cryptococcosis Cryptococcus neoformans
Pseudallescheriasis Pseudallescheria boydii
Zygomycosis (mucormycosis) Rhizopus, Mucor, Rhizomucor, Absidia, etc.
Hyalohyphomycosis Penicillium, Paecilomyces, Beauveria, Fusarium, Scopulariopsis, etc.
Phaeohyphomycosis Cladosporium, Exophiala, Wangiella, Bipolaris, Exserohilum, Curvularia
Figure 2 Tissue invasion by Candida albicans in kidney: (a) colony formation on tissue surface highlighted and (b) an
apsergilloma (fungal ball) caused by Aspergillus fumigatus (excised from lung).
new systemic antifungal agents. It is this area of antifungal drug discovery that is of particular challenge to the
medicinal chemist. Diagnosis of fungal infections and treatment are described in this chapter in more detail, together
with emerging targets and chemistry starting points for the discovery of new antifungal drugs.
7.14.1.3 Emerging Fungal Diseases

Results of many surveys have shown that yeasts are still the most frequent cause of systemic mycoses, particularly those
caused by Candida species. Indeed, Candida species account for 70–80% of invasive bloodstream fungal infections, and
collectively they represent the fourth most common nosocomial bloodstream infection.17 Among the Candida species
causing invasive infections, C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata account for about 80–90% of fungal
isolates encountered in the clinical laboratory.18 Aspergillus species are the predominant molds, with Scedosporium
species now accounting for 25% of mold infections other than aspergillosis in organ transplant recipients. Following
these pathogens, previously uncommon hyaline filamentous fungi (e.g., Fusarium species, Acremonium species,
Paecilomyces species, and Pseudallescheria boydii), dematiaceous filamentous fungi (e.g., Bipolaris species, Cladophialophora
bantiana, Dactylaria gallopava, Exophiala species, and Alternaria species) and yeast-like pathogens (e.g., Trichosporon
species, Blastoschizomyces capitatus, Malassezia species, and Rhodotorula rubra) are increasingly encountered as causing life-
threatening invasive infections that are often refractory to conventional therapies.19,20 On the basis of past and current
trends, the spectrum of fungal pathogens will continue to evolve in the settings of an expanding population of
immunocompromised hosts, selective antifungal pressures, and shifting conditions in hospitals and the environ-
ment.21,22 An expanded and refined drug arsenal combined with the further elucidation of pathogenesis and resistance
mechanisms, the establishment of in vitro/in vivo correlations, and, finally, the implementation of combination (and
possibly immunotherapies) will offer hope for substantial progress in prevention and treatment of these mycoses.22,23
These include resistant strains from common infection-causing fungi, emergence of azole-resistant and consequently
high mortality-associated Candida strains, and new species.24 Less common and emerging fungal pathogens are often
resistant to conventional antifungal therapy, and may cause severe morbidity and mortality in immunocompromised
hosts. Emerging pathogens are increasingly reported as causing invasive mycoses refractory to amphotericin B therapy,
although the new agents caspofungin (Cancidas) and voriconazole in particular have been shown to have impressive
potency against these species in vitro and in the clinic.25–30 Pfaller and Diekema provide an excellent review of new and
emerging fungal infections together with changes in epidemiology and treatment modalities.25
7.14.2 Pathogenesis of Major Fungal Diseases

As discussed above, the treatments for superficial and dermatophyte-associated infections are highly effective, well
tolerated, readily available, and in general address medical need. However, life-threatening systemic mycoses,
especially as caused by Candida and Aspergillus species, will be reviewed in detail in this chapter in light of their high
medical need, the challenges faced in their treatment, and since these are no longer considered as rare infections. To
put these specific mycoses in perspective, the crude mortality from invasive aspergillosis is around 85%, while that for
Candida bloodstream infections is 40%.14,25,31,32
7.14.2.1 Candida albicans and Candida Species

The ability of this otherwise commensal microorganism to cause disease is usually a consequence of host immuno-
compromisation rather than fungal virulence.15,33–35 In general, pathogenesis is associated with factors that enable
fungal adherence, tissue invasion, and growth. Adherence and persistence are necessary for the initiation of mycosis,36
for which, in the case of Candida, mannoproteins are believed to play a role, and indeed mannose transferase has been
identified as a virulence factor.37 To assist in the invasion of host tissues, proteases and phospholipase secretion by the
pathogen is enabled, and a degree of correlation exists between the proteolytic activity of the Candida species with
pathogenicity. Invasion is also associated with a switch from the generally noninvasive yeast to the invasive hyphal form
for C. albicans.34,35,37–41 Detailed reviews of fungal pathogen virulence factors have been recently published.1,35
The extreme virulence of Candida species reflects the diversity of disease states caused. These are briefly described
below. In addition to the text references above, comprehensive descriptions of Candida pathogenicity and disease states
can be found online.37a
C. albicans and, to a lesser extent, non-albicans species of Candida cause a variety of severe infection, including:
* esophageal candidiasis (frequently associated with AIDS and severe immunosuppression following treatment for
leukemia or solid tumors);
* gastrointestinal candidiasis;
* pulmonary candidiasis;
* peritonitis (usually from colonization of indwelling catheters used for peritoneal dialysis or gastrointestinal
perforation);
* urinary tract and renal candidiasis (pyelonephritis);
* meningitis;
* hepatic and hepatosplenic candidiasis;
* endocarditis, myocarditis, and pericarditis;
* candidemia (Candida septicemia) and disseminated candidiasis.
The complications associated with Candida infections is compounded by the emergence of non-albicans species as
both a colonizer and a pathogen. More than 17 different species of Candida have been identified as etiologic agents
of bloodstream infections, with approximately 95% of these being caused by four species: C. albicans, C. glabrata,
C. parapsilosis, and C. tropicalis.25 The greater efficacy of fluconazole as a prophylactic in transplant patients has indeed
led to a shift toward the less susceptible C. glabrata species as the predominant pathogen in such immunocompromised
patients, as well as more emerging opportunistic pathogens.25 The inherent resistance of a few non-albicans species to
commonly used systemic antifungals such as fluconazole and amphotericin B can pose a therapeutic challenge in the
management of candidaemia. Encouragingly, newer drugs, including broad-spectrum triazoles such as voriconazole, and
candins such as caspofungin, are emerging as therapeutic alternatives for non-albicans infection treatment, as discussed
elsewhere (see 7.15 Major Antifungal Drugs).
7.14.2.2 Aspergillus
Aspergillosis is a spectrum of diseases of humans (and animals) caused by members of the genus Aspergillus. Aspergillus
fumigatus is overwhelmingly the predominant pathogenic species. The type of disease and severity depends upon the
physiologic state of the host and the species of Aspergillus involved. The etiological agents are ubiquitous, and include
A. fumigatus, A. flavus, A. niger, A. nidulans, and A. terreus.
Numerous virulence factors have been associated with aspergillosis, including phospholipases and proteases to
facilitate adhesion and invasion,38,42 and the biosynthesis of factors that enable evasion of limited host defenses in the
immunosuppressed patient.41 As with Candida infections, the clinical manifestations resulting from Aspergillus virulence
are varied, although pulmonary infection and aspergilloma caused by the saprophytic colonization of preformed cavities
(Figure 2b) is the most common. Acute invasive pulmonary aspergillosis (usually a result of prolonged neutropenia,
especially in leukemia patients or in bone marrow transplant recipients) generates symptoms similar to acute bacterial
pneumonia. Dissemination can lead to tissue-specific infections. Abscesses may occur in the brain (cerebral asper-
gillosis), kidney (renal aspergillosis), heart (endocarditis, myocarditis), bone (osteomyelitis), and gastrointestinal tract.
Ocular lesions (mycotic keratitis, endophthalmitis, and orbital aspergilloma) manifest themselves as erythematous
papules or macules with progressive central necrosis.
7.14.3 Host Defenses

As discussed earlier, host defense against fungal infection usually ensures that mycoses are restricted to minor
superficial, nonlife-threatening cases, which are well addressed by current therapies. The rarity of systemic infections
prior to the dramatic increase in the immunocompromised patient population15,21 has highlighted the extensive role that
the immune system must play in keeping opportunistic fungi at bay, in particular C. albicans, other Candida species, and
A. fumigatus. A number of comprehensive reviews of host defenses against mycoses have been published.1,43
The stratified squamous epithelium of the skin normally functions as an effective barrier to Candida. Mechanical
breakdown is the most important factor for skin infections. However, mucosal surfaces are not so well defended against
colonization, although infection in this tissue is greatly influenced by the host’s status and factors such as diabetes,
pH/hormonal factors (vaginal thrush), and competition with endogenous flora. After mucosal and skin barriers are
penetrated by fungi, and in particular Candida species, it is the cellular immune system complemented by humoral
factors that restrict invasion.44–46 Indeed, it is the former in particular that is compromised by HIV and many immune-
compromising medical advances that has led to the massive increase in fungal infections. Host immune cell inter-
actions and immune responses are extremely complex, and multiple arms of the immune system play roles to combat
fungal pathogens, with distinct patterns that are dependent on the fungal species.47 Humoral factors such as
complement have been shown to play a role in restricting opportunistic fungal infection.45,48 Antibody-mediated
defenses are believed to play a relatively small role in host defense, whereas cell-mediated immunity is believed to be
the predominant arm of the host defense. This has been shown by animal studies, specific defects in the cell-mediated
immunity in patients, and the known selective cytotoxic effects of immune-compromising medications and HIV.49–54
The high incidence of Candida in leukemic patients is mainly attributable to neutropenia and decreased neutrophil
functionality.43,55 Phagocytosis by neutrophils, eosinophils, monocytes, and macrophages specifically represents the
most important defense mechanism against the invading pathogenic fungus.43,49,50,56 In vitro studies have indicated
the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens C. albicans and A. fumigatus.
However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling
in vivo remain largely undefined.57
7.14.4 Diagnosis of Fungal Infection

7.14.4.1 General
The diagnosis of systemic fungal diseases can present a major dilemma for the clinician. With the majority of suspected
infections in a given patient being bacterial in origin, relief by appropriate antibiotic use is a predominant course of
action. However, with immune-compromised patients in particular, the ever-increasing concern is that the causative
pathogen is a fungus. Given the speed of debilitation resulting from systemic mycoses, rapid diagnosis to enable a
timely decision is needed, although this is not always possible. In light of the high morbidity and mortality surrounding
systemic fungal infections, particularly those of Candida and Aspergillus, rapid administration even before confirmed
diagnosis is often the favored option, even when the administered agent has severe side effects, such as those
associated with amphotericin B. However, in recent years, the choice of antifungal drugs for systemic diseases has
increased with the more widespread use of fluconazole for C. albicans infections (in particular maintenance
therapy), to voriconazole (particularly for Aspergillus infections, and broad-spectrum use), and caspofungin (which is a
better-tolerated infused agent compared with amphotericin).58 A comprehensive general review of the diagnosis (and
management) of fungal infections has been published.3
Much has been achieved recently in the improvement of laboratory diagnosis of mycoses, such as advances in blood
culture systems, and the development of new biochemical assays, antigen detection assays, and molecular methodo-
logies.59 More standardized susceptibility testing guidelines provide for better therapeutic interventions. In an era of
economic cutbacks in healthcare, future challenges include the development of cost-effective and technically
simplified systems, which will provide for the early detection and identification of common and emerging fungal
pathogens. The conventional diagnostic methods such as culture are often slow and lack sensitivity and specificity,
resulting in additional alternative diagnostic assays. Among the most promising new techniques are the detection of
fungal DNA, metabolites (see following sections), and serology. Fungal DNA can be detected with high sensitivity and
specificity when performed with specimens from sterile sites such as blood.60,61 Polymerase chain reaction (PCR)
assays can be used to detect a broad range of fungal pathogens, and combined with species identification, although a
lack of standardization has restricted its use. The sero-diagnosis of invasive fungal infections has become an important
tool in the management of invasive fungal infections. Both serology and PCR can be used to monitor the response to
antifungal therapy.60–62 These newer approaches are generally used to help confirm the diagnosis of systemic infection;
however, their impact on mortality may be greatest when they are used to screen high-risk patients.61 While the
mainstay of infectious disease diagnosis remains in microbiological culture with sensitivity follow-up, significant steps
in providing rapid diagnosis in commercialized and standardized assays have been achieved.
7.14.4.1.1 Microscopy and culture of pathogenic fungi

Clinical samples for suspected fungal pathogens, in particular Candida and Aspergillus species, are varied, and include
skin and nail scrapings (usually for cutaneous mycoses); urine, sputum and bronchial washings; cerebrospinal fluid,
pleural fluid and blood; tissue biopsies from various visceral organs; and indwelling catheter tips (subcutaneous and
systemic infections). Direct microscopy of skin and, to a lesser extent, body fluid samples for fungal identification can
be achieved with staining (e.g., using 10% KOH and Parker ink or calcofluor white mounts), although direct microscopy
of sterile body fluids, such as cerebrospinal fluid, vitreous humor, joint fluid, and peritoneal fluid is relatively
insensitive, and positive culture is usually required to make a diagnosis. A positive culture from blood, or other sterile
body fluid, or tissue biopsy (usually on blood or Sabourauds agar plates) should be considered significant. Candida
colonies are usually white to cream colored, with a smooth, glabrous to waxy surface, whereas Aspergillus colonies and
mycelia are white, yellow, yellow-brown, brown to black, or green in color (see Figure 1). Direct microscopy to identify
pathogenic fungi in tissue samples is enabled by staining (e.g., cell wall staining using periodic acid, Gram stain, or
methenamine silver stain – see Figure 1). Most Candida species are also characterized by the presence of well-
developed pseudohyphae; however, this characteristic may be absent in certain species such as C. glabrata.
7.14.4.1.2 Diagnosis of fungal infection by metabolite detection

The fungal infection diagnostic kit Fungitell has been commercialized for hospital/clinical use. This serological assay is
for the qualitative detection of b-glucan in the serum of patients with symptoms of, or medical conditions predisposing
the patient to, invasive fungal infection, and as an aid in the diagnosis of deep-seated mycoses and fungemias. Fungitell
is sensitive to a few trillionths of a gram of (1,3)-b-D-glucan, and has been shown to provide significant advantages in
the diagnosis of invasive fungal infection alone and in combination with traditional blood culture methods.63,64 The
Fungitell assay does not detect certain fungal species such as the genus Cryptococcus, which produces very low levels of
(1,3)-b-D-glucan. This assay also does not detect the Zygomycetes, such as Absidia, Mucor, and Rhizopus, which are not
known to produce (1,3)-b-D-glucan.
Galactomannan, a component of the Aspergillus cell wall, is released during invasive disease, and can be detected in
blood, cerebrospinal fluid, and bronchial fluid65,66, where it has proved useful as a biomarker for infection. Exploitation
of this to enable a commercialized kit for diagnosis has been achieved via enzyme-linked immunosorbent assay
(ELISA) technology (Platelia Aspergillus). Many studies have validated this diagnostic kit, and report excellent
sensitivities and specificities.67,68 The degree of galactomannan antigenemia correlates with tissue fungal burden, and
the course of antigenemia in patients has been shown to correspond with clinical outcome and to have prognostic
value.69 The utility of such technology has resulted from years of extensive studies on circulating Candida antigens, in
particular mannan-based metabolites derived from the cell wall of Candida species.70,71 The Platelia Candida sandwich
ELISA represents a sensitive serological method, utilizing a monoclonal antibody that recognizes the sequences of
linked oligomannoses present in a large number of mannoproteins extracted from different Candida species. An issue
of concern regarding the Platelia Aspergillus antigen/antibody assay is the variable reactivity of different Candida
species.72
7.14.4.1.3 Diagnosis of fungal infection by gene sequence detection

The amplification of gene sequences unique to fungi is conceptually appealing, offering the potential for rapid, specific,
and sensitive diagnosis of systemic mycosis. Experimental models and clinical studies have shown PCR to be more
sensitive than culture for detection of candidaemia.73,74 Several PCR protocols have been developed utilizing different
extraction methods, amplification targets, and amplicon detection formats. These have been extensively reviewed.75
PCR-based detection of pulmonary-based infections may be more sensitive than traditional culture methods, and have
been successfully evaluated in bronchoalveolar lavage specimens from patients with pulmonary infection.76 Further
prospective studies have confirmed the utility of screening patients at high risk of systemic fungal infection using PCR.
Sensitivity and specificity values are encouraging, and PCR positivity precedes the development of clinical and
radiological signs and administration of empirical antifungal therapy in most patients.77
The gradual move toward dedicated custom PCR-based kits for the clinical diagnosis of systemic fungal infections
highlights the progress being made toward rapid and accurate pathogen identification.78 Indeed, kits have been com-
mercialized for PCR-based Candida detection in research samples. The Lightcycler (Roche) is intended as a research
tool, but highlights its potential in the use of PCR technology for fungal diagnostics. DNA preparations from blood
culture bottles, isolates from culture plates, and direct preparations from a wide range of specimens, such as urine,
swabs, bronchoalveolar lavage, and sputum, can be tested. Such advances may help to develop species-specific
antifungal drugs. For broad-spectrum diagnosis by this technology, amplification of a conserved region (e.g., 18S
ribosomal DNA), may represent the way forward.
7.14.5 Current Antifungal Drugs: Clinical Pharmacology and Treatment

Modalities
7.14.5.1 General
The antifungal drugs that are currently licensed or approaching this milestone are listed in Table 6. These have been
discovered and developed over many years, with only a few now being used extensively in clinical practice (see 7.15
Major Antifungal Drugs). Despite the number of drugs, the mechanisms by which they operate are very limited, with
disruption of the ergosterol function (direct or indirect) a dominant theme (see Figure 3). The use of the agents varies
according to the type of infection for which treatment is sought. Systemic, life-threatening fungal infections are an
ever-increasing medical problem faced by the immunosuppressed population. The arsenal of antifungal drugs for
treatment of these mycoses is gradually improving, in particular with the emergence of new-generation triazoles
and candins. However, limitations exist with the current range of antifungal drugs, in terms of an agent that combines
broad-spectrum fungicidality, predictive pharmacokinetics with manageable drug–drug interactions, high safety/
tolerance, and oral plus intravenous formulations. New targets and leads to increase future treatment options are
needed. This chapter is aimed at describing the causative pathogens of fungal infections (in particular systemic
mycoses), the current drugs for their treatment, and future directions for new drugs, for which medicinal chemists will
play a key role. In the following sections, the leading therapies will be discussed in more detail.
7.14.5.2 Superficial Mycoses

As described above (see Sections 7.14.1.2.1 and 7.14.1.2.3), these infections are generally mild, and are well addressed
by current therapies, which are mostly topical. A range of topical therapies that are available has been extensively
reviewed,2,3,15,79 with therapies dominated by azole and allylamine topical drugs, in particular terbinafine. Terbinafine
has well-documented activity when used both topically and systemically for infections of nails and skin. It is active
in vitro against a wide range of fungi, including dermatophytes, molds, and some yeasts.80 Terbinafine selectively
inhibits fungal squalene epoxidase (and is essentially inactive against human squalene epoxidase81), which converts
squalene into lanosterol (see Figure 3). Lanosterol is eventually converted into the functional sterol ergosterol.
Terbinafine leads to ergosterol deficiency and accumulation of intracellular squalene, which leads to a fungicidal
endpoint, particularly so for filamentous fungi.82 Highly effective therapy for skin infections is also achieved with azole
Table 6 Licensed and late-stage development antifungal drugs
Antifungal class (mode of action) Drug Route
Allylamines (squalene epoxidase) Amorolfine Topical
Butenafine Topical
Naftifine Topical
Terbinafine Oral, topical
Antimetabolite (DNA) Flucytosine Oral
Azoles (cytochrome P450 demethylase) Fluconazole Oral, intravenous
Itraconazole Oral, intravenous
Ketoconazole Oral, topical
Posaconazole Oral, intravenous
Ravuconazole Oral, intravenous
Voriconazole Oral, intravenous
Clotrimazole Topical
Econazole Topical
Miconazole Topical
Oxiconazole Topical
Sulconazole Topical
Terconazole Topical
Tioconazole Topical
Candins (glucan synthase inhibitors) Caspofungin Intravenous
Micafungin Intravenous
Anidulafungin Intravenous
Polyenes (ergosterol) Amphotericin B Intravenous
Amphotericin B lipid complex Intravenous
Amphotericin B colloidal dispersion Intravenous
Liposomal amphotericin B Intravenous
Amphotericin B oral suspension Oral
Liposomal nystatin Intravenous
Topical Nystatin Topical
Pimaricin Ophthalmic
Other systemic drugs Griseofulvin Oral
Other topical drugs Ciclopirox olamine Topical
Haloprogin Topical
Tolnaftate Topical
Undecylenate Topical
Primary metabolism
Acetyl-CoA
Early stage isoprenoid

biosynthetic pathway
Squalene
Squalene epoxidase Terbinafine
Squalene epoxide
Lanosterol
(nonfunctional sterol)
HO
Fungi Human
Amphotericin
Cytochrome
P450
demethylase
HO
HO
Ergosterol (functional Azoles Cholesterol (functional
sterol in fungi) sterol in humans)
Figure 3 Sterol biosynthesis pathway and target sites for antifungal drugs.
creams and pessaries, and oral fluconazole is effective for vaginal thrush.3 In light of these infections being in general
well addressed, and thereby the need for the involvement of the medicinal chemist to discover new agents being
reduced, the greater challenge of finding and prescribing antifungal drugs for the treatment of systemic infections by
Candida, Aspergillus, and emerging pathogens will be focused on here.
7.14.5.3 Systemic Mycoses

Treatment of invasive fungal infections is increasingly complex. Efforts to standardize treatments as far as practicable,
and set guidelines, have progressed in light of advances in diagnosis and, moreover, the greater availability, efficacy, and
overall safety of newer antifungal drugs. Comprehensive reviews of treatment modalities and clinical pharmacology of
currently available antifungal drugs have been published, and are very useful reference points.82–85
The treatment of systemic fungal infections has been an evolutionary process as newer agents have become available
and comparative trials for efficacy and safety have proceeded. Generally, amphotericin led the way as a broad-spectrum
intravenous agent, which has efficacy but very poor tolerance. Azole antifungals represent the next major advance and
have added to the antifungal arsenal. Early azoles such as ketoconazole showed significant side effects, and were rapidly
supplanted by fluconazole as a highly safe systemic antifungal, with good efficacy against the predominant pathogen
C. albicans. Further progress was made with broader-spectrum azole itraconazole, although this met with limitations due
to reduced tolerance and unpredictable pharmacokinetics relative to fluconazole. In more recent years the introduction
of a new generation of azoles and glucan synthase-inhibiting candin antifungals have substantially broadened
therapeutic options for clinicians. The profile of the leading antifungals amphotericin and fluconazole and new agents
are reviewed in more detail below.
7.14.5.3.1 Amphotericin
Amphotericin was first isolated from Streptomyces nodosus in 1955. It is an amphoteric compound composed of a
hydrophilic polyhydroxyl chain along one side and a lipophilic polyene hydrocarbon chain on the other (see 7.15 Major
Antifungal Drugs). This parenternal drug was the gold standard antifungal for neutropenic patients with invasive
candidiasis for many years. The three lipid formulations are liposomal amphotericin B (AmBisome), amphotericin B
lipid complex (Abelcet), and amphotericin B colloidal dispersion (Amphocil). These formulations are more expensive
than conventional amphotericin B, but generally have the advantage of less infusional and renal toxicity, and provide the
opportunity to administer higher doses of amphotericin B.86 Animal studies suggest similar efficacy between liposomal
amphotericin B and amphotericin B lipid complex, as do retrospective comparisons of patient outcomes.82
Amphotericin and polyenes in general operate by inhibiting sterol function in the fungal cell membrane, to exert an
antifungal effect (Figure 3; for a review, see Polak87). Amphotericin achieves functional ergosterol blockade by directly
binding this functional sterol, to induce dysfunctional membrane properties, including leakage of intracellular
potassium, magnesium, sugars, and metabolites, and then cellular death. The mechanism of action is the same for all
the preparations, and is due to the intrinsic antifungal activity of amphotericin B. Target binding is only moderately
selective with respect to cholesterol binding, in particular at cholesterol-rich sites such as the kidney tubules, which
underpins the mechanistic toxicity of amphotericin B.15,88 In addition to the poor tolerance, the pharmacokinetic
profile is poorly defined, and administration is limited to intravenous infusion. Amphotericin B is not bioavailable
following oral administration.87,88 Generally speaking, amphotericin B has a very broad range of activity, and is active
against most pathogenic fungi. Notable exceptions include the emerging pathogens Trichosporon beigleii and Fusarium
species.89,90 It is testament to the rapid progression and high mortality and morbidity of systemic fungal infections that
such a poorly tolerated drug as amphotericin has been the gold standard for antifungal therapy for so many years.
7.14.5.3.2 Fluconazole
Fluconazole is an extremely safe and effective drug for the treatment and prevention of C. albicans infections
(see 7.15 Major Antifungal Drugs). Fluconazole also has the advantages of oral and intravenous administration, and has
highly predictable pharmacokinetics.91,92 However, like all azoles, it is fungistatic and not fungicidal against C. albicans,
and has a very limited spectrum with no efficacy against molds. Furthermore, some Candida species are intrinsically
resistant to fluconazole. Almost all C. krusei are resistant, and approximately 50% of C. glabrata isolates are resistant or
have intermediate dose-dependent susceptibility to fluconazole.3,25 Until recently, oral fluconazole has been the drug of
choice for controlling oropharyngeal candidiasis in AIDS patients, and for prophylactic use (predominantly transplant
and cancer patients).93,94 The success of azoles and polyenes in addressing a high medical need has been significant,
despite their mutually exclusive limitations. Both drug classes operate by inhibiting sterol function in the fungal cell
membrane, to exert an antifungal effect (see Figure 3).87 However, azoles indirectly target ergosterol function via
fungal-specific inhibition of 14-sterol demethylase, to block the synthesis of functional ergosterol from nonfunctional
lanosterol. No significant effect on human/mammalian cholesterol biosynthesis is achieved in vivo as a result of this
activity, which underpins the mechanistic safety of azoles and fluconazole in particular.95,96
7.14.5.3.3 New antifungal drugs

The dominance of fluconazole and amphotericin B in the treatment of serious fungal infections has been recently
challenged by newer drugs. Indeed, the new antifungal agents voriconazole, caspofungin, and micafungin are rapidly
establishing a place in the clinician’s antifungal arsenal, and are now available for the treatment of systemic fungal
infections. Voriconazole is an extended-spectrum triazole that is fungicidal for many filamentous fungi, including
Aspergillus, Scedosporium, Fusarium, and Paecilomyces, and is active against all species of Candida.97,98 Clinical trials have
shown that treatment with voriconazole cleared Candida from the blood as quickly as amphotericin B, with a lower
incidence of treatment-related adverse events. Subsequently, it was approved for the first-line treatment of invasive
aspergillosis and as salvage therapy for fungal infections caused by the pathogens Scedosporium apiospermum and Fusarium
species. More recently, voriconazole was approved for use in treating esophageal candidiasis.98 This is a reflection of its
impressive activity against filamentous fungi, in particular Aspergillus, where it exerts fungicidal activity, as seen in a
variety of in vivo and in vitro models.99,100 Voriconazole is given either by the oral or the intravenous route. Unlike
fluconazole, voriconazole is not renally cleared in light of its higher lipophilicity. Clearance is cytochrome P450-
mediated. The effects of voriconazole on cytochrome P450-mediated metabolism means that clinicians must be aware
of drug–drug interactions. The clinical pharmacokinetics of voriconazole have been described in detail by Purkins
et al.101 Voriconazole represents the most advanced and widely used third-generation azole antifungal, with
posaconazole and ravuconazole (see 7.15 Major Antifungal Drugs) also showing promising broad-spectrum activity.
The most obvious rational starting point for a drug discovery program would be to target the cell wall of fungal
pathogens, in light of its essentiality for pathogen viability, and total absence in host cells, despite the commonality of
their eukaryotic origin. This approach has been aggressively targeted by drug researchers, as seen by the massive success
demonstrated with the penicillin and cephalosporin series of antibacterial antibiotics.102 Despite the complex nature of
fungal cell walls (a highly structured complex of mannan, b-glucan, and n-acetyl glucosamine (chitin)-based polymers103)
and their biosynthesis, highlighting a host of target areas for antifungal drug discovery, it is arguably disappointing that
relatively little success has been made (with one exception) in terms of novel drug series. Numerous attempts and
screens to find inhibitors of chitin synthases have been undertaken, and inhibitors such as the natural products
nikkomycin and poloxin have been discovered.104 However, these have not led to clinical candidates, and other more
amenable inhibitors to a drug discovery program have not been forthcoming. Similarly, the pradamicins, which bind to
mannans in the cell wall, have proven to be of limited value as an antifungal drug lead.102 These are far from being
antifungal drugs due to inherent limitations in potency and unfavorable physicochemical properties that limit their
systemic bioavailability. Despite the substantial effort of medicinal chemists and biotransformation scientists to improve
the pradamicins, little of therapeutic use has emerged from these starting points. One notable exception
in the search for new drugs that target the cell wall of fungi is the discovery, development, and successful com-
mercialization of the echinocandin and pneumocandin drugs (reviewed by Denning,105 and discussed in detail in
7.15 Major Antifungal Drugs). These agents inhibit the glucan synthase enzyme responsible for the b-glucan polymer, a
major constituent of the cell wall. Clinical trials have highlighted amphotericin-like efficacy, in empirical-based therapy,
with superior tolerance.86 The most successful of these to date is caspofungin (Cancidas), which is a semisynthetic
analog of a pneumocandin lead.106 Caspofungin has subsequently progressed through clinical development, and met
with considerable success for the empirical therapy of fungal infections in febrile neutropenic patients. Approval was
based on results from the largest prospective antifungal empirical therapy trial published to date in neutropenic patients,
where caspofungin was as effective as amphotericin (Ambisome) but with fewer side effects.86 This represents a
breakthrough in supplanting the gold standard amphotericin B with a safer agent for many deep-seated life-threatening
infections. Caspofungin is also active against aspergillosis,107 and has been used successfully in combination with
voriconazole for this life-threatening mycoses.108 Caspofungin is currently the most widely used glucan synthase
inhibitor for the treatment of serious fungal infections. Micafungin (Mycamine), has a very similar profile, and has been
approved for prophylaxis against Candida infection as well as esophageal candidasis. Other agents in this class are also
showing great promise. Clinical studies with anidulafungin (Eraxis) have been encouraging,109 where recent Phase III
studies showed it to be superior to fluconazole against invasive candidiasis, yet with a similar safety profile.
However, a limitation of caspofungin, and the candin class of antifungal drugs in general, is the lack of an oral route of
administration. Efforts by medicinal chemists and drug discoverers are ongoing to find new small molecule templates for
the inhibition of glucan synthase (e.g., see Onishi110), although this has currently met with limited success.
7.14.6 Antifungal Drug Discovery and Development: Future Directions and New
Modes of Action
7.14.6.1 Antifungal Compounds in Clinical Development
7.14.6.1.1 Isoleucyl-tRNA synthetase inhibitors (ITRS)
ITRS is the target of icofungipen (formerly PLD-118, see Figure 4), which is a highly novel clinical development
compound for the treatment of Candida infections.111,112 Icofungipen is a synthetic derivative of the naturally occurring
Isofungipen Cispentacin
H2N COOH H2N COOH

(a) (b)
Figure 4 (a) Isofungipen and (b) cispentacin.
b-amino acid cispentacin (derived from Bacillus cereus and Streptomyces setonii) that blocks isoleucyl-tRNA synthetase,
resulting in the inhibition of protein synthesis and growth of fungal cells. Icofungipen, like fluconazole, has the
advantage of renal clearance to drive highly predictive pharmacokinetics and avoid drug–drug interactions as a result
of hepatic cytochrome P450-based clearance. Like fluconazole, it is also a small, highly soluble and orally bioavailable
molecule. Although active against a number of Candida species, including fluconazole-resistant strains, it has only
moderate antifungal potency in vitro (minimum inhibitory concentration (MIC) ¼ 8–64 mg mL 1 with no fungicidal
activity). However, this compound showed promising efficacy in models of candidiasis (C. albicans), which was
similar to that seen with amphotericin B.112,113 Icofungipen showed a clear pharmacokinetic–pharmacodynamic
relationship, with no overt safety alerts, as shown by a well-tolerated profile against numerous toxicity markers. In light
of promising preclinical data, icofungipen has progressed to Phase II for oropharyngeal comparison with fluconazole,
where efficacy was seen, albeit less than that demonstrated for fluconazole, and with a more frequent dosing regimen.114
Unlike most natural-product antifungal leads, the small molecular size and simple structure of icofungipen make it
an attractive starting point for the medicinal chemist, since added substituents to increase potency may not
compromise the physicochemical properties, and thus hamper the favorable oral bioavailability and phamacokinetics.115
7.14.6.1.2 Sphingolipid biosynthesis inhibitors – isophosphorylceremide synthase (IPCS)

Sphingolipid biosynthesis, although not a fungal specific anabolic pathway, has attracted much interest for the discovery
of a broad-spectrum fungicidal agent. Blockade of the pathway is associated with rapid cell death, as demonstrated by a
number of natural product inhibitors of the pathway.116,117 Most importantly, one step in the pathway, the conversion of
phosphoinositol into the C-1 hydroxy group of ceramide by IPCS, is associated with extremely rapid fungal death when
inhibited, and is unique to fungi (Figure 5). Aureobasidin A (Figure 6), a cyclic depsipeptide produced by
Aureobasidium pullulans, is a potent inhibitor of IPCS from Candida and Aspergillus.118,119 The discovery of Abureobasidin
A was prompted by observations of very potent and rapid fungicidal activity (IC50 against IPCS in the nanomolar range,
and the MIC against Candida species in the sub-microgram per milliliter range),120–122 and subsequent identification
and characterization of resistance-encoding genes highlighting IPCS as the molecular target.123–125 The absence of the
target and lack of cytotoxicity prompted interest in this target, and, indeed, aureobasidin as a potential new antifungal
agent or starting point. Aureobasidin-based compounds have progressed from animal models of infection, through to
Phase I clinical assessment. An analog was shown to be superior to both fluconazole and amphotericin B in mice with
systemic candidiasis.121 Limitations in its spectrum (highly active against Candida species, but reduced activity against
Aspergillus) have been addressed at the preclinical stage by semisynthetic modifications, which have enabled activity
against Aspergillus through reduction of efflux susceptibility.118,126
7.14.6.2 New Antifungal Targets and Leads – Preclinical Discovery

The treatment of fungal infection, in particular systemic mycoses, is based upon the discovery and development of
compounds that inhibit a critical mechanism in the pathogen, but not in the host. Given the widespread penetration
associated with systemic mycoses, this represents a major challenge to medicinal chemists and drug discovery
scientists. The rationale for finding an essential target that is specific for the pathogen has existed for many years
following the historic discovery of antibiotic action against bacteria. Fungi poses an additional challenge in that, like
their human hosts, they consist of eukaryotic cell(s), and hence share high homology in their genome, and thereby
proteome and cellular machinery. The holy grail for a new antifungal drug that addresses the shortcomings of current
therapies is a low-dose fungicidal agent with high safety and tolerance that is administered by oral and non-oral routes,
and is active against the spectrum of fungi that can cause systemic infections. Some of the approaches and progress
toward the goal of finding appropriate new antifungal targets and leads are described below. The combined approaches
of synthetic, semisynthetic, and natural-product screening has been exceptionally important for this discovery of
Fatty acid biosynthesis
Serine
Palmitoyl-CoA
Ketodihydrosphingosine
Dihydrosphingosine
Fungi Humans
Phytosphingosine Sphingosine
Fatty acid
Ceramide Ceramide
Phosphatidyl inositol Phosphatidyl choline

catalyzed by IPCS
AureobasidinA
Inositol-P-ceramide Sphingomyelin
Mannosyl-inositol-P-ceramide Gangliosides
Mannosyl-(inositol)2-ceramide Cerebrosides
Essential cellular function

Figure 5 Sphingolipid biosynthesis pathway in fungal and human cells. The branchpoint in the pathway, with separate
subsequent enzyme reaction, provides scope for selective inhibition of the fungal pathway. The target for the fungicidal
compound aureobasidin A is highlighted.
O
N N
O N
O
O O
O O N
N O
O
N N
O N
O
Figure 6 Aureobasidin A.
antimicrobial agents. This approach has also led to the discovery of new antifungal targets of great potential. An
excellent review of natural-product antifungal leads, clinical candidates, and details of their mode of action has been
done by Vicente et al.117
7.14.6.2.1 N-Myristoyltransferase (NMT)

NMT catalyzes the co-translational, covalent attachment of myristate to the N-terminal glycine residue of a number of
eukaryotic proteins involved in cellular growth and signal transduction, including ADP-ribosylation factors (ARFs). The
catalytic cycle and site for known inhibitors is highlighted in Figure 7. NMT has been shown to be an essential enzyme
in all fungal species tested to date, including Candida species,127,128 and consequently has been targeted for antifungal
drug discovery. NMT isoforms have 50–80% amino acid sequence identity between human and fungal pathogens. NMT
is also believed to play an essential role in human cell viability and function, which does not make it an obvious
antifungal target. Peptide-based and depeptidized inhibitors129–131 showed NMT inhibition in fungal species,
and exerted a measurable inhibition of the function of this enzyme in whole fungal cells via the reduction of ARF
myristoylation.132 This inhibition was selective for C. albicans NMT over human, but did not impose sufficient potency
for progression as a drug candidate. The leading compounds appeared limited due to their partial peptidic nature,
leaving them prone to cleavage and reduced cell permeability. However, high-throughput screening based on
scintillation proximity assay technologies run at Pfizer133 identified the benzothiazole lead CP-123,457 (Table 7) as a
potent small molecule (IC50 C. albicans NMT ¼ 1.4 mM), with no measurable potency against the human isoform.
A medicinal chemistry campaign on this provided UK-370,485 (IC50 C. albicans NMT ¼ 40 nM) and UK-362,091
(Table 7). The latter compound had dramatically increased potency (IC50 C. albicans NMT ¼ 10 nM), which was
sufficient to drive whole-cell antifungal activity (C. albicans MIC ¼ 3.1 mg mL–1). However, this compound and
close analogs showed antifungal activity against C. albicans alone, with no activity against other pathogens, including
A. fumigatus and non-albicans species of Candida. In addition, mechanistic studies indicated that although 490%
inhibition of NMT activity in whole Candida cells was apparent at its MIC and greater, fungicidality was not achieved.
Crystal structures of fungal pathogen NMT have greatly helped in the design of inhibitors.134–136 The NMT active
site residues that were responsible for the high affinity of benzothiazole inhibitors specifically were identified by co-
crystallographic studies133 (Figure 8). Bioinformatic studies indicated key residue interactions that were responsible
for the narrow spectrum and human selectivity. These were the Phe339 and Ile111 residues in the NMT active site,
which formed strong hydrophobic interactions with the benzothiazole moiety of the inhibitor (Figures 9 and 10).
These interactions did not form in the NMT isoforms from Aspergillus and non-albicans species of Candida, which
explained the inactivity and the narrow spectrum of the inhibitors. Similar series to those discovered by Pfizer have
since been found with a similar narrow spectrum.137,138
7.14.6.2.2 Ribonucleic acid (RNA) polymerase (Pol) III

Traditionally, most agricultural and medicinal antifungal agents have been discovered through whole-cell antifungal
testing, with the eventual mode of action elucidation often accomplished later. This approach has been recently
implemented using high-throughput screening technologies with follow-up mode-of-action studies to identify new lead
compounds and targets for antifungal drug discovery. An example of this includes the discovery of an antifungal RNA
Pol III inhibitor.139 High-throughput screening of a large compound file was undertaken by measuring compound-
dependent inhibition of C. albicans growth (turbidometric readout). The benzothiophene UK-118,005 (Table 8)
mCoA
NMT/mCoA binary Peptide substrate (e.g., ARF)
complex
Target for benzothiazole
inhibitors
NMT Ternary complex
NMT-Myr-peptide
Myristoylated peptide CoA
Figure 7 The catalytic cycle of NMT and the site of inhibition for benzothiazole and peptide-based inhibitors.
Table 7 Benzothiazole inhibitors of NMT and antifungal properties
Compound Properties
H C. albicans NMT IC50 ¼ 1.4 mM

S N NH2 Human NMT IC504100 mM
O Antifungal MIC4200 mg mL 1 (all species)
O N
O
CP-123,457
O C. albicans NMT IC50 ¼ 40 nM

S Human NMT IC504100 mM
N H Antifungal MIC4200 mg mL 1 (all species)
N NH2
N
O
UK-370,485
C. albicans NMT IC50 ¼ 10 nM

H H Human NMT IC504100 mM
S N N
C. albicans MIC ¼ 3.1 mg mL 1
O
N O Non-albicans and Aspergillus MIC4200 mg mL 1
N
UK-362,091
Figure 8 Three-dimensional image of benzothiazole occupation of the Candida albicans NMT active site cleft (peptide-binding
site) as derived from x-ray diffraction of NMT-inhibitor co-crystals.
was found to have potent antifungal activity against Candida (all species) and Aspergillus, with a high therapeutic index
relative to human cell line cytotoxicity. A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to
identify the target of antifungal compounds. Three lines of evidence showed that UK-118,005 inhibited cell growth by
targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol
III, conferred resistance to the compound. Second, UK-118,005 rapidly inhibited tRNA synthesis in wild-type cells but
Tyr 225
Phe 115 IIe 111 WAT 762 Glu 109
Leu 415
WAT 887
WAT 898
Tyr 354
Phe 240
Leu 350 His 227

Thr 211
WAT 788 Leu 451
Phe 339 Asn 392
Figure 9 Two-dimensional representation of benzothiazole (UK-370,485) interactions with active site amino acid residues in
the Candida albicans NMT.
UK-370,485 − spectrum and selectivity
Phe339 − Candida albicans
lle111 − Candida albicans
Asp − Cryptococcus
Aspergillus
Candida resistant species
Human
Ser − Cryptococcus
Aspergillus
Candida resistant species
Human
Figure 10 Interaction of key amino acid residues of Candida albicans NMT with a benzothiazole highlighting inhibitor. Van der
Waals interaction of the benzothiazole moiety with the hydrophobic Ile111 and Phe339 residues of C. albicans NMT. The
equivalent polar residues (incompatible with respect to forming interactions with the benzothiazole) from other species are
highlighted.
Table 8 Antifungal profile of UK 118,005
Structure MIC (g mL 1)
C. albicans C. glabrata A. fumaigatus S. cerevisiae
6.3 3.1 6.3 0.78

N N
not in UK-118,005-resistant mutants. Third, in biochemical assays, UK-118,005 inhibited tRNA gene transcription
in vitro by the wild-type but not the mutant Pol III enzyme. Further examination showed UK-118,005 to inhibit
RNA Pol III transcription systems derived from C. albicans and human cells. The identification of these inhibi-
tors demonstrates that RNA Pol III can be targeted by small synthetic molecules, to exert a potent antifungal
effect. UK-118,005 showed broad-spectrum antifungal activity (MIC ¼ 3.1–12.5 mg mL–1 against Candida species and
A. fumigatus (Table 8). Unfortunately, this compound showed little selectivity for the fungal/human enzyme in
controlled kinetic assays, indicating the need for an extensive medicinal chemistry campaign to generate a fungal
selective inhibitor.
7.14.6.2.3 Acetyl-CoA carboxylase (ACCase)

ACCase catalyses the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA in a multistep reaction (Figure 11).
This is the first committed step in fatty acid synthesis, is rate-limiting for the pathway, and is tightly regulated.
Inhibition of ACCase in fungi results in fatty acid depletion, leading to rapid cell death due to membrane
dysfunction.140,141
ACCase is a universal enzyme, and has been researched in detail from a number of species. It has been particularly
well studied in plants, as ACCase is the target site for numerous highly successful herbicides. Herbicidal ACCase
inhibitors show great potency against grass ACCase activity, but do not inhibit mammalian, fungal, or even broadleaf
plant ACCase.142 This underpins their success as nontoxic and selective grassweed killers, and highlights ACCase as
having scope for selective inhibition with a lethal effect on the target species. The discovery of soraphen A (Figure 12),
a potent fungal ACCase inhibitor (IC50E30 nM) with broad spectrum and extremely rapid fungicidal activity (MIC
against Candida species in the sub-microgram per millilter range) triggered considerable interest in ACCase as a target
for antifungal drug discovery.141 It is therefore not surprising that gene knockout studies show ACCase to be an essential
enzyme in fungi.143,144
In light of the precedent for selective inhibition of ACCase and its association with extremely potent fungicidal
activity, high-throughput screening for this and other targets in the lipid pathway to find tractable chemical lead matter
are being pursued.145
7.14.6.2.4 Fungal protein biosynthesis inhibitors

Protein synthesis has long been considered as an attractive target in the development of antimicrobial agents, in light
of the widespread use of antibacterial antibiotics that target the specific areas of this process. However, application of
this idea to the field of antifungal therapy is not an easy task, due to the eukaryotic rather than prokaryotic nature of
−
ATP + HCO3 ADP + Pi
Biotin carboxylase
BCCP BCCP-COO−
Carboxyl transferase ----------------Target of Soraphen A
Acetyl-CoA Malonyl-CoA
Figure 11 Acetyl-CoA carboxylase (ACCase) catalysis of malonyl-CoA formation from acetyl-CoA. Catalysis is a multistep
process, taking place at different catalytic sites of a single multifunctional protein. The reaction proceeds via the ATP-dependent
carboxylation of a biotin group at the biotin carboxylase domain of ACCase, with the transfer of this (by a biotin carboxyl carrier
peptide – BCCP) to the carboxyl transferase domain of ACCase.
OMe
OH
O O
O OH
OH
OMe
Figure 12 Soraphen A.
H3C CH3
R O
O OH O
HO O HO O
CH3 CH3
OHC COOH O CH3 O CH3
(a) (b) (c)
Figure 13 Antifungal sordarins. (a) Core template, (b) parent sordarin (R group), and (c) GR1305402 (R group).
fungi, and therefore the great degree of similarity between the fungal and mammalian protein synthesis machineries.
Two soluble elongation factors show some fungal specificity: EF3, a factor that is required by fungal ribosomes only, and
EF2, which has been demonstrated to possess at least one functional distinction from its mammalian counterpart. The
sordarins are the most important family of antifungal agents acting at the protein synthesis level. Compounds in this
class inhibit in vitro translation in C. albicans, C. tropicalis, C. kefyr, and C. neoformans, to varying degrees.117 The lack of
activity of the sordarins against C. krusei, C. glabrata, and C. parapsilosis, in comparison with their extremely high levels of
potency against C. albicans, suggests that these compounds have a highly specific binding site, which may also be the
basis for the greater selectivity of these compounds in inhibiting fungal, but not the mammalian, protein synthesis. The
most advanced inhibitors of fungal protein biosynthesis are analogs of the natural product lead sordarin lead, GR135402
(see Figure 13).146,147 Its spectrum of activity includes C. albicans, C. tropicalis, and C. neoformans, where impressive
antifungal activity has been observed in vitro (MICo1 mg mL 1 in many cases), but not in other Candida or Aspergillus
species, which severely restricts its potential as a quality lead. Efficacy has been seen in animal models,147 although at
high dose and predominantly via nonoral routes, reflecting the potential for rapid clearance and limited oral
bioavailability.
7.14.7 Further Reading

The textbook edited by Warnock and Richardson3 and the two volumes edited by San-Blas and Calderone1,148 are
convenient and comprehensive reference points for many aspects of fungal infections and antifungal drugs. In addition,
there are a number of useful web resources,37,149,150 and Chapter 7.15 should be consulted.
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Biography
Patrick Dorr gained his BSc in microbiology from Sheffield University, UK, in 1987, and PhD from Kent University,
UK, in 1990, following research in bacterial metabolism in Professor Chris Knowles’ laboratory. A subsequent move to
industry resulted in 5 years at Rhone-Poulenc, researching into new targets for pesticide discovery. In 1995, he moved
to Pfizer GRD in Sandwich, as a team leader in antifungals discovery, undertaking research in many aspects of drug
discovery, from new target validation and lead seeking to efficacy studies in infection models. He is currently leading
the CCR5 and HIV entry preclinical teams for the discovery of new antiviral drugs.
& 2007 Elsevier Ltd. All Rights Reserved Comprehensive Medicinal Chemistry II
No part of this publication may be reproduced, stored in any retrieval system or transmitted ISBN (set): 0-08-044513-6
in any form by any means electronic, electrostatic, magnetic tape, mechanical, photocopying,
recording or otherwise, without permission in writing from the publishers ISBN (Volume 7) 0-08-044520-9; pp. 419–443

7.14 Fungi and Fungal Disease

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7.

14 Fungi and Fungal Disease

7.14.1 Medically Important Fungi and Major Fungal Diseases 420

7.14.1 Medically Important Fungi and Major Fungal Diseases

7.14.1.2 Human Pathogenic Fungi and Disease States

7.14.1.2.1 Superficial mycoses

7.14.1.2.2 Subcutaneous mycoses

7.14.1.2.3 Cutaneous mycoses

Table 1 Diseases and causative organisms of superficial mycoses

Disease Causative organism

Pityriasis versicolor (Figure 1a) Malassezia furfur (a lipophilic yeast)

Tinea negra Exophiala werneckii

White piedra Trichosporon beigelii

Black piedra Piedraia hortae

Disease Causative organism Presentation

Sporotrichosis Sporothrix schenckii

Chromoblastomycosis Fonsecaea, Phialophora, Cladosporium

Phaeohyphomycosis Cladosporium, Exophiala, Wangiella,

Mycotic mycetoma Pseudallescheria, Madurella,

Subcutaneous zygomycosis (including Basidiobolus ranarum, Conidiobolus

Rhinosporidiosis Rhinosporidium seeberi

Disease Causative organism Presentation

Dermatophytosis. Dermatophytes (Microsporum,

Candidiasis of skin, mucous Candida albicans and related species

Dermatomycosis Nondermatophyte moulds (Hendersonula

7.14.1.2.4 Dimorphic systemic mycosis

Table 4 Dimorphic systemic mycoses

Disease Causative organism Presentation

Histoplasmosis Histoplasma capsulatum

Coccidioidomycosis Coccidioides immitis

Blastomycosis Blastomyces dermatitidis

Paracoccidioidomycosis Paracoccidioides brasiliensis

7.14.1.2.5 Opportunistic systemic mycoses

Table 5 Opportunistic systemic mycoses

Disease Causative organism

Candidiasis Candida albicans and other Candida spp.

Aspergillosis Aspergillus fumigatus

Cryptococcosis Cryptococcus neoformans

Pseudallescheriasis Pseudallescheria boydii

Zygomycosis (mucormycosis) Rhizopus, Mucor, Rhizomucor, Absidia, etc.

Hyalohyphomycosis Penicillium, Paecilomyces, Beauveria, Fusarium, Scopulariopsis, etc.

Phaeohyphomycosis Cladosporium, Exophiala, Wangiella, Bipolaris, Exserohilum, Curvularia

7.14.1.3 Emerging Fungal Diseases

7.14.2 Pathogenesis of Major Fungal Diseases

7.14.2.1 Candida albicans and Candida Species

7.14.3 Host Defenses

7.14.4 Diagnosis of Fungal Infection

7.14.4.1.1 Microscopy and culture of pathogenic fungi

7.14.4.1.2 Diagnosis of fungal infection by metabolite detection

7.14.4.1.3 Diagnosis of fungal infection by gene sequence detection

7.14.5 Current Antifungal Drugs: Clinical Pharmacology and Treatment

7.14.5.2 Superficial Mycoses

Table 6 Licensed and late-stage development antifungal drugs

Antifungal class (mode of action) Drug Route

Allylamines (squalene epoxidase) Amorolfine Topical

Terbinafine Oral, topical

Antimetabolite (DNA) Flucytosine Oral

Azoles (cytochrome P450 demethylase) Fluconazole Oral, intravenous

Itraconazole Oral, intravenous

Structure MIC (g mL 1)