Antifingal drug discovery TOP ARTICLE SUPPLEMENT CONTENTS COMMENTARY: The GPI anchor pathway: a promising antifungal target? Future Medicinal Chemistry Vol. 8 Issue 12 COMMENTARY: How can we bolster the antifungal drug discovery pipeline? Future Medicinal Chemistry Vol. 8 Issue 12 REVIEW: Strategies in the discovery of novel antifungal scaffolds Future Medicinal Chemistry Vol. 8 Issue 12 REVIEW: Fungal biofilm composition and opportunities in drug discovery Future Medicinal Chemistry Vol. 8 Issue 12 Powered by
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Antifingal drug discoveryTOP ARTICLE SUPPLEMENT
CONTENTSCOMMENTARY: The GPI anchor pathway: a promising antifungal target? Future Medicinal Chemistry Vol. 8 Issue 12
COMMENTARY: How can we bolster the antifungal drug discovery pipeline? Future Medicinal Chemistry Vol. 8 Issue 12
REVIEW: Strategies in the discovery of novel antifungal scaffolds Future Medicinal Chemistry Vol. 8 Issue 12
REVIEW: Fungal biofilm composition and opportunities in drug discovery Future Medicinal Chemistry Vol. 8 Issue 12
A key challenge with developing antifungal drugs is that both humans and fungi are eukaryotic organisms; as a result, antifungal therapeutics may have substantial toxicity due to inhibition of closely related human targets. Compounding this challenge, the principal clinically relevant fungal pathogens Aspergillus fumigatus and Candida albicans diverged from a common ancestor 1 billion years ago [1], underscoring their substantial genetic and pathogenic diversity and the dif-ficulty of new antifungal agents achieving broad-spectrum activity. Identifying thera-peutics that can both overcome this diversity as well as remain well tolerated in human subjects has proven elusive. As a result, there are only three mechanistically distinct classes of antifungal therapeutics that are currently employed to treat serious life-threatening infections due to both yeast and molds.
There have been multiple ‘calls to action’ for the development of new antifungal therapeu-tics and many discussions of what the optimal properties of the ‘ideal’ therapeutic might be: 1) activity against fungi that are intrinsically resistant to currently marketed antifungal agents; 2) broad-spectrum activity, especially versus a wide array of molds that are difficult to diagnose in the clinic; 3) no cross resistance with current antifungals; 4) the ability to bol-ster the immune response to combat mycoses; and 5) fungicidal activity [2,3]. Within this framework, recent work on inhibitors of gly-cosylphosphatidylinositol (GPI) biosynthesis
will be considered for their potential as new entrants in the antifungal armamentarium.
With the increased prevalence of drug-resis-tant species of both yeasts and molds as caus-ative pathogens of invasive, life-threatening infections, there have been renewed efforts to identify novel, broad-spectrum antifungal agents that can provide therapeutic benefit to improve clinical outcomes. GPI biosynthe-sis is a conserved and essential process that is required for cell wall biogenesis in both yeasts and molds [4,5]. The role of GPI in anchoring proteins to the outer wall of fungi underscores its function in maintaining cell wall integ-rity [6]. Due to the prevalence of this pathway in fungal species, GPI biosynthesis presents an attractive target for the development of a broad-spectrum antifungal compound that is also a cell wall active agent. Indeed, numerous cell wall anchored proteins have been identified that rely on the GPI post-translational modifi-cation for cell surface localization (Saccharo-myces cerevisiae has ∼60) and chemical genet-ics approaches have proven highly successful in identifying these GPI-anchored proteins as well as cognate small molecule inhibitors of GPI biosynthesis [7,8]. The structures of known GPI pathway inhibitors described herein as well as a representation of the GPI precursor and enzymatic steps of GPI biosynthesis are shown in Figure 1. However, as orthologous GPI biosynthetic enzymes exist in man, fungal specificity of such agents remains an important consideration.
“The activity of Gwt1 inhibitors as well as Mcd4 inhibitors versus a broad range of yeast and molds and lack of observed cross-resistance with existing therapeutics should encourage further investigation for
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One antifungal drug target identified by chemi-cal genetics that is part of the GPI pathway is Gwt1, an enzyme that is required for the assembly of GPI-anchored proteins that are later attached to the fungal cell wall [6]. This enzyme has been further characterized as an acyltransferase that is required for the produc-tion of acylated GPI. Importantly, acylated GPI forms a required anchor that attaches mannoproteins to the cell wall of yeasts including C. albicans [7,9]. This target
has been explored by the pharmaceutical company Eisai Co. Ltd because of: 1) low homology (<30% amino acid sequence identity) with the closest mammalian ortholog, PIG-W; 2) the critical role of Gwt1 in maintaining the integrity of the fungal cell wall; 3) the role of Gwt1 in enabling fungal cell adhesion to host mucosal surfaces; 4) the potential to avoid cross-resistance with existing antifungal therapeutics [10]. A challenge with producing a rationally designed small molecule Gwt1 inhibitor is
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The GPI anchor pathway: a promising antifungal target? Commentary
due to the lack of 3D structural elucidation of the target, likely because Gwt1 has an estimated 13 transmembrane domains [9]. As a result, the drug discovery effort at Eisai Co. started with a chemical library screen employing a newly developed reporter assay for localization of the S. cerevisiae GPI-anchored mannoprotein, Cwp2, to the cell wall. After screening their corporate library, the hit compound 1-[4-butyl-benzyl-isoquinoline] (BIQ1) was identified [7]. Analog design was then based on (BIQ1) as an inhibitor of Gwt1 [7], and these analogs were refined by phenotypic screening for target engagement and a ntifungal activity both in vitro and in vivo [11].
This extensive effort yielded a lead compound, E1210 (now known as APX001A), that has shown promising broad-spectrum activity against both yeasts and molds, and was well tolerated when administered to mice at therapeutic doses [10]. Importantly, E1210 exhibited no measurable inhibition of PIG-W, the clos-est human ortholog of fungal Gwt1. E1210 also exhib-its >40% oral bioavailability in multiple mammalian species [12]. Moreover, this compound has shown prom-ising activity versus fungal pathogens that have proven challenging to treat with the existing antifungal arma-mentarium, including Mucorales, Fusarium solani and Scedosporium prolificans. In addition, the compound maintained activity versus strains of Candida spp. or Aspergillus spp. that are broadly resistant to azoles, echinocandins or amphotericin B [13,14].
A second effort to target GPI biosynthesis has been recently described and employed chemically induced haploinsufficiency screening to identify small molecule inhibitors of fungal growth [8]. Briefly, the technique used a library of C. albicans heterozygous deletion mutants consisting of about 5400 heterozygote deletion mutants and covering >90% of the fungal genome [15,16]. The response of this mutant library to the presence of sub-MIC concentrations of inhibitor candidate compounds was then characterized by an abundance assay employ-ing DNA microarray analysis. This analysis yielded the relative abundance of specific mutants after compound treatment, and reflects the sensitivity or insensitivity of a mutant to the molecule tested. Although this screen did not specifically target the GPI pathway, small molecules were identified that targeted Gwt1 (G884 and G365) as well as a second GPI pathway enzyme, Mcd4 (M743 and M720) [8]. Mcd4 was previously characterized as an ethanolamine phosphotransferase [17]. Interestingly, the Mcd4 inhibitors, a novel natural product scaffold (M743) as well as a semisynthetic derivative (M720) resulting from the screen revealed a broader spectrum of antifungal activity compared with the Gwt1 inhibitors tested. However, the Gwt1 inhibitors showed lower cyto-toxicity relative to the Mcd4 inhibitors, likely because the identified Mcd4 inhibitors also exhibit inhibition of
GPI biosynthesis in mammalian cells [17]. Nonetheless, due to the broad spectrum in vitro activity of the Mcd4 inhibitors as well as demonstrated in vivo efficacy with-out signs of acute toxicity in a murine infection model of candidiasis, Mcd4 appears to merit further explora-tion to identify potent inhibitors that have improved s electivity toward fungal cells versus mammalian cells.
Another recent report identified the small molecule, gepinacin, as a Gwt1 inhibitor [18], originally selected as a false positive from a screen for heat shock protein inhi-bition. Gepinacin exhibits broad-spectrum antifungal activity, albeit at higher concentrations than E1210 or Mcd4 inhibitors. Interestingly, the report also highlights the potential of Gwt1 inhibitors in enabling recognition of fungi by the immune system [18]. The immune system has multiple pathways for mounting a response to fungal infections including complement, antibody and innate immune Toll-like receptors [19]. Notably, Dectin-1 serves as a pattern recognition receptor that recognizes the pathogen-associated ligand β-(1,3)-glucan within the fungal cell wall. However, fungal cells naturally evade this strategy of host immune recognition via the formation of an outer GPI-derived mannoprotein layer at the cell surface, effectively masking the presence of their underlying β-(1,3)-glucan within the cell wall [19]. Candida albicans treated with sub-MIC levels of any of the above-described GPI inhibitors exposes this under-lying β-(1,3)-glucan [8,18]. Consequently, GPI inhibitor-treated C. albicans incubated with a macrophage cell line results in a marked increase in the secretion of TNF-α, an important cytokine associated with systemic inflam-matory responses [8,18]. Notably, the enhanced immune system recognition provoked by gepinacin persisted in the presence of both the yeast (nonfilamentous) and hyphal (filamentous) forms of C. albicans [18]. In contrast, echinocandins are able to provoke TNF-α secretion only in the hyphal form of C. albicans [19]. Although the clini-cal implications of these observations are unknown, the potential for Gwt1 inhibitors to enable better immune recognition of pathogenic fungi is intriguing.
An additional aspect of the promise of GPI biosyn-thesis inhibitors in treating fungal infections is their demonstrated synergy with echinocandins. In contrast with cancer chemotherapy and antibacterial regimens, combination therapeutic strategies for invasive fungal infections are uncommon. The main exception is the use of flucytosine with amphotericin B for the treatment of cryptococcal meningitis, which has become the standard of care. A primary reason for the dearth of combina-
“...the promise of GPI biosynthesis inhibitors in treating fungal infections is their demonstrated
synergy with echinocandins.”
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Commentary Mutz & Roemer
tion approaches has been the historical demonstration of indifferent or antagonistic interactions between clini-cally used antifungals such as amphotericin B and azoles or between echinocandins and azoles [20,21]. In contrast, the Gwt1 inhibitor E1210 has demonstrated in vitro synergy with multiple echinocandins versus numerous strains of A. fumigatus and Aspergillus flavus [22]. In addi-tion, combinations of the two drug classes in vivo have demonstrated efficacy greater than either drug alone [22]. This synergy makes mechanistic sense because in the absence of a coating of GPI-anchored mannoproteins, the β-glucan layer of the cell wall is directly exposed to the echinocandin, affording better access to the echino-candin drug target, β-(1,3)-glucan synthase. For reasons that are not clear, the presence of synergy was less consis-tent between E1210 and azoles or echinocandins versus different species of Candida. Importantly, there was no observation of antagonism between E1210 and azoles, echinocandins and amphotericin B versus all yeasts and molds tested. Finally, Gwt1 and Mcd4 inhibitors dem-onstrate strong chemical synergy with each other, par-alleling the synthetic lethality of genetic mutations to these targets in S. cerevisiae, suggesting an alternative strategy to consider such inhibitors as novel combination agents [8].
Although inhibitors of GPI biosynthesis offer poten-tial as broad-spectrum therapeutics, a possible short-coming is the fungistatic activity of the molecules as described in recent literature reports [8,14]. However, for E1210, there was a substantial postantifungal effect (PAFE), which is a measurement of how long it takes the test compound-treated fungus to regrow after removal of the test compound for in vitro studies. PAFE was also measured in vivo by assessing regrowth after a single dose of test compound [23]. For C. albicans, the in vitro PAFE was 3.9 h at 16 × MIC (0.13 μg/ml) and 11 h in vivo after a single oral dose of 10 mg/kg in a neutropenic mouse model of invasive candidiasis. For comparison, flucon-azole has no significant in vitro PAFE in C. albicans [24]. One rationale for the significant PAFE of E1210 is that the target, Gwt1, is part of a multistep, essential pathway
in producing GPI anchors, and this defect in the path-way stresses the endoplasmic reticulum. For example, GPI-anchored protein Gas1 is normally transported to the outer cell wall in yeast. In the presence of gepinacin, the Gas1 precursor accumulates in the endoplasmic retic-ulum at unusually high levels [18]. In addition, gepina-cin induces a considerable unfolded protein response and this also creates stress on the fungi [18]. Therefore, the observed PAFE may be due to the time required for Gwt1 inhibitor-treated fungal cells to recover from these defects in protein trafficking and cellular stress responses.
In summary, targeting GPI biosynthesis may offer considerable potential to develop novel, broad-spec-trum antifungal therapeutics. The challenge of devel-oping these GPI targets has likely stemmed from the lack of 3D structural elucidation of the component proteins. In the case of both Gwt1 and Mcd4, a struc-tural challenge is posed by their multiple transmem-brane topologies, thereby complicating a protein crys-tallization effort to assist the rational design of more potent and specific leads. Despite this challenge, mul-tiple groups have discovered highly potent, selective hit and lead compound inhibitors of these targets. Indeed, in the case of Gwt1, cognate inhibitors display potent broad-spectrum antifungal activity lacking appreciable cross-activity against its closest human ortholog, PIG-W. The activity of Gwt1 inhibitors as well as Mcd4 inhibitors versus a broad range of yeast and molds and lack of observed cross-resistance with existing thera-peutics should encourage further investigation for novel antifungal leads targeting GPI biosynthesis.
Financial & competing interests disclosureM Mutz is a founder and shareholder of Amplyx Pharmaceu-
ticals. T Roemer is an employee of Merck & Co. The authors
have no other relevant affiliations or financial involvement
with any organization or entity with a financial interest in or fi-
nancial conflict with the subject matter or materials discussed
in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this
manuscript.
References1 Cowen LE, Singh SD, Kohler JR et al. Harnessing Hsp90
function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc. Natl Acad. Sci. USA 106(8), 2818–2823 (2009).
2 Roemer T, Krysan DJ. Antifungal drug development: challenges, unmet clinical needs, and new approaches. Cold Spring Harb. Perspect. Med. 4(5), pii: a019703 (2014).
3 Denning DW, Bromley MJ. Infectious disease. How to bolster the antifungal pipeline. Science 347(6229), 1414–1416 (2015).
4 Leidich SD, Drapp DA, Orlean P. A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis. J. Biol. Chem. 269(14), 10193–10196 (1994).
5 Li H, Zhou H, Luo Y, Ouyang H, Hu H, Jin C. Glycosylphosphatidylinositol (GPI) anchor is required in Aspergillus fumigatus for morphogenesis and virulence. Mol. Microbiol. 64(4), 1014–1027 (2007).
6 Umemura M, Okamoto M, Nakayama K et al. GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast. J. Biol. Chem. 278(26), 23639–23647 (2003).
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The GPI anchor pathway: a promising antifungal target? Commentary
7 Tsukahara K, Hata K, Nakamoto K et al. Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly. Mol. Microbiol. 48(4), 1029–1042 (2003).
8 Mann PA, Mclellan CA, Koseoglu S et al. Chemical genomics-based antifungal drug discovery: targeting glycosylphosphatidylinositol (GPI) precursor biosynthesis. ACS Infect. Dis. 1(1), 59–72 (2015).
9 Sagane K, Umemura M, Ogawa-Mitsuhashi K, Tsukahara K, Yoko-O T, Jigami Y. Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p. J. Biol. Chem. 286(16), 14649–14658 (2011).
10 Hata K, Horii T, Miyazaki M et al. Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis. Antimicrob. Agents Chemother. 55(10), 4543–4551 (2011).
11 Eisai R&D Management Co., Ltd, Tokyo (JP): US 769,882 B2 (2010).
12 Hata K, Yamamoto E, Okubo M. Physiochemical properties and nonclinical pharmacokinetics of E1211, a water-soluble prodrug of E1210. Presented at: 51st Interscience Conference of Antimicrobial Agents and Chemotherapy (ICAAC) IL, USA, 17–20, September 2011.
13 Pfaller MA, Duncanson F, Messer SA, Moet GJ, Jones RN, Castanheira M. In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Aspergillus spp. determined by CLSI and EUCAST broth microdilution methods. Antimicrob. Agents Chemother. 55(11), 5155–5158 (2011).
14 Miyazaki M, Horii T, Hata K et al. In vitro activity of E1210, a novel antifungal, against clinically important yeasts and molds. Antimicrob. Agents Chemother. 55(10), 4652–4658 (2011).
15 Roemer T, Xu D, Singh SB et al. Confronting the challenges of natural product-based antifungal discovery. Chem. Biol. 18(2), 148–164 (2011).
16 Xu D, Jiang B, Ketela T et al. Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans. PLoS Pathog. 3(6), e92 (2007).
17 Hong Y, Maeda Y, Watanabe R et al. Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol. J. Biol. Chem. 274(49), 35099–35106 (1999).
18 Mclellan CA, Whitesell L, King OD, Lancaster AK, Mazitschek R, Lindquist S. Inhibiting GPI anchor biosynthesis in fungi stresses the endoplasmic reticulum and enhances immunogenicity. ACS Chem. Biol. 7(9), 1520–1528 (2012).
19 Wheeler RT, Kombe D, Agarwala SD, Fink GR. Dynamic, morphotype-specific Candida albicans beta-glucan exposure during infection and drug treatment. PLoS Pathog. 4(12), e1000227 (2008).
20 Drogari-Apiranthitou M, Mantopoulou FD, Skiada A et al. In vitro antifungal susceptibility of filamentous fungi causing rare infections: synergy testing of amphotericin B, posaconazole and anidulafungin in pairs. J. Antimicrob. Chemother. 67(8), 1937–1940 (2012).
21 Johnson MD, Macdougall C, Ostrosky-Zeichner L, Perfect JR, Rex JH. Combination antifungal therapy. Antimicrob. Agents Chemother. 48(3), 693–715 (2004).
22 Watanabe N-A, Horii T, Miyazaki M, Hata K. In vitro activity of E1210 and in vivo activity of E1211, a water-soluble prodrug of E1210, in combination with other antifungals. Presented at: 51st Interscience Conference of Antimicrobial Agents and Chemotherapy (ICAAC) IL, USA, 17–20, September 2011.
23 Horii T, Okubo M, Miyazaki M, Hata K, Watanabe N-A. In vivo pharmacodynamic correlates of success for E1210 treatment of disseminated candidiasis. Presented at: 50th Interscience Conference of Antimicrobial Agents and Chemotherapy (ICAAC). MA, USA, 12–15 September 2010.
24 Anil S, Ellepola AN, Samaranayake LP. Post-antifungal effect of polyene, azole and DNA-analogue agents against oral Candida albicans and Candida tropicalis isolates in HIV disease. J. Oral Pathol. Med. 30(8), 481–488 (2001).
FutureMedicinalChemistry
part of
Commentary
How can we bolster the antifungal drug discovery pipeline?
First draft submitted: 1 June 2016; Accepted for publication: 10 June 2016; Published online: 27 July 2016
Keywords: antifungals • combination • drug design and development • fungicides • mycoses • nanoparticles • resistance
Fungi are simple eukaryotic organisms that have colonized diverse environments around the planet. There are approximately 2 mil-lion different species of fungi on the Earth [1]. Their coexistence with other organisms can vary between mutually beneficial mutualism, commensalism and parasitism. Higher fungi have a long history of use in national cuisines, brewery, viticulture and folk medicine. Inves-tigations of isolated secondary metabolites of higher fungi as well as microfungi have resulted in the discovery of bioactive com-pounds as potential lead structures for the subsequent design and development of new drugs and other biologically active agents. Yeasts and other fungal species are also used in biotechnology for production or biotrans-formation of various agents used in medi-cine [2] as well as for synthesis of n anoparticles within green nanotechnology [3].
Fungi are ubiquitous in nature and vital for recycling of nutrients contained in organic matter. The vast majority of the known fungal species is strict saprophytes, and based on the above-mentioned facts they are very useful. However, some of them can attack humans, animals and plants; it is estimated that 270,000 fungal species are associated with plants, and 325 are known to infect humans [1,4]. Fungi are part of the skin and mucosal microflora of each person. Nevertheless, they can cause diseases varying from unpleasant superficial and cutaneous forms via subcutaneous complicated forms
to systemic frequently fatal diseases. Diseases caused by colonization, proliferation and sporulation of fungi in tissues or body fluids are known as mycoses, and invasive fungal infections are the most fatal [5].
It is estimated that about 1.2 million peo-ple worldwide suffer from fungal diseases, but a substantial part of these infections are invasive or chronic, and such fungal infec-tions are difficult to diagnose and treat. Annually, 1.5 to 2 million people die from fungal infections worldwide, which is more than from malaria or tuberculosis. Genera Candida (C. albicans, parapsilosis, krusei, glabrata, tropicalis) and Malassezia furfur cause mycoses the most frequently from yeast micro-organisms. C. glabrata, the sec-ond most frequently isolated Candida in the EU (>10%) and in the USA (>20%) in the last decade, represents a risk due to its high resistance to fluconazole, voriconazole and echinocandins. Filamentous fungi, such as Trichophyton, Epidermophyton and Micros-porum spp. are the most frequent etiological species of superficial mycoses [1,6].
Invasive fungal infections constitute life-threatening diseases especially for immuno-compromised population, in other words, patients with AIDS, diabetes mellitus, epi-dermal and skin lesions, burns, malnutri-tion, patients undergoing anticancer chemo-therapy, transplantation, long-term corticoid therapy or wide-spectrum antibacterial che-motherapeutics. There is also a danger of
“Vaccines with antifungal effects seem to hold significant promise for the future.”
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Commentary Jampilek
development of invasive fungal infections in case of surgery, catheterization, hemodialysis and parenteral nutrition patients. Very old age and preterm birth are risk factors. The most frequent fungal pathogens caus-ing high mortality are Candida, Aspergillus, Fusarium, Cryptococcus and Pneumocystis. The emerging global threat is the increasing resistance of these species to clinically used antifungals; even cross-resistant, mul-tidrug- or totally resistant strains to all clinically used antifungals can be found and constitute a serious p roblem [5,6].
Approved antifungal drugs & their new analoguesHuman fungal infections generally receive less atten-tion than viral or bacterial diseases; however, mortal-ity from invasive fungal infections is very high, often exceeding 50%. Antifungals are drugs that destroy or prevent the growth of fungi (yeasts, moulds). They can be divided into two main classes: nonspecific antifungals, and site-specific antifungals. Nonspecific antifungals (disinfectants and antiseptics) are applied for superficial/local treatment of skin or mucosa. Although the first specific antimycotic was approved in the 1950s, and since then there has been a signifi-cant increase in the number of antifungal drugs used in clinical practice, the treatment of invasive fungal infections is restricted due to the limited number of systemically administered drugs [1,6,7].
Clinically used site-specific antifungal drugs can be classified according to the mode of action and their chemical structure as follows: drugs affecting ergos-terol, drugs interacting with cell wall (glucan syn-thesis inhibitors – echinocandins, pneumocandins), inhibitors of transport processes (ciclopirox) and inhibitors of nucleic acid synthesis (flucytosine), pro-tein synthesis (tavaborole) and microtubules synthesis (griseofulvin). Most of the drugs target and inhibit ergosterol-synthesizing enzymes (azoles, thiocarba-mates, naphthylmethylamines, phenylpropylmor-pholines) or bind to ergosterol in the cell membrane (polyene antimycotics) [7,8]. Unfortunately, most of drugs have been approved for the treatment of myco-ses of nails, skin and mucosa especially due to their narrow therapeutic window, limited bioavailability and drug resistance. Only 5 triazoles (fluconazole, isavuconazole, itraconazole, posaconazole, voricon-azole), 3 echinocandins (anidulafungin, caspofungin, micafungin), 1 polyene macrolide (amphotericin B), 1 naphthylmethylamine (terbinafine), 1 pyrimidine (flucytosine) and 1 benzofuran (griseofulvin) have
been approved for the treatment of systemic fungal infections [8].
Some new antifungals have been recently approved for treatment of human mycoses, or have been in clinical trials, for example, new azoles (lanosterol 14-α-demethylase inhibitors) such as dapaconazole (Zilt® [Biolab Sanus Farmaceutica, Sao Paulo, Brazil]), efinaconazole (Jublia® [Valeant Pharmaceuticals, Que-bec, Canada]), luliconazole (Luzu® [Medicis Pharma-ceutical, AZ, USA], Lulicon® [Pola Pharma, Tokyo, Japan]) [8], albaconazole (UR-9825), VT-1161 [9] and VT-1129 – a promising candidate for systemic therapy [10]. Also new glucan synthesis inhibitors ASP9726 and biafungin (CD101) from the class of echinocandins have been subject to clinical trials [9]. An orally active derivative of triazole enfumafungin, SCY-078 (MK-3118), is at the early stage of clinical development. Although it possesses the same mecha-nism of action as other echinocandins, it demonstrates enhanced efficacy for most echinocandin-resistant isolates of C. albicans and C. glabrata as well as for Aspergillus spp. [9]. It is important to note that also 5-[4-(sulfonyl)piperazin-1-yl]-2-arylpyridazin-3(2H)-ones (SCH A–D) and 1-pyrrolidinyl-pyridobenzimid-azole-4-carbonitriles (D11-2040, D21-6076) [11] are investigated as new nonechinocandin β-(1,3)- and β-(1,6)-D-glucan synthesis inhibitors.
Emerging resistanceMechanisms of resistance to antifungals differ among groups of drugs mainly due to the mode of action of each class of antifungals; however, in general resistance can be classified as follows: natural resistance (micro-organisms lack the target structure of the drug, and all isolates of the species are resistant; e.g., resistance of some nonalbicans Candida sp. to azoles, amphotericin B); acquired resistance (micro-organisms obtain the ability to resist the activity of the drug to which it was previously susceptible); clinical resistance (therapeutic failure caused by drug pharmacokinetics, drug–drug interactions, patient immunity). The acquired resis-tance is the most serious. This can result from the mutation of genes involved in normal physiological processes and cellular structures, from the acquisition of foreign resistance genes or from a combination of these two mechanisms [5]. Mechanisms of antifungal resistance can be classified as follows: changes in anti-fungal transport – in other words, a decrease of effective drug concentration (efflux pumps overexpression or influx decrease; resistance to azoles, naphthylmethyl-amines and flucytosine); changes in the target struc-ture (enzyme alterations or deficiency/overproduction of some structural components; resistance to azoles, polyenes, echinocandins and naphthylmethylamines);
“The emergence of antifungal drug resistance calls for the urgency to design new antifungals.”
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How can we bolster the antifungal drug discovery pipeline? Commentary
the use of compensatory mechanisms (metabolic enzyme alterations, metabolic bypasses, toxic-product tolerance; resistance to azoles, polyenes, flucytosine); biofilm formation (complex mechanisms resulting in total changes of micro-organism p roperties; resistance to azoles, polyenes) [5,12].
Changes in transport inside/outside the cell are the most frequent mechanism of resistance. Influx condi-tioned resistance is caused by facilitated diffusion of drugs to fungal cells and changes in the composition of the membrane. Drug efflux is connected with over-expression of transport proteins that are able to bind structurally and functionally unrelated compounds, and their stimulation can cause development of mul-tidrug resistance or cross-resistance. Both well-known ATP binding cassette and major facilitator superfamily transporters can be found in the fungal cell membrane. Thus, considerable attention is devoted to understand their structure, function and regulation in order to design/find blockers/modulators of these transporters and sensitize again resistant strains to clinically used drugs [12].
New trends in design of antifungal drugsThe emergence of antifungal drug resistance calls for the urgency to design new antifungals. The discovery procedure of agents with a new mode of action is rela-tively long and risky; therefore, the approach of the first-choice can be preparation of nanoparticles/nano-formulations of existing drugs. Nanomaterials repre-sent a noteworthy alternative for treatment and mitiga-tion of infections caused by resistant pathogens, which are unlikely to develop resistance to nanomaterials. In contrast to conventional drugs, nanomaterials exert efficiency through various mechanisms; in addition to the drug activity itself, they show ‘intrinsic effects’, such as damaging membrane morphology, disruption of transmembrane energy metabolism and the mem-brane electron transport chain, generation of reactive oxygen species, etc. In addition, application of nano-formulations enhances the bioavailability of active substances (specific nanoformulations also provide a controlled release system or targeted biodistribution), and the route of administration can be modified. An increase of the efficacy of individual agents can be also ensured by fixed-dose drug combinations or anti-fungal-active matrices. The US FDA approved some nanoformulations of amphotericin B, for example, Abelcet® (Cephalon Ltd., Harlow, UK), AmBisome® (Gilead Sciences Inc., Uxbridge, UK; Astellas Pharma, IL, USA), Amphotec® (Three Rivers Pharmaceuticals LLC, PA, USA; Kadmon Pharmaceuticals LLC, NY, USA) and Fungizone® (Bristol-Myers Squibb, NY, USA) . Nanoformulations of other antifungal drugs
or antifungals conjugated with metal or metal oxide nanoparticles for reinforcement of their effect have been investigated. In addition, nanoformulations of silver, gold, copper, iron or zinc have been extensively tested [13].
A different approach consists in combining anti-fungal drugs, especially azoles and amphotericin B, with other known drugs or newly designed molecules (e.g., aminoglycosides, antiprotozoals, antipsychot-ics, calcium channel blockers, berberine, milbemycin, calcineurin inhibitors, HSP90 inhibitors, etc.) that together demonstrate synergistic antifungal properties. These synergistic combinations help to enhance or restore antifungal efficiency of drugs against resistant fungal strains. The main mechanisms of these syner-gistic effects seem to be perturbation of membrane, disturbance of intracellular ion homeostasis, inhibition of efflux pumps, inhibition of the activity of enzymes required for fungal survival and biofilm formation inhibition [14–19].
The most valuable approach to drug R&D is a ratio-nal design of new entities from new chemical classes influencing new targets, in other words, with new mechanisms of action. However, this process is the most expensive, lengthy and uncertain as to the outcome. Also design of new entities from new chemical classes influ-encing known targets or modification of known entities to impact new targets can be used, although it can be stated that drugs obtained by these two approaches have limited possibilities to solve this critical situation with increasing resistance. One of the reasons, why the R&D process of new antifungals is so complex, is the fact that the eukaryotic nature of a fungal cell is very similar to that of a human cell. Therefore, it is very important to search for antifungal agents, the mechanism of action of which targets the specific structure of the fungal cell.
Pharmaceutical industry is closely connected with agrochemical, especially pesticide producers. Some classes of new modern agricultural fungicides can be used for design of structurally new antifungal drugs, since many of them meet criteria of lead-likeness and/or drug-likeness, such as the Lipinski’s Rule of Five and the Carr’s Rule of Three. Most newly marketed fungicides have physicochemical parameters within the lead-like range for drugs. In addition, modern fungicides target active sites with high specificity and affinity (in nano-molar [or lower] concentrations); they are subjected to lead optimization and thus fulfill other requirements of lead chemistry such as tractability in structure–activ-ity relationships and lack of reactivity or promiscuous
“The mechanism of action of the majority of existing antifungals used in clinical practice is
associated with the cell wall.”
1396 Future Med. Chem. (2016) 8(12) future science group
Commentary Jampilek
binding. The similarity of drugs and pesticides was sup-ported by comparison of the frequency of occurrence of structural fragments in the two types of compounds. Extensive toxicology evaluation, including mammalian toxicology assays, is routinely performed during the whole discovery and development process [7]. Commer-cial agricultural fungicides are classified according to their target sites by the international Fungicide Resis-tance Action Committee (FRAC) [20]. From the FRAC classification, it is evident that fungicides show much higher diversity in their chemical structures and modes of action than antifungals. Also as in the case of anti-fungal drugs, among agricultural fungicides inorganic, organometallic or organic site-specific fungicides or multi-site fungicides can be found. The most interesting groups of modern specific-target fungicides that could accelerate the process of identification of new modes of action and leads/lead-like structures for a pharmaceuti-cal pipeline to control human fungal pathogens include, for example, inhibitors of adenosin-deaminase, cellulose synthase, 3-keto reductase within ergosterol biosynthe-sis, DNA/RNA synthesis, trehalase- and inositol-syn-thesis, respiration (especially cytochrome bc1), biosyn-thesis of melanin in cell wall or agents affecting signal transduction, mitosis and cell division, lipid s ynthesis and membrane integrity [7,20].
The mechanism of action of the majority of exist-ing antifungals used in clinical practice is associated with the cell wall. It remains a major objective for the development of other potential antifungals, because it contains components specific for fungal cells. Another possible way in the development consists in the inhi-bition of transition to the fibrous form by dimorphic pathogens, which leads to a reversible change from a saprophytic to a pathogenic form. Potential antifun-gal active compounds are also host defense peptides and cationic antimicrobial peptides due to the low risk of resistance to them. New targets can also include agents affecting biosynthesis of chitin, glycosylphos-phatidylinositol, glucosylceramide, heme; affecting virulence and mitochondrial functions, generat-ing of oxygen radicals; or inhibiting dihydroorotate d ehydrogenase.
Renewed nikkomycin Z, a competitive chi-tin synthase inhibitor lacking mammalian toxicity with effect against Coccidioides spp. is under clini-cal trial [9]. E1210 (3-[isoxazol-5-yl]pyridin-2-amine derivative) is an orally active broad-spectrum inositol acyltransferase inhibitor with high potency against Candida, Aspergillus and Fusarium suitable for treat-ment of disseminated candidiasis and pulmonary aspergillosis [11]. Glycosylphosphatidylinositol inhibi-tors include Gwt1 inhibitors (e.g., gepinacin [G642], G365 and G884 with an effect against C. albicans
and A. fumigatus) and Mcd4 inhibitors (e.g., M720 with tetradecahydroindeno[5′,6′:4,5]cycloocta[1,2-c]pyran-2[1H ]-one scaffold and potency against Can-dida sp. and A. fumigatus) [21]. Fungicidal acylhydra-zone derivatives such as BHBM and D0 inhibit syn-thesis and transport of glucosylceramide and show high potency against Cryptococcus neoformans and Pneumocystis jeroveci [22]. Compound SM21 (2,6-di-tert-butyl-4-{(E)-2-[4-(dimethylamino)phenyl]ethe-nyl}pyranium) with effect against multidrug-resistant Candida spp. and Candida biofilms is an agent influ-encing virulence, which inhibits change morphology between yeast and filamentous forms [11]. Ilicicolin H (4-hydroxypyridin-2[1H ]-one derivative) isolated from Gliocadium roseum, an inhibitor of mitochon-drial cytochrome bc1 reductase, with activity against Candida spp., A. fumigatus, and Cryptococcus spp. is limited in application due to high plasma protein binding [23]. On the other hand, hydroxyarylpyr-azoles, for example, ME1111, were discovered as selec-tive inhibitors of succinate-coenzyme Q reductase (or respiratory Complex II) of Trichophyton sp. [9]. Fun-gistatic arylamidine T-2307 showed activity against Candida spp., C. neoformans and Aspergillus spp. This compound causes a collapse of the mitochon-drial membrane potential and is in the 1st phase of clinical trials [9]. Thiofene derivative of sampagine, an alkaloide isolated from Cananga odorata inhibit-ing generation of heme and initiating production of free oxygen radicals, showed high activity against A. fumigatus and C. neoformans [24]. F901318 from the group of orotomides – compounds with unique mode of action – is dihydroorotate dehydrogenase (essen-tial enzyme for pyrimidine synthesis) inhibitor in the first Phase of clinical trials that demonstrates signifi-cant activity against Aspergillus spp. [9]. Compound VL-2397 (ASP2397) is a new intravenously adminis-tered fungicidal antifungal with an unknown mecha-nism of action. The siderophore-mediated uptake of VL-2397 to fungal cells causes high selectivity of this compound. It was isolated from Acremonium spp. and shows excellent efficiency against multidrug resis-tant A. fumigatus and C. glabrata. The first Phase of c linical t rials is being prepared [10].
Antimicrobial peptides may be considered as interest-ing therapeutic agents. They are cationic endogenous polypeptides produced by metazoans and causing mem-branolysis of negatively charged surface microbial mem-branes. They are effective against multidrug resistant pathogens and do not have any potential for develop-ment of resistance, nevertheless due to their instability, limited bioavailability and high price they have not been registered so far. The following antimicrobial peptides effective especially against Candida sp. and Aspergillus
www.future-science.com 1397future science group
How can we bolster the antifungal drug discovery pipeline? Commentary
spp. are in the second Phase of clinical trials: (CKPV)2
Vaccines with antifungal effects seem to hold signifi-cant promise for the future. They could be useful for prevention and a decrease of morbidity and mortality and can help to reduce societal costs. Possible indica-tions include various candidiases of ordinary as well as immunosuppressed patients. Prophylactic recombinant vaccines such as NDV-3, PEV-7 and rHyr1p-N are in clinical trials [9].
In conclusion, beside the design of structurally new antifungals based on new targets (single- or multi-site antifungal agents), promising strategies to combat anti-
fungal drug resistance seem to be the design of efflux inhibitors, various chemosensitizers, inhibitors of pH signaling pathways, biofilm formation, fi lamentation and virulence as well as genome-wide studies.
Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement
with any organization or entity with a financial interest in or fi-
nancial conflict with the subject matter or materials discussed
in the manuscript. This includes employment, consultancies,
honoraria, stock ownership or options, expert testimony,
grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this
manuscript.
References1 Gauthier GM, Keller NP. Crossover fungal pathogens:
the biology and pathogenesis of fungi capable of crossing kingdoms to infect plants and humans. Fungal. Genet. Biol. 61, 146–157 (2013).
2 Aly AH, Debbab A, Proksch P. Fifty years of drug discovery from fungi. Fungal. Divers. 50, 3–19 (2011).
3 Alghuthaymi MA, Almoammar H, Rai M et al. Myconanoparticles: synthesis and their role in phytopathogens management. Biotechnol. Biotechnol. Equip. 29(2), 221–236 (2015).
4 Fisher MC, Henk DA, Briggs CJ et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484(7393), 186–194 (2012).
5 Sanglard D. Emerging threats in antifungal-resistant fungal pathogens. Front. Med. 3, 11 (2016).
6 Denning DW, Bromley MJ. How to bolster the antifungal pipeline. Science 347(6229), 1414–1416 (2015).
7 Jampilek J. Potential of agricultural fungicides for antifungal drug discovery. Exp. Opin. Drug Dis. 11(1), 1–9 (2016).
11 Wong SS, Samaranayake LP, Seneviratne CJ. In pursuit of the ideal antifungal agent for Candida infections: high-throughput screening of small molecules. Drug Discov. Today 19(11), 1721–1730 (2014).
13 Jampilek J, Kralova K. Impact of nanoparticles on living organisms and human health. In: Encyclopedia of Nanoscience and Nanotechnology. Nalwa HS (Ed.). American Scientific Publishers, CA, USA, (2017) (In Press).
14 Spitzer M, Griffiths E, Blakely KM et al. Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole. Mol. Syst. Biol. 7, 499 (2011).
15 Calderone R, Sun N, Gay-Andrieu F et al. Antifungal drug discovery: the process and outcomes. Future Microbiol. 9(6), 791–805 (2014).
16 Liu S, Hou Y, Chen X et al. Combination of fluconazole with non-antifungal agents: a promising approach to cope with resistant Candida albicans infections and insight into new antifungal agent discovery. Int. J. Antimicrob. Agents 43(5), 395–402 (2014).
17 Shrestha SK, Fosso MY, Garneau-Tsodikova S. A combination approach to treating fungal infections. Sci. Rep. 5, 17070 (2015).
18 Pierce CG, Chaturvedi AK, Lazzell AL et al. A novel small molecule inhibitor of Candida albicans biofilm formation, filamentation and virulence with low potential for the development of resistance. NPJ Biofilms Microbiomes 1, 15012 (2015).
19 Liu S, Yue L, Gu W et al. Synergistic effect of fluconazole and calcium channel blockers against resistant Candida albicans. PLoS ONE 11(3), e0150859 (2016).
21 Mann PA, McLellan CA, Koseoglu S et al. Chemical genomics-based antifungal drug discovery: targeting glycosylphosphatidylinositol (GPI) precursor biosynthesis. ACS Infect. Dis. 1(1), 59–72 (2015).
22 Mor V, Rella A, Farnoud AM et al. Identification of a new class of antifungals targeting the synthesis of fungal sphingolipids. mBio 6(3), e00647–15 (2015).
23 Singh SB, Liu W, Li X et al. Antifungal spectrum, in vivo efficacy, and structure–activity relationship of ilicicolin H. ACS Med. Chem. Lett. 3(10), 814–817 (2012).
24 Jiang Z, Liu N, Dong G et al. Scaffold hopping of sampangine: discovery of potent antifungal lead compound against Aspergillus fumigatus and Cryptococcus neoformans. Bioorg. Med. Chem. Lett. 24(17), 4090–4094 (2014).
The development of next-generation antifungal agents with novel chemical scaffolds and new mechanisms of action is vital due to increased incidence and mortality of invasive fungal infections and severe drug resistance. This review will summarize current strategies to discover novel antifungal scaffolds. In particular, high-throughput screening, drug repurposing, antifungal natural products and new antifungal targets are focused on. New scaffolds with validated antifungal activity, their discovery and optimization process as well as structure–activity relationships are discussed in detail. Perspectives that could inspire future antifungal drug discovery are provided.
First draft submitted: 23 January 2016; Accepted for publication: 13 May 2016; Published online: 27 July 2016
Keywords: antifungal drug resistance • antifungal lead compounds • drug repurposing • fungal biofilm inhibitors • invasive fungal infections • new antifungal scaffolds • strategies in antifungal drug discovery
It is estimated that there are approximately 600 species of human fungal pathogens. Among them, approximately 20 species cause more than 99% of human fungal infections, which can be classified into superficial fungal infection and invasive fungal infection (IFI). IFIs are often life-threatening and associate with high mortal-ity in immunocompromised hosts such as patients with AIDS and patients undergo-ing organ transplants or anticancer chemo-therapy [1,2]. Candida albicans (mortality rate: 20–40%), Cryptococcus neoformans (mortality rate: 20–70%), and Aspergillus fumigatus (mortality rate: 50–90%) are the most c ommon pathogens of IFIs [3,4].
Clinically, antifungal agents for the treatment of life-threatening IFIs (Figure 1) mainly include amphotericin B (AmB), azoles (e.g., fluconazole, itracon-azole and voriconazole), echinocandins (e.g., caspofungin, micafungin and anidu-lafungin) and 5-fluorocytosine (usually used as adjunctive therapy) [5,6]. AmB is a polyene antibiotic that targets on fungal cell membrane by selective interaction with ergosterol [7]. Although AmB is still consid-
ered as the ‘gold standard’ for some severe infections [8], it has serious nephrotoxicity and many other side effects [9]. Extensive efforts have been made to reduce the tox-icity of AmB including the development of lipid formulations and synthesis of new derivatives [7,10]. Azole antifungal agents are inhibitions of CYP51 in fungal cell membrane and are widely used as the first line of antifungal therapy [11]. However, their antifungal potency has been signifi-cantly reduced because of severe drug resis-tance [12–14]. Development of new triazole is still an active area. For example, isavucon-azole has been approved by the US FDA in 2015 for treatment of invasive aspergillosis and invasive mucormycosis [15] in adults. Echinocandins are the newest class of anti-fungal agents, which act by inhibition of GS in fungal cell wall [16]. Although echi-nocandins are fungicidal with good selec-tivity, they cannot be orally administrated because of their complex lipopeptide struc-tures [17]. To address the limitation, several small molecule GS inhibitors have been discovered [18]. However, none of them is under clinical evaluation.
Figure 1. Chemical structures of antifungal agents for the treatment of invasive fungal infections.
1. Amphotericin B 2. Fluconazole 3. Voriconazole
4. Isavuconazole
5. Itraconazole
6. Posaconazole
7. Caspofungin 8. Micafungin
9. Anidulafungin
Polyene Azoles
X = O R =
X = C R =
Echinocandins
O
HO
O O
NH2OH
OH
O
OHOH
HO
OHOH
OH
OHOHO
F
OH
NNN
N N
N
F
F
OH
NN
N F
CH3
NN
F
F
OH
NN
N F
ON
O
CH3
CH3 CH3
N
O
OHN
H3C S
N
CN
SOO
HO O N N NN
N
OR
OX
OCH2
CH2
NN
N
Cl
Cl
HO
HO
NH
O
N
HO
OH
OHO
NH
NO
CH3
OH
HOO
HN
NH
NH
OOH
H2N
O
NH
OOH
H2NH2CH2C
NaO3S
HN
O
N
HO
OH
OHO
NH
NO
CH3
OH
HOO
HN
HO
NH
OOH
O
NH
OH
H3C
HO
O
NO
H2NOCH3C
H3C(H2C)4O
HO
NH
O
N
HO
OH
OHO
NH
NO
CH3
OH
HOO
HN
HO
NHO
OH
O
NH
OH
H3C
H3C
C
O
O(CH2)4CH3
1436 Future Med. Chem. (2016) 8(12) future science group
Review Liu, Wang, Su, Zhang & Sheng
In recent years, the incidence and mortality of IFIs has been increasing dramatically due to the increas-ing number of immunocompromised hosts and limi-
tations of current antifungal therapy. First, clinically available antifungal agents are very limited and far from satisfaction [19]. New drugs are expected to
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Strategies in the discovery of novel antifungal scaffolds Review
overcome their drawbacks, such as limited-potency, narrow-spectrum, nonoptimal pharmacokinetics, drug-related toxicity and drug–drug interactions. Second, drug resistance has been observed for all of the three classes of antifungal agents [20]. Although several new triazoles (e.g., albaconazole [21]) and echinocandins (e.g., aminocandin [2]) are under clinical or precilinical trials, these ‘me too’ drugs with the same mechanism of action cannot solve the resistance problem fundamentally. Third, infections caused by new fungal pathogens, such as hyaline moulds (Fusarium and Scedosporium), zygomycetes as well as opportunistic yeast-like fungi (Trichospo-ron and Rhodotorula), are also increasing in recent years. They are generally more difficult to diagnose and treat and are associated with greater mortality. Therefore, there is an emergent need to discover and develop new generation of antifungal agents with novel chemical scaffolds and fungal-specific mode of action.
An ideal antifungal agent should have fungicidal activity, good selectivity toward a fungal-specific tar-get, a broad-spectrum, low risk to develop resistance, reasonable pharmacokinetic (PK) and pharmacody-namic (PD) profile, and low toxicity or side effects. They could serve as guidelines for discovery and development of new generation of antifungal agents. However, the progress in antifungal drug develop-ment is relatively slow, particularly lagging behind the development of antibacterial agents. The main difficulty is attributed to the eukaryotic nature of fungal cells, which share high similarity with human counterparts. Thus, the discovery of new chemical scaffolds acting on fungal-specific targets is highly desirable.
In 2011, new lead compounds in antifungal drug discovery were reviewed by our group [22]. Herein we will primarily focus on current strategies to dis-cover novel antifungal scaffolds. Moreover, examples of antifungal lead compounds were mainly selected from recent publications (2011–2015). In particular, new antifungal scaffolds whose antifungal activity has been fully validated by different assays and/or a combination of in vitro and in vivo models will be discussed in detail.
Screening-based strategies to discover novel antifungal scaffoldsHistorically, the most common approach for antifun-gal drug discovery is to screen large compound librar-ies (synthetic small molecules or natural products). Clinically available antifungal agents, namely AmB, triazoles and echinocandins, were all originally dis-covered by screening for their ability to inhibit the
growth of fungal pathogens prior to know their mode of action. In recent years, phenotypic screening con-tinues to be a major approach to discover novel anti-fungal scaffolds. This section will mainly focus on screening methods and antifungal scaffolds derived from libraries of synthetic small molecules. Screening of natural products will be discussed in the ‘Natural product-based strategies to discover novel antifungal scaffolds’ section.
Compound library for antifungal screeningThe efficiency of screening-based antifungal drug discovery largely depends on the quality of com-pound library and antifungal assay. Recently, the number of compound libraries has been increasing dramatically, which offers opportunities for novel antifungal drug discovery. These compound libraries are commercially available (e.g., SPCES, ChemDiv, Maybridge), freely accessed (e.g., NIH Libraries Pro-gram) or privately owned (in-house libraries of phar-maceutical companies or research groups). Struc-tural diversity (particularly scaffold diversity) and drug-likeness (favorable physicochemical properties) are widely regarded to be the main characteristics of a high-quality compound library. Highly efficient synthetic methods, such as diversity oriented syn-thesis (DOS) [23,24], were proven to be fruitful tools to construct high-quality compound libraries and provide a valuable source of biologically active lead compounds. For example, our group reported a new synthetic method, divergent organocatalytic cascade approach (DOCA), to build compound libraries with good scaffold diversity and drug-likeness [25]. The DOCA-derived library was assayed for antifun-gal activities and two compounds (10 and 11) dis-played antifungal activities against C. albicans and C. neoformans with minimum inhibitory concentra-tion (MIC) values ranging from 8 μg/ml to 32 μg/ml (Figure 2) [25]. Notably, their scaffolds are totally d ifferent from the reported antifungal compounds.
New antifungal scaffolds derived from traditional assaysCurrently, there are two standards, the Clinical and Laboratory Standards Institute (CLSI) and the Euro-pean Committee on Antimicrobial Susceptibility Testing (EUCAST), for in vitro susceptibility testing of the selected fungal pathogens [26,27]. Both of them use traditional broth or microbroth growth inhibi-tion method and measure the microbial growth by optical density of the culture. The standard assays are well-accepted protocols to evaluate the in vitro anti-fungal activities. Also, they are effective tools to high-throughput screening of compound libraries. For
Figure 2. Chemical structures and in vitro antifungal activity of new antifungal scaffolds derived from structurally diverse compound library. MIC: Minimum inhibitory concentration.
10 11
C. albicans MIC = 16 ug/ml
C. neoformans MIC = 8 ug/ml
C. albicans MIC = 32 ug/ml
C. neoformans MIC = 32 ug/ml
O
O
O
O
H
O
S
HN
O
OO
O
O
H
O
S
HN
O
O
Figure 3. Chemical structures and in vitro antifungal activity of new antifungal scaffolds derived from traditional assays. MFC: Minimum fungicidal concentration; MIC: Minimum inhibitory concentration.
C. albicans MIC = 1.6 ug/ml
C. neoformans MIC = 1.6 ug/ml
A. fumigatus MIC = 12.5 ug/ml
12
SN
O
ON
CH3
C. albicans MFC = 12.5 ug/ml
A. fumigatus MIC ≤3.1 ug/ml
13
O
N
O
HN
O
1438 Future Med. Chem. (2016) 8(12) future science group
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example, new 1,2-benzisothiazolinone (12) [28,29], 2-(2-oxo-morpholin-3-yl)-acetamide (13) [30] scaf-fold were identified by screening of compound librar-ies, which showed potent antifungal activities with a broad spectrum (Figure 3).
Our group performed an antifungal screen of an in-house library and identified β-carboline scaffold 14 as a new antifungal agent (Figure 4) [31]. Then, a series of carboline derivatives (15) were designed and synthesized to investigate the structure–activity relationship (SAR). As a result, the optimized com-pound 16 showed improved antifungal activity and was comparable to fluconazole (Figure 4) [31]. Inter-estingly, compound 16 showed several promising features as a novel antifungal lead compound. First, it exhibited good fungicidal activity against both flu-conazole-sensitive and fluconazole-resistant C. albi-cans cells [31]. Second, compound 16 was also proven to be a good inhibitor of C. albicans biofilm forma-tion and hyphal growth, highlighting the potential to overcome fluconazole-related and biofilm-related
drug resistance [31]. Third, it showed good synergis-tic activities in combination with fluconazole. Last, compound 16 had weak inhibitory activity against CYP enzymes, indicating that it had low potential to cause drug–drug interactions [31]. Preliminary mechanism studies revealed that the carboline lead compound might act on fungal cell wall [31].
The development of new assays and discovery of new antifungal scaffoldsLimitations of traditional antifungal assays mainly include poor correlation between fungal growth and optical density for some species (e.g., Aspergillus) and inability to identify fungal biofilms inhibitors. Moreover, they are unable to distinguish between molecules with fungistatic and fungicidal activities. Recently, a number of antifungal screening assays were reported to address the limitations of traditional methods [32]. For example, adenylate kinase-based [33] and alamarBlue-based [34] high-throughput screening assays have been developed to the specific discovery of fungicidal agents. Rabjohns et al. screened 1280 small-molecule compounds using the alamarBlue-based assay and identified a potential rhodanine scaffold (17) that exhibited fungicidal activity in the low micromolar range (Figure 5) [34]. Compound 17 showed good fungicidal activity against C. neo-formans with low toxicity. However, it needs further structural optimization to exert efficacy in the mouse cryptococcal meningitis model because of its low half-life (t ½ = 10 min) in mice.
There are three different morphological forms (i.e., yeast, pseudohyphae and hyphae) of Candida cells. During the fungal infection, the first step is adhesion to surfaces, and a subsequent yeast-to-hyphal morphology transition is a major virulence that leads to tissue invasion and biofilm formation. The strong ability of Candida cells to form biofilms is an important reason for the emergence of high levels of resistance to most clinically used antifun-gal agents [35–38]. Thus, fungal biofilm inhibitors have low potential for the development of resistance and represent a new area to develop novel antifun-gal therapies [39]. XTT and alamarBlue-based assays using metabolic activity as reporters were developed for high-throughput screening against C. albicans biofilms [40,41]. For example, the alamarBlue-based assay was successfully used to identify 1,3-benzo-thiazole scaffold (18) as potent C. albicans biofilm inhibitors [40]. Another high-throughput screening discovered the diazaspiro-decane scaffold (19) to be potent inhibitors of biofilm formation and filamen-tation [42]. Compound 19 showed good inhibitory activity against C. albicans biofilms (IC
50 = 2.7 μM)
Figure 4. Chemical structures, in vitro antifungal activity and optimization strategy of new carboline antifungal scaffolds. MIC: Minimum inhibitory concentration.
Figure 5. Chemical structures of antifungal scaffolds identified by new assays.
C. albicans MIC = 32 ug/ml
C. neoformans MIC = 8 ug/ml C. albicans MIC = 2 ug/ml
C. neoformans MIC = 1 ug/ml
14
15 16
N
N
O
N
NR3
R2
X
R1
n
N
HN
O
Cl
F
1817 19 Filastatin (20)
SM21 (21) CAPE (22)
NH
S
O S
S
N
N
OH
Br
N
N
H3C
N
NO
H3C
Cl
N
O
N
NO2
HO
HO
O
O
O
ClO4
N
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Strategies in the discovery of novel antifungal scaffolds Review
with low toxicity (CC50
= 104.1 μM). Moreover, it inhibited C. albicans filamentation without affecting overall growth under planktonic conditions. Inter-estingly, resistance was not developed after repeated exposure to compound 19. The lead compound also showed in vivo efficacy in clinically relevant murine models of invasive and oral candidiasis.
Moreover, new assays for high-throughput screen-ing of adhesion [43] and yeast-to-hyphal [44] inhibitors were also developed. Fazly et al. screened a library of 30,000 small molecules and identified the 1-benzoyl-4-phenylpiperazine scaffold that inhibited adhesion of
C. albicans to cultured human epithelial cells [43]. The lead compound (named filastatin, 20) could effec-tively inhibit C. albicans biofilm formation and hyphal morphogenesis. Consistent with its in vitro activities, filastatin showed in vivo antifungal activity in a nem-atode model of C. albicans infection. More recently, Wong et al. identified a series of inhibitors of yeast-to-hypha transition from a collection of over 50,240 small molecules. Among the hits, SM21 (21) bearing a 4-(dimethylamino)styrylpyrylium scaffold exhib-ited highly potent antifungal activity both in vitro and in vivo [44]. The MICs of SM21 against the Candida
Figure 6. Chemical structures of representative antifungal scaffolds from known drugs.
Figure 7. Chemical structures of three old drugs as new inhibitions of Candida albicans biofilm formation. IC50: Inhibitory activity of biofilm formation.
Thioridazine (23)
C. neoformans MIC = 16 ug/ml
Amiodarone (24)
C. neoformans MIC = 8 ug/ml
S
NH3CS
NCH3
I
ON
I
O
C4H9
O
Therapeutic use:
Inhibition of biofilm formation:
Auranofin (25)anti-inflammatory agent
IC50 = 6.1 uM IC50 = 2.0 uM IC50 = 6.7 uM
Benzbromarone (26)anti-gout agent
Pyrvinium Pamoate (27)antihelminthic agent
Br
Br
OH
O
O
HO
HO-
O
O
O
-O
O SAu
OO P
O
O
O
O
O
O
2 Pamoate
1440 Future Med. Chem. (2016) 8(12) future science group
Review Liu, Wang, Su, Zhang & Sheng
and other fungal species ranged from 0.2–6.25 μg/ml. Moreover, it was still active against resistant Candida spp. with MIC values in the range of 0.5–1 μg/ml. Besides inhibition of yeast-to-hypha transition, SM21 also inhibited biofilm formation and displayed low toxicity to human cells. In the oral candidiasis mouse model, oral rinses containing 200 μg/ml SM21 could significantly reduce tongue lesions, whose ef ficacy of SM21 was better than that of nystatin.
Besides the in vitro assays, Mylonakis’s group devel-oped a whole-animal high-throughput antifungal assay using Caenorhabditis elegans as the model hosts [45,46]. The Candida-mediated C. elegans assay allows high-throughput in vivo screening of compound libraries for both antifungal activities and toxicity. The in vivo assay has been used to identify several new antifungal lead compounds. Among them, caffeic acid phenethyl ester (CAPE, 22) exhibited antifungal activity in a murine model of candidiasis [45].
Drug repurposing-based strategies to discover novel antifungal scaffoldsThe concept of ‘drug repurposing’ is the application of known drugs to new diseases, which has emerged
as an promising approach to accelerate drug develop-ment [47]. As compared with traditional drug devel-opment, a significant advantage of drug reposition-ing is that the toxicology and pharmacology of the marked drugs have already been well-established. As a result, the risk of failure for development of an old drug with a useful new indication could be significantly reduced. Several studies have identified a number of ‘nonantifungal’ drugs with antifungal activity by screening libraries of marketed drugs [48]. The antifungal activity and/or synergic effects with fluconazole of rapamycin [49], sertraline [50] and tamoxifen [51] have been reviewed by Krysan’s group [48]. The same group also screened the Prest-wick library of 1,120 off-patent drugs to identify drugs with fungicidal activity toward C. neofor-mans [52]. A total of 31 hits were detected and 15 of them were newly identified. A large portion of the hits shares common structural features, namely a hydro-phobic moiety (labeled red in Figure 6) linked to a basic amine (labeled blue in Figure 6) [52]. Another interesting feature of them is the ability of crossing the blood–brain barrier, highlighting their potential application as anticryptococcal agents. Two repre-
Figure 8. Chemical structures and antifungal activity of seven newly identified antifungal scaffolds derived from marketed drugs. MIC0.3: Minimal concentration of drug resulting in 30% growth inhibition.
Strategies in the discovery of novel antifungal scaffolds Review
sentative hits, thioridazine (23, MIC = 16 μg/ml) and amiodarone (24, MIC = 8 μg/ml), were active against intraphagocytic C. neoformans and were additive or synergistic to fluconazole [52]. Prelimi-nary mechanism studies revealed that they acted by directly binding C. neoformans calmodulin [52].
Siles et al. screened Prestwick Chemical Library containing 1200 FDA-approved, off-patent drugs for biofilm inhibitors [53]. Three old drugs, aurano-fin (anti-inflammatory agent, 25, IC
50 = 6.1 μM),
benzbromarone (antigout agent, 26, IC50
= 2.0 μM) and pyrvinium pamoate (antihelminthic agent, 27, IC
50 = 6.7 μM) were validated as potent inhibitors
of C. albicans biofilm formation (Figure 7). How-
ever, their antifungal efficacy was not reported in this work. Interestingly, another study identified that a combination of pyrvinium pamoate and flu-conazole was effective toward azole resistance in C. albicans [54].
More recently, Stylianou et al. screened a library of 844 drugs (approved or in clinical trial) for anti-Candida activity based on the EUCAST guidelines [55]. Seven drugs, namely amonafide (28), tosedostat (29), megestrol acetate (30), melengestrol acetate (31), stanozolol (32), trifluperidol (33) and haloperidol (34), were newly identified to possess antifungal activ-ity (Figure 8). Their antifungal activities were validated by three individual assays and different Candida spp.
1442 Future Med. Chem. (2016) 8(12) future science group
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Box 1. Selected examples of new antifungal scaffolds from natural products.
Compound
Source: Hyalis argentea var. latisquamaCandida albicans MIC50 = 15.6 μg/ml
Cryptococcus neoformans MIC50 = 15.6 μg/ml
O
H H
O
O
O
35
12
34 5
6
78
9
10
11
12
∆8,9 7,11, ∆
[60]
O
OHO
O
OH
36
Source: Guignardia sp. and Guignardia mangiferaeC. albicans MIC = 15.5 μg/ml
[61,62]
O O
O
O
OH
37
Source: Guignardia sp.C. albicans MIC = 87.5 μg/ml
[61,63]
Source: Guignardia sp. and Coniothyrium sp.C. albicans MIC = 24.3 μg/ml
O O
38
O
OH OH
[61,64]
Box 1. Selected examples of new antifungal scaffolds from natural products (cont).
Source: Pseudallescheria boydii SNB-CN73C. albicans MIC = 2 μg/ml
Candida parapsilosis MIC = 16 μg/mlAspergillus fumigates MIC = 16 μg/ml
[71]
Box 1. Selected examples of new antifungal scaffolds from natural products (cont.).
Compound
N
HO
O
47
Source: Beilschmiedia alloiophyllaC. albicans MIC = 8 μg/ml
[72]
O
OHOH O
48
Source: Fimetariella sp.A. fumigates MIC = 5 μg/ml
[73]
O OO
O
O
49
Source: Clausena excavataCandida tropicalis MIC = 39 μg/ml
[74]
50
Source: Dimocarpus longan Lour.C. parapsilosis MIC = 7.81 μg/ml
C. albicans MIC = 7.81 μg/mlC. neoformans MIC = 15.63 μg/ml
O
O O
OH
HO
O
OH
OH
[75]
Figure 9. Scaffold hopping and structural simplification of antifungal natural product sampangine. MIC: Minimum inhibitory concentration; SAR: Structure–activity relationship.
Scaffoldhopping
Remove A ringand SAR
Sampangine (51)
C. albicans MIC = 0.5 ug/ml
C. neoformans MIC = 2 ug/ml
A. fumigatus MIC = 16 ug/ml
C. albicans MIC = 4 ug/ml
C. neoformans MIC = 0.25 ug/ml
A. fumigatus MIC = 1 ug/ml
C. albicans MIC = 0.5 ug/ml
C. neoformans MIC = 0.5 ug/ml
A. fumigatus MIC = 4 ug/ml
C. albicans MIC = 0.5 ug/ml
C. neoformans MIC = 0.25 ug/ml
A. fumigatus MIC = 2 ug/ml
Remove B ringand SAR
ZG-20-41 (54)
ZG-20-07 (53)ZG-20 (52)
D C B
A
N
N
O
N
N
O
S
N
O
O
SO2N
O
O
SO2N
N
1444 Future Med. Chem. (2016) 8(12) future science group
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clinical isolates. The results showed that the antifungal activities of these drugs were comparable to those of the clinically available antifungal agents (e.g., flucon-azole, amorolfine). Moreover, the antifungal activity of auranofin that was previously identified as a biofilm inhibitor was validated in this study (C. albicans, MIC = 0.68 μg/ml). Up to now, about 20 old drugs were reported to have antifungal activity, which could serve as pharmacologically attractive scaffolds for further development. However, in-depth evaluation and par-ticularly structural optimization of them is still rare, which limited their therapeutic application as novel antifungal agents. Subsequent work should aim to optimize their antifungal properties and reduce their original functions.
Natural product-based strategies to discover novel antifungal scaffoldsNatural products are rich sources of novel antifungal agents [56,57]. Polyenes and echinocandins, two major classes of antifungal agents, are originated from natural products [56]. Natural product-based discov-ery of novel antifungal agents is becoming an active research area due to their structural diversity [22,57]. Recent examples of antifungal natural products (35–50) are summarized in Box 1. However, the challenges of natural product-based antifungal dis-covery mainly include the structural and stereo-
chemical complexity, difficulty in chemical synthesis and structural modifications, and unfavorable physi-cochemical properties for drug development [58]. To tackle these problems, substitution optimization and scaffold hopping present a promising strategy to improve the antifungal activity, PK/PD profile of natural products [59]. Moreover, elucidation of the structural requirements for antifungal activity and structural simplification of the natural scaffold are effective methods to discover highly potent but less complex scaffolds. The following section will i ntroduce our efforts in this field.
Scaffold hopping & structural simplification of antifungal natural product sampangineSampangine (51), an azaoxoaporphine alkaloid extracted from the stem bark of Cananga odorata, showed broad spectrum antifungal activity against various human fungal pathogens including C. albicans, C. neoformans and Aspergillus fumigatus [76,77]. Although several A- and B-ring-substituted [78] and hetero analogs [79] of sam-pangine derivatives have been reported, further drug development was hampered because of poor water solu-bility and lack of in vivo antifungal efficacy. In order to address the problems, we performed a scaffold hopping study of sampangine using heterocycles to replace the D-ring (Figure 9). The thiophene derivative ZG-20 (52) revealed improved activity agianst C. neoformans (MIC
Figure 10. Chemical structures, structural optimization process and antifungal activity of inhibitors of fungal cell wall GPI biosynthesis. MIC: Minimum inhibitory concentration.
Screening of compound library
Gepinacin (56)BIQ (55)
C. albicans MIC = 1.56 ug/ml
A. fumigatus inactive
C. albicans MIC = 0.1 ug/ml
A. fumigatus MIC = 1.56 ug/ml
C. albicans MIC = 0.05 ug/ml
A. fumigatus MIC = 0.78 ug/ml
C. albicans MIC = 0.004 ug/mlA. fumigatus MIC = 0.03 ug/ml
C. albicans MIC = 0.39 ug/ml
A. fumigatus MIC = 3.13 ug/ml
57 58 59
optimization
Optimization
E-1210 (60)
N HN
O
O
O
H3CO
N
NH
OS O F
N
NH
OS O F
NH2N
NH
OS O
NH
ON
ON
N NH2
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Strategies in the discovery of novel antifungal scaffolds Review
= 0.25 μg/ml) and A. fumigatus (MIC = 2 μg/ml) and enhanced water solubility (solubility: 48 μg/ml) [80]. Inspired by the results, the simplification of sampang-ine scaffold was further investigated [81]. Interestingly, A-ring removed analog ZG-20-07 (53, MIC range: 0.125 μg/ml–2 μg/ml) and A, B-ring removed analog ZG-20-41 (54, MIC range: 0. 5 μg/ml to 4 μg/ml) showed excellent antifungal activity against a variety of fungal pathogens. Moreover, ZG-20-07 (solubility: 38.6 μg/ml) and ZG-20-41 (solubility: 65.2 μg/ml) showed significantly improved water solubility than sampang-ine (solubility: 12.6 μg/ml). Interestingly, they exhib-ited fungicidal activity on both fluconazole-sensitive and fluconazole-resistant C. albicans. In a Caenorhabdi-tis elegans–C. albicans infection model, ZG-20-07 and ZG-20-41 showed good in vivo antifungal activity with low toxicity and could effectively protect the C. albicans
infection. In addition, both of them inhibited C. albi-cans biofilm formation, cellular surface hydrophobicity (CSH) and yeast-to-hypha morphological transition of C. albicans in a dose-dependent manner, highlighting their potential as good lead c ompounds for the treat-ment of resistant fungal infections.
Target-based strategies to discover novel antifungal scaffoldsClinically available antifungal agents mainly target on ergosterol in cell membrane (AmB), CYP51 in fun-gal cell membrane (azoles) and GS in fungal cell wall (echinocandins). Among them, only the crystal struc-ture of Saccharomyces cerevisiae CYP51 was solved [82]. Our group used computational approaches to structure-based rational design new triazole CYP51 inhibitors and nonazole inhibitors [83,84]. For GS, the discovery of
1446 Future Med. Chem. (2016) 8(12) future science group
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new inhibitors, particularly small molecule inhibitors, still mainly depends on high-throughput screening [18]. On the other hand, the development of new antifungal drugs based on new targets is highly desirable due to the limitations of current antifungal agents and the emer-gence of severe resistance [85]. Over the past few years, important progress has been made in fungal genom-ics, which provide a good opportunity to identify novel antifungal targets [86]. The genomes of important fun-gal pathogens, such as C. albicans, C. neoformans and A. fumigatus, have been released and functional proteins that are essential for fungal growth are important source of antifungal drug targets. In particular, targets that are conserved among fungal pathogens and lack a human counterpart are highly promising for drug discovery and development. Moreover, a druggable antifungal target should be able to bind drug-like compounds, which needs to be v alidated by medicinal chemistry efforts.
Currently a variety of new antifungal targets are already available, which can be classified into four types according to their location and functions in fungal cells. They are fungal cell wall targets (e.g., chitin synthase, and mannoprotein), fungal cell membrane targets (e.g., inositol phosphocer-
amide synthase), DNA and protein synthesis targets (e.g., N-myristoyltransferase, aminoacyl-tRNA syn-thetase, elongation factor, secreted aspartic protein-ase, topoisomerase) and signal transduction pathway targets (e.g., calcineurin, electron transport chain). Most inhibitors acting on these targets have been reviewed by our group and others [22,87–89]. Herein only recent progress of new antifungal targets, new scaffolds as well as discovery and optimization s trategies was highlighted.
GPI inhibitorsGPI-anchored proteins, a kind of cell wall mannopro-teins, are required for the adhesion of pathogenic fungi to human epithelium [90]. Due to the importance of GPI-anchored proteins in fungal cell wall biosynthesis and maintenance of homeostasis, designing inhibitors targeting this biosynthetic pathway has the advantage of reducing adverse effects in human cells. Up to now, there are two scaffolds reported to inhibit fungal GPI biosyn-thesis (Figure 10) [91,92]. 1-(4-Butylbenzyl)isoquinoline (BIQ, 55) is the first GPI-anchored protein inhibitor discovered by means of a yeast cell-based screening [91]. BIQ has moderate inhibitory activity against C. albicans
Figure 11. Chemical structures and antifungal activity of AHAS inhibitors. MIC: Minimum inhibitory concentration.
61 62
Chlorimuron ethyl (63) 64
Phenotypic screening and chemogenomic profiling strategy
Target-based screening and optimization strategy
S. cerevisiae AHAS IC30 = 4.2 uM
C. albicans MIC50 = 1 ug/ml
C. neoformans MIC50 = 4 ug/ml
A. fumigatus MIC50 = 4 ug/ml
S. cerevisiae AHAS IC30 = 9.2 uM
C. albicans AHAS Ki = 7 nM
C. albicans MIC50 = 2 uM
C. albicans AHAS Ki = 3.8 nM
C. albicans MIC50 = 0.6 uM
S. cerevisiae AHAS IC30 = 4.9 uM
C. albicans MIC50 >128 ug/ml
C. neoformans MIC50 = 0.5 ug/ml
A. fumigatus MIC50 = 4 ug/ml
N
N
N
NS
O
O
NH
N
N
N
NS
O
O
NH
O
O
S
HN
HN
O N
N Cl
OCH3
O O
OCH3
O
O
S
HN
HN
O N
N I
O O
Figure 12. Chemical structures and binding mode of dihydrofolate reductase inhibitors. (A) Chemical structures and antifungal activity of fungal dihydrofolate reductase inhibitors; (B) the binding mode of a 2,4-diaminopyrimidine propargyl inhibitor with Candida albicans dihydrofolate reductase. The figure was generated on the basis of the crystal structure obtained from Protein Data Bank (PDB code: 4HOF). DHFR: Dihydrofolate reductase; IC50: Inhibitory activity of biofilm formation; MIC: Minimum inhibitory concentration.
AB
CN
NH2N
NH2
OCH3
N
NH2N
NH2 N
Meta-linked
65
C. albicans DHFR IC50 = 30 nM
C. glabrata DHFR IC50 = 28 nM C. glabrata DHFR IC50 = 5.5 nM C. glabrata DHFR IC50 = 27 nM
C. albicans DHFR IC50 = 20 nM C. albicans DHFR IC50 = 49 nMMIC > 100 ug/ml MIC = 1.6 ug/ml MIC = 0.39 ug/ml
MIC = 12.5 ug/ml
E32 E36
M33
I9
NAPDH
I112
S61
I62
P63
F66
MIC = 0.39 ug/ml MIC = 0.2 ug/ml
66 67
Para-linked
N
NH2N
NH2
OCH3
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Strategies in the discovery of novel antifungal scaffolds Review
(MIC = 1.56 μg/ml) and was inactive against A. fumiga-tus [93]. However, chemical modifications of BIQ did not yield a more potent compound. Inspired by another screening hits with a nicotine amide scaffold, quinoline (57), pyridine (58) amide and azaindole (59) derivatives were designed, which showed improved antifungal activ-ity and broader spectrum [93]. SAR analysis revealed that the pyridinyl amine scaffold was essential for excellent antifungal activity and further optimization of the side chain led to the discovery of E1210 (60) as a preclinical candidate [94]. E1210 showed excellent in vitro antifun-gal activity against a variety of fungal pathogens [95–97]. Moreover, E1210 was orally active in various fungal infection models including disseminated candidiasis, oropharyngeal candidiasis, pulmonary aspergillosis, and disseminated fusariosis [98]. Importantly, E1210 also showed good efficacy in the treatment of azole-resistant and echinocandin-resistant C. albicans infections [98,99]. Another scaffold, phenoxyacetanilide (gepinacin, 56), was identified by antifungal screening and target char-acterization [92]. Gepinacin is a GPI acylation inhibitor
and selectively inhibited an important acyltransferase (Gwt1) that is essential for the biosynthesis of fungal GPI anchors. Gepinacin showed highly selective antifungal activity against C. albicans, C. glabrata and A. terreus as well as resistant C. albicans isolates. Gepinacin is a useful probe for studying the mechanisms of Gwt1 inhibitors and its further evaluation and structural optimization studies are still required to know the a ntifungal ef ficacy of this antifungal scaffold.
AHAS inhibitorsAHAS or ALS is the first enzyme in the biosynthe-sis of branched-chain amino acids [100]. AHAS is highly conserved in fungi without human counter-part, which is a potential target for the development of selective antifungal agents [88]. In 2013, two scaf-folds were identified as fungal AHAS inhibitors by different strategies (Figure 11) [101,102]. Richie et al. used high-throughput phenotypic screening in com-bination with chemogenomic profiling strategy to discover triazolopyrimidine-sulfonamide scaffold
Figure 13. Chemical structures, antifungal activity and optimization strategies of β-1,6-glucan inhibitors. MIC: Minimum inhibitory concentration.
Optimization ofC1–C4 substitution
Scaffold hopping
Removal ofbenzene coreD75-4590 (68)
C. glabrata MIC = 1 ug/ml
C. albicans MIC >128 ug/ml
D11-2040 (69) D21-6076 (70)
7271 73
C. glabrata MIC = 0.016 ug/ml
C. glabrata MIC = 0.063 ug/ml MIC = 0.25 ug/ml MIC = 1 ug/ml
C. albicans MIC >32 ug/ml
C. glabrata MIC = 0.125 ug/ml
C. albicans MIC >32 ug/ml
NN
NC
N
NN
NN
NC
N
N
NN
NC
NH
N1
2
34
N N
NH3C
CN
N
N
N
NH3C
CN
N
N
N N
H3C
CN
N
N
1448 Future Med. Chem. (2016) 8(12) future science group
Review Liu, Wang, Su, Zhang & Sheng
as S. cerevisiae AHAS inhibitors [102]. Compounds 61 and 62 had broad-spectrum in vitro activity, no significant cytotoxicity, and low protein binding. In another study, Lee et al. used C. albicans AHAS assay to screen eight commercial sulfonylurea herbicides. Chlorimuron ethyl (63) is the most potent compound in both AHAS inhibition assay (K
i = 7 nM) and anti-
fungal activity assay (C. albicans MIC50
= 2 μM). Structural optimization of 63 led to the discovery of the iodine analog 64 with improved activity (AHAS K
i = 3.8 nM, C. albicans MIC
50 = 0.6 μM). In both
studies, the binding mode of inhibitors with AHAS were analyzed on the basis of the crystal structure of S. cerevisiae AHAS. However, structure-based design and optimization of AHAS inhibitors have not been reported up to now.
DHFR inhibitorsDihydrofolate reductase (DHFR), a key enzyme in thymidine synthesis, is a traditional antitumor target. In recent years, targeting DHFR has also proven to be an effective strategy in antimicrobial drug discovery. There are several important differences in the active site between human and Candida species, which provides basis for the design of selective inhibitors. Although fungal DHFR inhibitors have been reported [103], a major problem is the poor correlation between DHFR inhibitory activity and antifungal activity.
Anderson’s group reported a 2,4-diaminopyrimi-dine propargyl scaffold as Candida DHFR inhibitors (Figure 12) [104–106]. Moreover, compounds containing a para-linked biphenyl moiety (66) had a broader spec-trum than the corresponding meta-linked compounds (65). The binding modes of this class of inhibitors with C. albicans and C. glabrata DHFR were confirmed by determining crystal complexes [107]. The pyrimidine ring of the inhibitor forms conserved hydrogen bond-ing and hydrophobic interactions with Glu32, Ile9, Phe36, Met/Ile33 and Ile121 (Figure 12). The propargyl linker and biphenyl moiety form hydrophobic and van der Waals interactions with DHFR. The crystal struc-tures also revealed that additional hydrophobic func-tionality substituents to the para position of the distal C-ring may enhance the potency of enzyme inhibition and selectivity alter the physicochemical properties. On the basis of the binding model, a series of new para-linked compounds were designed, most of which inhib-ited both C. albicans and C. glabrata DHFR and had dual antifungal activity. For example, compound 67 showed MIC values lower than 0.5 μg/ml against both C. albicans and C. glabrata.
Inhibitors of β-1,6-glucan synthesisβ-1,6-glucan is an important fungal cell wall compo-nent [108]. Researchers from Daiichi Sankyo (Japan) developed a cell-based assay for screening inhibitors of
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Strategies in the discovery of novel antifungal scaffolds Review
cell wall components and identified the first inhibitor (D75-4590, 68) of β-1,6-glucan synthesis [109]. D75-4590 acted by targeting Kre6p and showed moderate activities against a variety of C andida species including fluconazole-resistant strains, but was inactive toward C. neoformans and Aspergillus species. Subsequently, D75-4590 was optimized to improve the antifungal activ-ity and physicochemical properties (Figure 13). First, medicinal chemistry efforts were focused on the C1-C4 substitutions on pyridobenzoimidazole scaffold [110]. The SAR revealed that the C3-methyl group, C4-cyano group and a terminal amine in C1-side chain were essen-tial for antifungal activity. In contrast, the introduction of hydrophobic group to the C-2 position and and cyclic amine in the C1 side chain enhanced antifungal activ-ity. Compound D11-2040 (69) showed excellent activ-ity against C. glabrata (MIC = 0.016 μg/ml) [110,111]. Using the scaffold hopping strategy, D11-2040 was further optimized by inserting a nitrogen atom in the phenyl part of pyridobenzoimidazole scaffold. The resulting compound D21-6076 (70) had improved physicochemical properties and showed good in vivo efficacy in the C. glabrata infection model [112]. Interest-ingly, although compound D21-6076 was poorly active to inhibit the growth of C. albicans in the in vitro assay, it still showed protective effects in the C. albicans infec-tion model. Mechanism studies revealed that compound D21-6076 may act by inhibiting the invasion process of C. albicans [112]. To further optimize the water solubility and metabolic stability, a structural simplification strat-egy was used to design bicyclic derivatives (i.e., triazo-lopyridines, imidazopyridines, and pyrazolopyridines, 71–73) [113]. After the removal of a phenyl group, the triazolopyridine scaffold (71) showed improved water solubility and retained the excellent activity against C. glabrata [113]. However, in vivo results of these b icyclic scaffolds have not been reported.
ConclusionIn summary, current strategies for the discovery of novel antifungal scaffolds were reviewed. High-throughput screening (HTS) of compound library is still a use-ful and effective tool to identity new antifungal com-pounds. However, the hit rate of HTS is relatively low. Drug repurposing is a special kind of HTS using mar-keted drugs as compound library. As compared with traditional HTS hits, the drug hits possess better physi-cochemical properties and higher safety. Even though, medicinal chemistry optimization are necessary to improve the antifungal activity and reduce the origi-nal therapeutic effects. Natural products are important source of antifungal lead compounds. Their complex structures and unfavorable physicochemical proper-ties always limit further development. Identification
and validation of new antifungal targets can greatly promote the drug discovery and development process. Thus, it is highly challenging to find a fungal-specific target and also a selective antifungal agent. Fortunately, important progress has been made in fungal genom-ics. Genome-scale analysis, comparative genomics and bioinformatics approaches have been used to find out potential targets unique to fungal cells [85,114]. Struc-tural biology is also of great importance to determine the 3D structures of targets and thus facilitate the fol-lowing structure-based drug discovery. However, crys-tal structures for most targets located in fungal cell wall or cell membrane still remain unknown. Currently, most of the new antifungal scaffolds were still discov-ered by traditional ‘compound-centric’ approach. In the post genomics-era, combining the ‘compound-centric’ and ‘target-centric’ strategies can accelerate the drug discovery process.
Future perspectiveAlthough a number of screening hits with whole-cell antifungal activity have been reported, subsequent medicinal chemistry studies, such as SAR, lead opti-mization and mode of action, are still highly desirable. Moreover, these novel antifungal scaffolds can also be used chemical probes as to identify antifungal targets by chemical genetics approaches. To fight against drug resis-tance, more efforts need to focus on new types of chemi-cal scaffolds with fungicidal activity and new mode of action. The journey of a new chemical scaffold from conception to clinical application is a long one and it is estimated that most of the new antifungal agents mar-keted in the following years are still from existing classes (e.g., new triazoles and echinocandins). Even though, the application of new strategies, such as genomics-based target identification, new screening models, structural biology and rational drug design into antifungal drug discovery will accelerate the p rocess of new antifungal drug development.
Financial & competing interests disclosureResearch conducted by the authors in this area is supported
by the National Natural Science Foundation of China (Grant
81573283, 21502224), the 863 Hi-Tech Program of China
(Grant 2014AA020525), Science and Technology Commission
of Shanghai (Grant 13431900301), the Shanghai ‘ShuGuang’
Project (Grant 14SG33) and Youth Foundation of Second Mili-
tary Medical University (grant 2014QN06). The authors have
no other relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the
manuscript apart from those di sclosed.
No writing assistance was utilized in the production of this
manuscript.
1450 Future Med. Chem. (2016) 8(12) future science group
Review Liu, Wang, Su, Zhang & Sheng
ReferencesPapers of special note have been highlighted as: • of interest; •• of considerable interest
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Executive summary
The urgent need to develop novel antifungal agents• There is an emergent need to discover and develop new generation of antifungal agents with novel chemical
scaffolds and fungal-specific mode of action due to high incidence and mortality of invasive fungal infections and lack of effective antifungal agents.
Discovery of novel antifungal scaffolds by high-throughput screening• High-throughput screening is a major approach to discover novel antifungal scaffolds. The screening efficiency
largely depends on the quality of compound library and antifungal assays.• A number of in vitro (e.g., adenylate kinase-based and alamarBlue-based methods) and in vivo
(e.g., Caenorhabditis elegans whole-animal model) assays have been developed to improve the quality of screening hits.
Discovery of novel antifungal scaffolds by drug repurposing• A number of ‘nonantifungal’ drugs were identified to possess antifungal activity by screening drug libraries.
Several of them, such as tamoxifen, amiodarone, pyrvinium, haloperidol and melengestrol, showed potent activity and need to be further optimized.
Discovery of novel antifungal scaffolds from natural products• Natural products are featured as good structural diversity and are rich sources novel antifungal scaffolds. Due
to structural complexity and unfavorable physicochemical properties, antifungal natural products need to be further optimized to improve potency and drug-likeness.
• Scaffold hopping and structural simplification of antifungal natural product sampangine led to the discovery of two novel scaffolds with facile chemical synthesis, potent antifungal activity and good water solubility.
Discovery of novel antifungal scaffolds based on new targets• New inhibitors of glycosylphosphatidylinositol-anchored proteins, acetohydroxyacid synthase, dihydrofolate
reductase and β-1,6-glucan synthesis showed potent antifungal activity.Future perspective• New strategies, such as genomics-based target identification, new screening models, structural biology and
rational drug design, will be applied to antifungal drug discovery and accelerate the drug development process.
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Strategies in the discovery of novel antifungal scaffolds Review
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FutureMedicinalChemistry
part of
Review
Fungal biofilm composition and opportunities in drug discovery
Courtney Reichhardt1, David A Stevens2,3 & Lynette Cegelski*,1
1Department of Chemistry, Stanford
University, Stanford, CA 94305, USA 2Department of Medicine,
Division of Infectious Diseases and
Geographic Medicine, Stanford
University, Stanford, CA 94305, USA 3California Institute for Medical Research,
Biofilm infections are exceptionally recalcitrant to antimicrobial treatment or clearance by host immune responses. Within biofilms, microbes form adherent multicellular communities that are embedded in an extracellular matrix. Many prescribed antifungal drugs are not effective against biofilm infections owing to several protective factors including poor diffusion of drugs through biofilms as well as specific drug–matrix interactions. Despite the key roles that biofilms play in infections, there is little quantitative information about their composition and structural complexity because of the analytical challenge of studying these dense networks using traditional techniques. Within this review, recent work to elucidate fungal biofilm composition is discussed, with particular attention given to the challenges of annotation and quantification of matrix composition.
First draft submitted: 19 February 2016; Accepted for publication: 27 May 2016; Published online: 3 August 2016
AimThe majority of micro-organisms, including fungi, exist in nature as multicellular com-munities called biofilms [1]. Traditionally, most microbes have been studied as planktonic organisms. However, research into biofilm communities and biofilm composition and physiology is expanding given the recognition of biofilms as major contributors to microbial persistence and virulence in infectious dis-eases. Indeed, it is estimated that up to 80% of human microbial infections are biofilm-related [2]. Biofilms can be recalcitrant to anti-microbial treatment and can avoid clearance by host immune responses [1–5]. Both Can-dida albicans and Aspergillus fumigatus form biofilms, and these biofilms are involved in a range of human infections, including lethal fungal infections among immunosuppressed individuals [6–9]. C. albicans contributes to hos-pital-acquired infections and fouling of medi-cal devices including catheters [5,6,10]. Biofilms formed by the opportunistic filamentous fun-gus A. fumigatus are also implicated in asper-
gilloma and invasive pulmonary aspergillosis (IPA) [11,12]. Additionally, both C. albicans and A. fumigatus have been found to co-infect with Pseudomonas aeruginosa in lung infections of patients with cystic fibrosis [13,14]. In a study of the prevalence of fungal species in the sputum of adult patients with cystic fibrosis, A. fumiga-tus was isolated from the sputum from 45.7% of patients in the study and C. albicans from 75.5% of patients [13]. Several additional fun-gal species are known to form biofilms and contribute to human infections [15–18].
A defining feature of a biofilm is the extracellular matrix (ECM), which is a self-produced, typically noncrystalline material that encases microbial cells [19,20]. The ECM is rich in biopolymers and can contain pro-teins, polysaccharides, lipids, nucleic acids, and other molecules [21] that can interact with one another and the cellular surface to form a robust, protective network [3,22,23]. ECM composition varies across species and even growth conditions [19,22], yet the ECM composition of many biofilms remains
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unknown [19,21]. Functionally, the ECM can serve as a protective barrier against chemical and biological anti-microbial agents including many prescribed antifungal drugs (Figure 1A) [5,19–21]. In some instances, ECM can contribute to antifungal resistance by binding to anti-fungals, thereby preventing access to their intended target at the surface or within fungal cells [16,24–29]. As such, a better understanding of ECM composi-tion is key to understanding the specific mechanisms of antifungal resistance exhibited by biofilm formers. Antifungal resistance has been studied extensively for C. albicans, and in some cases Candida biofilms have been found to be capable of withstanding antifungal concentrations that are 1000-fold higher than those that inhibit planktonic cells [5,30–32]. The presence of β-1,3-glucans in the ECM partially can explain this remarkable level of resistance as β-1,3-glucans can interact with a number of antimicrobials [33–37]. For example, radio-labeled fluconazole was found to be sequestered by ECM, which was correlated with the quantity of β-1,3-glucans in the matrix [30]. β-1,3-glucans can also interact with amphotericin B, flucy-tosine and echinocandins [30]. Interestingly, biofilm resistance to echinocandins is lower as compared with other antifungals, and it has been hypothesized that this may be because echinocandins impair β-1,3-glucan synthesis [30]. Indeed, mutants unable to pro-duce the enzymes that transfer glucan from the cell to the extracellular milieu demonstrated reduced ECM glucan levels and decreased resistance to antifungal therapy [27]. Other ECM components, including extra-cellular DNA, also modulate drug resistance in bio-films produced by C. albicans and A. fumigatus, and in some instances, treatment of biofilms with DNase can reduce antifungal resistance [30,38–42]. A. fumigatus biofilms similarly exhibit decreased susceptibility to azoles, echinocandins and polyenes [43–45], including to amphotericin B. In addition, the co-administration of amphotericin B with alginate lyase, an enzyme that can degrade some exopolysaccharides within bio-films, was found to enhance the antifungal activity of amphotericin B against A. fumigatus biofilms, possibly by disrupting the polysaccharide component of the ECM and thus permitting drug access to hyphae [46]. These examples help to demonstrate the importance of having knowledge of ECM composition to provide insight into inhibitor modes of action and, ultimately, for developing strategies to combat the antifungal resistance exhibited by biofilms.
The ECM is often noted as a permeability barrier to antifungals that contributes to decreased drug suscep-tibility, although exceptions have been reported. One study utilized a disk diffusion assay to determine the C. albicans biofilm permeability of four antimicrobi-
als: flucytosine, fluconazole, amphotericin B, and vori-conazole [25]. Drug penetration through the biofilm failed to kill the cells, which suggested the existence of drug resistance mechanisms in addition to poor antifungal penetration for Candida biofilms. How-ever, a previous study showed that the growth condi-tions (shaking above 30 rpm) used can inhibit ECM formation for C. albicans, as documented by scanning electron microscopy (SEM). Thus, further analysis is needed to probe effects on drug penetration [21,25,48]. Resistance can also be conferred to biofilm cells through the community’s harboring of persister cells (Figure 1B) [24,29,49–51]. Persister cells are metaboli-cally dormant cells that exhibit tolerance to multiple antifungals including amphotericin B, azoles, and chlorhexidine [30,49,50]. Finally, biofilm microbes also upregulate the production of efflux pumps to confer enhanced antibiotic resistance relative to microbes not associated with biofilms (Figure 1C) [28,29,52]. Increased expression of efflux pumps during biofilm growth has been observed for several Candida spp. and A. fumiga-tus, and resistance to azoles, drugs that disrupt ergos-terol synthesis, is frequently due to increased expres-sion of efflux pumps [30,53,54].
The formidable survival traits of biofilms combined with our dwindling pipeline of effective antifungals make it imperative that we undertake in-depth anal-yses to improve upon our understanding of biofilms and ECM composition and function [55–57]. Indeed, standard antimicrobial susceptibility tests are still per-formed to examine the efficacy of single drugs using planktonically grown cultures, yet the biofilm state is often more relevant [3,24]. Early descriptions of bio-films were often limited to ‘slime’, and this simplistic description masked the complexity of biofilms and slowed the design of biofilm inhibitors and interven-tions [20,58]. The poor solubility and lack of crystallin-ity of biofilms and ECM render these materials espe-cially challenging to examine using many traditional biochemical and biophysical techniques [19,20,59,60]. Despite these challenges, recent studies have developed the necessary tools to measure and define quantitative parameters of biofilms and the ECM [3,61,62]. Here we review two approaches to annotate ECM composi-tion with specific attention given to C. albicans and A. fumigatus, the most well-studied fungal systems and common causative agents of fungal infections in hospitalized patients. The first approach imple-ments an arsenal of traditional biochemical methods to identify important ECM components, including identification via immunoassays [63–66]; and the sec-ond approach relies upon solid-state nuclear magnetic resonance (NMR) analysis of the intact ECM to pro-file the atomic-level contributions of unique classes of
Fungal biofilm composition & opportunities in drug discovery Review
biomolecules to the ECM [47]. Solid-state NMR uses the nuclear spin property to obtain information about chemical composition and structure, and can be used to examine any heterogeneous and insoluble materi-als, including whole cells and biofilm communities, and is not restricted to the study of soluble materials as many conventional methods are. The strengths and limitations of the two methods will be compared, and at the conclusion of this review, we discuss how these approaches might be used in the context of drug dis-covery to provide improved opportunities to effectively treat fungal infections.
Immunoassays to identify A. fumigatus ECM constituentsSpecific biomolecules can be identified using a range of indicator dyes, antibodies, and lectins [67]. Alter-natively, the ECM constituents within each macro-molecular class can be annotated through systematic biochemical analysis without prerequisite knowledge of putative ECM composition [65]. The first approach, using indicator dyes and antibody- and lectin-conju-gated dyes, has been useful in fluorescence micros-copy studies to identify and spatially resolve biofilm constituents [23,32,68–70], and recent advances in super-resolution microscopy have permitted the tracking of single proteins and polysaccharides during biofilm
growth [71]. These studies have taught us that many ECM constituents appear to play complementary architectural roles and that localization of components within a biofilm can vary over time. It is important to note, however, that an inherent limitation of these studies is that they require prior knowledge of the b iofilm molecules of interest.
Immunoassays, which use antibodies that recognize specific molecules in order to identify or track the pres-ence of that molecule, were useful to validate the pres-ence of specific ECM polysaccharides and proteins in A. fumigatus biofilm, including biofilms formed in vitro and in vivo [63,64]. Early work by Beauvais et al. revealed that the colony surface of A. fumigatus grown under aerial static conditions contained a hydrophobic ECM that bound hyphae in a manner that closely resembled in vivo ‘fungal balls’ [63]. Additionally, as predicted for biofilm, cultures grown under these conditions exhib-ited enhanced resistance to polyene antifungals relative to those grown under non-ECM-producing conditions (shaken, submerged). Interestingly, gentle mechani-cal removal of the ECM did not modify resistance to nystatin, which suggested that the A. fumigatus ECM may only reduce mycelial accessibility of some poly-ene antifungals, and in some cases additional biofilm-specific mechanisms may be responsible for antifungal resistance. Immunoassays and subsequent gas chroma-
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tography identified galactomannan and α-1,3-glucans as A. fumigatus ECM components.
Glucose was identified as a major ECM component of in vitro A. fumigatus biofilms by monosaccharide analysis of digested ECM. Although the authors con-trolled for the possibility of contaminating glucose from the growth medium by culturing in glucose-free medium, it is important to recognize the general limi-tations of conditions used for monosaccharide analysis in many applications. To emphasize this, we highlight an example from work with the opportunistic human pathogen Pseudomonas aeruginosa. Until recently, Pel, which is an important extracellular polysaccharide of P. aeruginosa, was mistakenly believed to be a glucose-rich molecule. This misidentification was due in part to the recalcitrance of Pel to isolation and standard preparatory hydrolysis conditions for monosaccharide compositional analysis [23]. Using harsher hydrolysis conditions for monosaccharide compositional analysis, Pel was determined to be a polymer of N-acetylglu-cosamine and N-acetylgalactosamine [23]. This assign-ment was supported by the reactivity of Pel to antibod-ies raised against poly-β-1,6-N-acetylglucosamine and chitosan (poly-β-1,4-N-acetylglucosamine) as well as binding to N-acetylgalactosamine-specific lectins.
Protein quantification assays also have limitations in their application to biofilm and ECM samples. The overall protein concentration of in vitro A. fumigatus ECM was found to be 2% (w/w) by the bicinchoninic acid (BCA) assay. However, this assay can incorrectly estimate protein concentration, as the complex molec-ular environment of the ECM can contain biomole-cules such as glucose and lipids that can cause interfer-ing absorbance in the presence of the BCA reagent or may limit accessibility of the protein peptide bonds to the BCA reagent (specifically Cu2+ ions) [72–74]. The major antigens, DppV, catalase B and Asp f1, were identified by immunoblot, and the hydrophobic nature of the colony suggested the presence of hydrophobic proteins [75]. However, the contributions and identities of other potential proteins were not determined.
Despite the technical limitations of the above meth-ods and inability to generate a complete accounting of the A. fumigatus ECM, the assays allowed an informa-tive comparison between A. fumigatus ECM samples formed during host invasion, which was observed to depend upon whether the aspergillosis was localized (aspergilloma) or invasive (IPA) [64]. ECM was present in both pathological settings. However, hyphae in the IPA were separated and surrounded by a thinner layer of ECM relative to that of an aspergilloma. Asper-gilloma appeared as a ball of strongly agglutinated hyphae, which was devoid of host cells and surrounded by a dense ECM network. Whole-biofilm immunoas-
says revealed that galactosaminogalactan (GAG) and galactomannan were major polysaccharides observed in the ECM, and α-1,3-glucan was only detected in aspergilloma at the periphery of the ECM. In vivo labeling with anti-GAG antibody was more intense as compared with in vitro labeling, suggesting that GAG was a major component of ECM produced during the development of A. fumigatus in tissues of patients with either aspergilloma or invasive aspergillosis. Later, it was observed that GAG mediates adherence to host cells, serves to control the host immune response by shielding β-glucans, and is a virulence factor that is required for biofilm formation [76–78]. The prevalence of GAG was later supported by studies using atomic force microscopy showing that GAG was highly exposed and able to serve as a fungal adhesin [79]. The biosynthesis of GAG has also been under study with efforts to understand the mechanism of deacety-lation of residues in GAG, for example [80,81]. Immu-nocytochemical assays performed with antibodies raised against the three major secreted antigens men-tioned above (DppV, catalase B, and Asp f1), showed that these antigens were not embedded in the in vivo ECM [64]. Thus, the immunoassays of putative poly-saccharide and protein components permitted com-parative analysis of the ECM from in vitro and in vivo A. fumigatus biofilms and demonstrated that each sys-tem included many similar components such as GAG, galactomannan, α-1,3-glucans, and melanin. In addi-tion, some components, such as the antigenic proteins, were enriched in the in vitro ECM but not detected in the in vivo ECM [63,64]. These studies highlight the strengths of using immunoassays to detect particular predicted ECM components. However, this approach is limited to studying only predicted components as well as those for which specific antibodies are available. Furthermore, using this approach, it is not possible to quantify the contributions of each component in the total ECM.
Macromolecular screening for C. albicans ECM compositionMany of the biochemical methods available to iden-tify ECM components were collectively implemented in a recent single study of C. albicans biofilm by Zar-nowski et al. (Figure 2) [65]. In this study, the research-ers first profiled the contributions to the crude ECM of each of the macromolecular classes: protein, poly-saccharide, lipid, and nucleic acid. This initial profil-ing was performed using a combination of spectro-photometric and colorimetric assays. Specifically, the protein enrichment was determined to be 55% (w/w) using a BCA assay; the carbohydrate content was determined to be 25% (w/w) using the phenol–sul-
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furic acid method; the nucleic acid concentration was determined to be 5% (w/w) using the absorbance of the crude ECM at 260 nm; and, finally, the remain-ing 15% (w/w) was assigned to lipid. The crude ECM was then processed in order to further characterize specific C. albicans ECM constituents using additional biochemical techniques. Specific proteins were iden-tified using proteomic mass spectrometry following enzymatic digestion of crude matrix material. Most of the 565 identified proteins play a role in metabolism or in the production and modification of polysaccha-rides. Interestingly, when the proteomic analysis was repeated using in vivo biofilms formed in a rat catheter model, the majority of identified ECM proteins were mammalian, suggesting that host factors may greatly impact biofilm composition, a topic that warrants fur-ther study. Sequence analysis found that much of the nucleic acid was noncoding. Lipids were profiled using gas chromatography, and nearly all of the lipids were found to be glycerolipids.
Further analysis of the carbohydrate contributions to the ECM primarily relied upon monosaccharide analysis and solution NMR following purification and fractionation steps. Initially, size-exclusion chroma-tography identified both high-molecular weight and low-molecular-weight fractions that constituted 38.3 and 61.7% of the total carbohydrate, respectively. Monosaccharide analysis concluded that the high-molecular weight fraction was primarily mannose, while the low-molecular-weight fraction was mostly arabinose. 1D and 2D solution NMR analysis of the abundant neutral carbohydrate fractions supported the presence of both mannan and glucan residues, and the most-abundant mannan polysaccharide was inves-tigated using small-angle x-ray scattering. While 2D correlative NMR methods were not able to identify a linkage between the mannan and glucan residues, a novel mannan–glucan complex was predicted based upon the constant ratio of each in chromatography fractions as well as colocalization of anti-mannan and anti-glucan antibodies observed in confocal imag-ing of in vitro biofilms. This colocalization also was observed in biofilms formed in vivo. As previously mentioned, β-1,3-glucan can bind to antifungals. However, only small amounts of matrix β-1,3-glucan were observed via immuno-TEM, suggesting that additional ECM components are likely able to seques-ter antifungals. This concept was explored using a solution-state 1H NMR binding assay, in which line-broadening of matrix peaks suggested an interaction of the matrix with fluconazole. It was also determined that multiple ECM components were necessary for drug binding, which was further supported in a later study [82].
This meticulous study provided new information about the relative abundance of the different macromol-ecules in C. albicans ECM that could be digested and accessed, and used this information to predict the pres-ence of a novel exopolysaccharide complex [65]. How-ever, many biofilms are resistant to the solubilization that is required for such solution-based analyses, and harsh degradative conditions are often used to attempt to overcome this challenge and generate some material for analysis [83,84]. Furthermore, in addition to the previ-ously stated limitations of the BCA assay to determine ECM protein concentration, the phenol–sulfuric acid method can incorrectly estimate the enrichment of car-bohydrate. For the phenol–sulfuric acid method to be quantitative, the standard samples must contain repre-sentative portions of the types of monosaccharides found in the experimental sample, which is difficult to predict for complex carbohydrate mixtures such as ECM [85]. As a consequence of the limitations of these solution-based assays, estimates of contributions of proteins, polysac-charides, and other biomolecules can be dramatically misrepresented depending on the efficiency of the solu-bilization and the extent of material loss during sample processing, and extreme care should be taken when interpreting results from such analyses. These limita-tions, coupled with the importance of defining biofilm matrix composition as well as comparing biofilms and the influence of potential inhibitors, have encouraged the development of new approaches to help transform more qualitative biofilm descriptors into quantitative parameters of molecular composition [58,59].
Solid-state NMR to quantify A. fumigatus ECM compositionWe recently developed an approach that utilizes solid-state NMR to quantitatively characterize and define biofilm and ECM composition [61,86]. Solid-state NMR, in general, permits analysis of the entire, intact ECM without preparatory chemical or enzymatic processing. There is no intrinsic size or mass limit as in solution NMR, which requires high-molecular tumbling rates to achieve high-resolution NMR spectra [61]. More specifi-cally, and as reviewed in more detail in the direct con-text of biofilm and ECM characterization, solid-state NMR employs magic-angle spinning to mechanically spin samples of multicellular biofilm communities or isolated ECM to help achieve the necessary resolution to obtain quantitative spectra of these heterogeneous and insoluble systems [61,62]. Solid-state NMR has been applied to study other similarly complex and insoluble systems such as bacterial whole cells and cell walls [87–91] and intact plant leaves [92]. In 2013, we reported the first quantitative determination of the chemical composition of intact ECM of a microbial biofilm by using solid-
Figure 2. Schematic representation of a protocol employed to characterize the composition of Candida albicans biofilm. This methodology relies upon extensive processing of the biofilm and extracted matrix. Differences in solubilization efficiency of ECM components can result in compositional estimates that may vary widely from prep-to-prep [65]. An advantage of this approach, which uses an integrated effort, is the identification of specific matrix parts in the ECM, although there are caveats with quantification of these parts. BCA: Bicinchoninic acid; ECM: Extracellular matrix; GC: Gas chromatography; HPLC: High-performance liquid-chromatography; LC–MS/MS: Liquid chromatography–mass spectrometry; NMR: Nuclear magnetic resonance; SAXS: Small-angle x-ray scattering.
C. albicans bio�lm
Matrix isolation
Crude ECM
Carbohydrateanalysis
Proteinanalysis
Lipidanalysis
Nucleic acidanalysis
Absorbanceat 260 nm
Solvent extractionand methylation
Colorimetricdetermination
BCAassay
Approximatecarbohydrate
contentApproximate
proteincontent
ApproximateDNA
content
Steroldetermination
Fatty acididenti�cationPuri�cation and
fractionation steps:size exclusion
chromatography,anion exchange,and gel �ltration
Chemical analysis:GC, NMR, and SAXS
GC analysis
Extractionand GC–MS
Cloned andsequencedEnzymatic
digestionSolid-phase extraction,
HPLC, and massspectrometry
Proteomicmass spectrometry
(LC–MS/MS)
Prostaglandincharacterization
Proteinidenti�cation
Monosugar identityand linkage determination
Determined thateDNA is noncoding
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state NMR, biochemical analysis, and electron micros-copy [86]. These initial experiments were performed on an important human pathogenic bacterium, the uro-pathogenic E. coli (UPEC) strain UTI89. We discovered that the spectral additional of just two components, the functional amyloid protein curli and a modified form of cellulose, completely recapitulated the 13C NMR spec-trum of intact UTI89 ECM. Thus, we determined that UTI89 ECM is 85% curli and 15% modified cellulose by mass. However, this bottom-up approach used for analysis of UTI89 required separate samples of puta-tive ECM components, which are not readily known or available for many biofilm systems [61].
To overcome this limitation, we developed an alternative top-down solid-state NMR approach that permits spectroscopic dissection and annotation of complex material and can be applied to biofilms or other multicomponent systems for which there is less known (or even nothing known) about poten-tial constituents [61]. We first developed this method to examine Vibrio cholerae ECM composition, and as discussed below, subsequently implemented the top-down approach in Aspergillus fumigatus [61,83]. This approach uses two types of 1D solid-state NMR experiments: cross-polarization magic-angle spinning (CPMAS) [93,94] and rotational-echo double-resonance
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(REDOR) [95]. The 13C CPMAS spectrum contains information about the quantities of carbon types, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics) and aliphatics, present in the ECM. By ensuring full incorporation of 15N into the matrix via defined growth medium, it is possible to further annotate the carbon pools accord-ing to C–N and C–P couplings using both 13C{15N} and 13C{31P}REDOR NMR experiments [61,83].
We applied this top-down solid-state NMR meth-odology to quantify the ECM composition of the fun-gal A. fumigatus biofilm grown in RPMI 1640 nutrient medium (selected because of its optimum for growing mammalian cells) [47]. The 13C CPMAS spectrum of A. fumigatus ECM showed contributions from a range of biomolecules (Figure 3A). To use CPMAS to quantify the number of nuclei at corresponding chemical shifts relative to others in a spectrum, differences in cross-polarization (CP) efficiency and relaxation must be taken into account. Such differences can arise due to variations in local dynamics and in the spatial distributions of 1H nuclei in the sample that are coupled to the carbons. By way of experimental detail, we monitored the A. fumiga-tus ECM 13C CPMAS intensities as a function of CP con-tact time, and observed a typical ‘exponential rise–expo-nential decay’ behavior that was extrapolated to zero contact time to quantify relative spin numbers. Together the 13C CPMAS peaks due to polysaccharide anomeric and ring-sugar carbons, which are chemical shift resolved and thus were able to be uniquely assigned, accounted for approximately 43% of the total carbon mass.
To annotate the remaining 57% of the carbon mass, we capitalized on the unique C–N and C–P couplings that are present in different biomolecules (Figure 3B & C). For example, the 13C spectrum of the ECM contained a peak near 173 ppm that was attributed to carbonyl carbons. Several types of biomolecules contain carbonyls, but all peptide carbonyl carbons are directly bonded to nitrogen (Figure 3C). Thus, by using 13C{15N}REDOR as a spec-troscopic filter to select only carbonyls that are directly bonded to nitrogen, we could determine an upper limit of the amount of protein carbonyls. Through determin-ing the quantitative CPMAS peak intensities of different carbon types as described above, we discovered that the carbonyl peak accounted for 12% of the total 13C spec-trum of A. fumigatus ECM. Using 13C{15N}REDOR, we found that only 76% of the carbonyl carbons were directly bonded to nitrogen. Thus, at most, only 9% (76 of 12%) of total ECM carbon could be attributed to peptide carbonyls of proteins. The percentage of possible α-carbons was similarly determined to be 7% (includ-ing α-carbons of both glycine and other amino acids). Amino acids contain an average of 5.4 carbons each, and so three additional carbons can be generally attributed
to protein. These carbons mostly would be observed in the upfield aliphatic region of the 13C CPMAS spectrum. Together, this analysis supports that approximately 40% of the total carbon mass can be attributed to protein. The aromatic region of the 13C CPMAS spectrum accounts for an additional 3% of the total carbon mass, which was partially attributed to melanin.
The remaining 14% of the 13C CPMAS spectral area contains contributions from carbonyls and aliphatics (excluding directly nitrogen-bonded carbons), includ-ing a sharp peak at 33 ppm that is characteristic of CH
2
groups typically found in lipids and accounts for 7% of the total spectral area. Spectral selection of portions of these regions by 13C{31P}REDOR also suggested that the ECM contained some phospholipid. Thus, A. fumigatus ECM contained at least 7% lipid (due to the character-istic peak at 33 ppm) and up to 14% lipid by carbon mass. Taken together, the A. fumigatus ECM produced under these growth conditions was approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components and up to 14% lipid.
Thus, atomic-level parameters of the intact isolated A. fumigatus ECM were measured and defined using the top-down solid-state NMR method. In addition, other valuable compositional parameters of the ECM constitu-ents were obtained. The 15N CPMAS spectrum showed that most of the ECM nitrogen is present in protein (appearing as an amide peak at 119 ppm). We were also able to detect low abundance nitrogen-containing motifs likely present as modifications to ECM constituents, for example, as amino or N-acetyl modifications. The 13C{15N}REDOR results suggested that some of the exo-polysaccharides were N-acetylated based upon the pres-ence of peaks attributed to N-acetyl methyl groups and the observed proximity of the nitrogen to some of the sugar-ring carbons. This finding is consistent with some of the previously identified extracellular polysaccharides produced by A. fumigatus including GAG. N-acetyl groups also could occur as part of the N-acetylglucos-amine groups present in N-linked glycosylation of ECM proteins. The possibility that some of the extracellular proteins were glycosylated was explored using traditional biochemical analysis. SDS-PAGE analysis of the ECM showed several bands corresponding to SDS-soluble proteins, and these bands were excised and subjected to proteomic mass spectrometry. The SDS-soluble pro-teins were identified as catalase B and Asp f2. Consistent with the NMR spectral detection of N-acetyl groups, these two proteins are reported to be N-linked glycosyl-ated. Digestion with peptide-N-glycosidase F (PNGase) resulted in cleavage consistent with glycosylation of Asp f2 in this ECM sample.
Importantly, the overall solid-state NMR approach provides absolute carbon intensities that can be com-
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pared across samples, whether due to different biofilm formers or the influence of external stimuli such as antibiofilm agents. We developed an approach to finely annotate the specific carbon types and to obtain a quan-titative accounting of these pools in the biofilm ECM. Even quick inspection of the comparative 1D CPMAS spectra would permit facile identification of whether any significant differences were present among a sample set. Coupled with electron microscopy of intact biofilms and the isolated ECM material as well as biochemical analy-ses for the specific identification of some biofilm parts, we believe that this integrated approach with solid-state NMR analyses provides one of the most robust and powerful ECM characterization approaches available.
ConclusionConnections between ECM composition and func-tion are crucial to understanding the fundamental molecular basis for: fungal biofilm physiology, the
recalcitrance of biofilm infections to antifungal treat-ment, and antifungal resistance. Robust and reliable methods to define and/or quantitatively compare ECM composition are crucial for driving these con-nections. The two major approaches to annotate ECM composition described in this review provide comple-mentary details of ECM composition and connections with function. Solid-state NMR is uniquely suited to profile chemical composition of complex, insoluble sys-tems like ECM [62]. The top-down solid-state NMR method described above can be applied to the study of any biofilm, including mixed species or in vivo-derived biofilms, and, importantly, does not require any pre-requisite knowledge of biofilm composition [47,61,62,83]. The approach first involves the annotation of the fun-damental carbon and nitrogen compositional pools, for example, quantifying the prevalence of carbonyls and methyls, etc. It also takes advantage of poten-tially unique internuclear couplings to further dissect
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ECM composition and to specify the types of carbon-yls present, for example. The analysis is performed on the intact ECM and provides parameters not acces-sible from solution-based biochemical methods that require solubilization of ECM parts, wherein much of the ECM is often not analyzed due to its recalcitrance to digestion and dissolution. Furthermore, solid-state NMR analysis permits one to simultaneously observe and quantify multiple macromolecular species and a complete analysis can be performed on the same sam-ple. The advantages of the solid-state NMR approach thus include the nondestructive nature of the analysis and the ability to quantify the types of nuclei present in a sample. A disadvantage, is the limited sensitivity of NMR and the relatively large sample sizes and/or long spectral acquisition times required to achieve high sig-nal-to-noise spectra using natural abundance 13C sam-ples. Isotopically labeled samples improve sensitivity, although it is attractive that 13C profiling, for example, does not require labeling and can be performed using 13C at natural abundance. NMR correlation experi-ments provide additional selectivity to specify the spe-cific nature of carbon pools that are annotated through 1D spectra. These experiments require isotopic enrich-ment, for example, as we demonstrated with 15N-incor-poration in a customized RPMI 1640 medium for the analysis of A. fumigatus ECM composition described above [47]. Thus, it is important to choose the right tool for the experiment, taking into account the problem-solving advantages and the technical limitations that can include sample solubility, processing requirements, and sample size.
Future perspectiveIdeally, the traditional biochemical methods applied to solubilized parts of the ECM should be integrated with quantitative compositional profiling by solid-state NMR. The NMR analysis provides a total snapshot of molecular contributions to the intact ECM and the prevalence of molecules in the ECM, such as the relative abundance of proteins versus polysaccharides, including an analysis of types of chemical modifica-tions present in the samples. Yet, while each protein has a unique NMR signature, identification of indi-vidual proteins in the ECM is best performed using proteomic mass spectrometry and immunoassays. Similarly, the identities of lipids and small, soluble polysaccharides can be determined using solution-based biochemical methods on the individual purified components in isolation from one another. In addi-tion to the study of complex, biomolecular mixtures, solid-state NMR can be applied to study in detail the isolated ECM components such as the high-molecular-weight polysaccharide fractions identified in the pre-
viously described C. albicans study [65], and does not require harsh, degradative conditions prior to analysis. In this way, solid-state NMR has been used to quanti-tatively determine structural information of similarly noncrystalline and insoluble bacterial [88,90,91,96] and plant cell walls [97–100].
Solid-state NMR also is well suited for the study of drug–matrix interactions, with inspiration from stud-ies that mapped atomic-level interactions of the anti-biotic [19F]oritavancin with S. aureus cell walls (both in isolated cell walls and in the whole-cell context) by measuring several 19F-13C and 19F-15N distances between drug and specific cell-wall sites [87]. In the realm of antifungals, solid-state NMR approaches have been used to examine the possible mechanism(s) of action of amphotericin B in lipid vesicle systems used as surrogates for fungal membranes. Most struc-tural studies have characterized the pore-forming assemblies of amphotericin [101], yet very recent solid-state NMR work examined samples prepared with phospholipids, ergosterol and amphotericin, and showed that amphotericin B could extract ergosterol out of phospholipids, and serve as a type of ergosterol sponge [102]. These types of structure-focused NMR approaches are ripe for identifying specific ECM com-ponents that bind to antifungals and for mapping the interactions between the ECM and 19F-labeled drugs, for example. Together, solid-state NMR methods enable the quantification of the abundance of bio-molecules in a biofilm and can be used to determine the ways in which the components could be interact-ing with antifungals to contribute to the antifungal r esistance often exhibited by biofilms.
The ECM is able to inhibit the access of some anti-fungals to fungal cells in a biofilm and contributes to the challenge of treating biofilm-associated infections. The potential mechanisms of ECM-mediated antifun-gal recalcitrance include both matrix–matrix interac-tions and matrix–drug interactions. For example, the matrix could be self-associating to form an impen-etrable barrier to drugs. Alternatively, the matrix could either bind to drugs or enzymatically inactivate the drugs, both of which prevent antifungals from reach-ing their intended target at the surface of or within the fungal cell. The ECM composition plays a key role in dictating the possible mechanisms that biofilms employ to decrease susceptibility to antifungals. In the past, knowledge of ECM composition has given rise to more effective strategies to treat biofilm-involved fungal infections such as the coadministration of amphoteri-cin B with alginate lyase to degrade exopolysaccharides and enhance the antifungal activity of amphotericin B against A. fumigatus biofilms [46]. As knowledge of biofilm composition, including composition of mixed
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Executive summary
Background• Biofilm infections are recalcitrant to antimicrobial treatment and clearance by host immune responses.• Many prescribed antifungal drugs are not effective against biofilm infections owing to several protective
factors including poor diffusion of drugs through biofilms as well as specific drug–matrix interactions.• Both Candida albicans and Aspergillus fumigatus form biofilms, and these biofilms are involved in a range of
human infections, including lethal fungal infections among immunosuppressed individuals.• A defining feature of biofilms is the extracellular matrix (ECM), which is a self-produced, noncrystalline
material that encases microbial cells. ECM is typically rich in biopolymers and can contain proteins, polysaccharides, lipids, nucleic acids, and other molecules.
• The formidable survival traits of biofilms combined with our dwindling pipeline of effective antifungals make it imperative that we undertake in-depth analyses to improve upon our understanding of biofilms.
• The two approaches, traditional biochemical methods and a newly developed solid-state NMR method, can be used to annotate ECM composition and provide complementary perspectives on ECM composition and function.
Immunoassays to identify A. fumigatus ECM constituents• Immunoassays were useful to validate the presence of specific ECM polysaccharides and proteins
in A. fumigatus biofilm, including both in vitro and in vivo biofilms.• Immunoassays and subsequent gas chromatography identified galactomannan and α-1,3-glucans as
A. fumigatus ECM components.• The major antigens, DppV, catalase B and Asp f1, were identified by immunblot in the in vitro ECM but were
not detected in the in vivo ECM.Macromolecular screening for C. albicans ECM composition• The ECM of Candida albicans was examined and the contributions of each of the macromolecular classes were
profiled.• This initial profiling was performed using a combination of spectrophotometric and colorimetric assays, and
the ECM was determined to be 55% (w/w) protein, 25% (w/w) carbohydrate, 5% (w/w) nucleic acid, and 15% (w/w) lipid.
• Carbohydrate characterization primarily relied upon monosaccharide analysis and solution nuclear magnetic resonance (NMR) following purification and fractionation steps, and both high-molecular-weight and low-molecular-weight fractions were identified.
• Many biofilms are resistant to the solubilization that is required for such solution-based analyses, and harsh degradative conditions are often used to overcome this challenge which can lead to misrepresentations of ECM composition.
Solid-state NMR to quantify A. fumigatus ECM composition• A solid-state NMR approach was developed that permits analysis of the entire, intact ECM without
preparatory chemical or enzymatic processing.• Solid-state NMR does not require high tumbling rates in solution (solution NMR) or homogeneous samples,
and provides quantitative information about composition.• This solid-state NMR approach uses two types of 1D NMR experiments: cross-polarization magic-angle
spinning and rotational-echo double-resonance.• Spectral dissection using solid-state NMR determined that the A. fumigatus ECM was approximately 40%
protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid.• In addition to the atomic-level parameters of A. fumigatus ECM gained using this top-down solid-state NMR
method, general characteristics of the ECM constituents were obtained including glycosylation of extracellular proteins and types of modifications of exopolysaccharides.
Opportunities in drug discovery• Measurements of ECM composition are crucial to understanding fungal biofilm physiology, the recalcitrance
of biofilm infections to antifungal treatment, and antifungal resistance.• Integrated approaches employing electron microscopy, biochemical methods including proteomics analyses,
together with solid-state NMR compositional measurements would allow for comprehensive characterization and elucidation of the generation of quantitative parameters of ECM composition, enabling comparisons with samples from organisms treated with antifungal and antibiofilm agents.
• Solid-state NMR approaches can be used to map the ECM binding sites and bound conformations of candidate therapeutics in isolated ECM and in intact cells.
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species and in vivo biofilms increases, we believe that effective strategies to treat biofilm-involved fungal infections will also improve. Knowledge of the ECM composition provides evidence of which mechanisms could be in play for a particular biofilm, and further-more, provides routes to overcome the matrix-medi-ated antifungal resistance. These routes may involve combined antifungal and anti-virulence approaches. For example, the biophysical and biochemical ways in which the ECM is able to act as a drug barrier could be taken into account when designing an antifungal drug so that the drug meets size, charge, and reactiv-ity requirements to be able to pass through the bio-film and impact the fungal cells. Complementary anti-virulence approaches could block matrix–matrix or matrix–drug interactions, either through co-admin-istration of additional drugs or through mechanical
disruption. Specific knowledge of those matrix–matrix and matrix–drug interactions is crucial to the design of such therapeutics. Thus, compositional and molecular-level descriptions of the ECM should help to drive the development of strategies to eradicate biofilm-associ-ated infections and develop more effective antifungal treatments.
Financial & competing interests disclosureThe authors acknowledge support from the National Science
Foundation CAREER Award, NSF Grant Number 1453247. The
authors have no other relevant affiliations or financial involve-
ment with any organization or entity with a financial interest
in or financial conflict with the subject matter or materials dis-
cussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this
manuscript.
ReferencesPapers of special note have been highlighted as: • of interest
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