Invited review
Fungal Lanosterol 14α-demethylase: A target for next-generation antifungal design

https://doi.org/10.1016/j.bbapap.2019.02.008Get rights and content

Highlights

  • Lanosterol 14α-demethylase (LDM) is the target of the azole antifungals.

  • Avoidance of target- and efflux-mediated antifungal resistance is needed.

  • LDM ligand occupy a buried active site, substrate entry & product exit channels.

  • Azole inhibitors bind via the heme, the polypeptide & water-mediated hydrogen bonds.

  • LDM structures & yeast-based tools can identify next-generation azole fungicides.

Abstract

The cytochrome P450 enzyme lanosterol 14α-demethylase (LDM) is the target of the azole antifungals used widely in medicine and agriculture as prophylaxis or treatments of infections or diseases caused by fungal pathogens. These drugs and agrochemicals contain an imidazole, triazole or tetrazole substituent, with one of the nitrogens in the azole ring coordinating as the sixth axial ligand to the LDM heme iron. Structural studies show that this membrane bound enzyme contains a relatively rigid ligand binding pocket comprised of a deeply buried heme-containing active site together with a substrate entry channel and putative product exit channel that reach to the membrane. Within the ligand binding pocket the azole antifungals have additional affinity determining interactions with hydrophobic side-chains, the polypeptide backbone and via water-mediated hydrogen bond networks. This review will describe the tools that can be used to identify and characterise the next generation of antifungals targeting LDM, with the goal of obtaining highly potent broad-spectrum fungicides that will be able to avoid target and drug efflux mediated antifungal resistance.

Section snippets

The impact of fungi on human health, food security and ecosystems

Of the estimated 2.2 - 3.8 million fungal species [1], most appear to be saprophytes that make available critical nutrients by assisting the decay of detritus in forests, soil and the sea. Many fungal species are commensals that cohabit with host plants or animals while humans have exploited some species to make palatable foods, produce biotechnological products and gain insight into basic biology [2]. Some commensal or opportunistic fungi may become significant pathogens when hosts are

The need for new azole antifungals

There is increasing need for more effective antifungals that prevent patient discomfort caused by superficial infections and patient deaths due to disseminated disease. Therapy with allylamines, using terbinafine to target dermatophyte squalene monooxygenases, often suppresses rather than cures superficial infections of the skin, and toenails. Azole drugs are also used to treat these difficult infections. Azoles and polyenes are widely used to treat superficial infections of mucosal membranes

Experimental models used to study LDM structure and function

Obtaining in depth structural and functional knowledge of fungal LDM was initially limited because the protein is bound to the membrane via an N-terminal transmembrane helix and was therefore thought to be difficult to purify and crystallize. These problems have been overcome by expressing the catalytic domain in Escherichia coli [9,10] or by expressing full-length fungal enzymes in S. cerevisiae [6].

Reaction mechanism

The LDM enzyme reaction involves 3 cycles of reduction that removes a water molecule bound to the haem, and the formation activated haem-oxygen (FeIV=O) complexes that sequentially modify the lanosterol 14-methyl group to an alcohol, an aldehyde and then introduces a 14-15 double bond and releases formate. The reaction uses as substrate lanosterol, the electrons generated from 3 molecules of NADPH by NADPH-cytochrome P450 reductase, 3 protons, 3 O2 molecules and generates

Murine models

Mouse models have been used as the gold standard in determining responses of fungal diseases to drug treatment. Murine models are available using oral, lung, vaginal, pulmonary or bloodstream infections [[171], [172], [173]]. There are numerous reports on the testing of new antifungals as well as existing ones in combination with modulators [141,174,175], or on drug delivery [176]. Because murine models are expensive, they are primarily used to test advanced drug candidates as a prelude to

The value of structural analysis

There are numerous challenges in determining the crystal structures of membrane proteins. Obtaining sufficient amounts of enzyme for purification often requires heterologous overexpression of a recombinant protein in a suitable host such as E. coli or S. cerevisiae. Solubilising the protein from membranes requires appropriate physiochemical conditions together with a detergent that enables retention of bioactivity during purification and is compatible with crystallization [215]. In some cases a

Basic research into ligand binding and the impact of mutations in the LBP

The most recent generation of azole drugs are potent antifungals with affinity in the <100 nM range. While posaconazole and possibly isavuconazole and VT-1598 have broad-spectrum activity, VT-1161 and VT-1129 are not effective against moulds [227]. The most widely used azole drugs are subject to a variety of drug resistance mechanisms including innate resistance to fluconazole in A. fumigatus, innate resistance to fluconazole and voriconazole in the mucormycetes, as well as the acquisition of

Summary

The discovery of antifungals that target LDM is at an important cross-roads. This discovery process can now be structure-directed with the support of a range of physiological and biochemical tools. The high-resolution structures of LDM from the model yeast S. cerevisiae, and from three of the most prominent fungal pathogens, have elucidated features that define the binding of lanosterol and the several azole drugs and agrochemicals within the LBP. This enables structure-directed discovery to

Acknowledgements

This research was supported by grants to BCM from the Marsden Fund of the Royal Society of New Zealand (UOO1004) and the Health Research Council of New Zealand (HRC of NZ 13/263 and 16/232).

References (233)

  • D. Sears et al.

    Candida auris: An emerging multidrug-resistant pathogen

    Int J Infect Dis

    (2017)
  • R. Rajasingham et al.

    Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis

    Lancet Infect Dis

    (2017)
  • F. Tekaia et al.

    Aspergillus fumigatus: saprophyte or pathogen?

    Curr Opinion Microbiol

    (2005)
  • P.E. Verweij et al.

    Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use?

    Lancet Infect Diseases

    (2009)
  • P.A. Greenberger

    Allergic bronchopulmonary aspergillosis

    J Allergy Clin Immunology

    (2002)
  • E. Snelders et al.

    Genotype–phenotype complexity of the TR 46/Y121F/T289A cyp51A azole resistance mechanism in Aspergillus fumigatus

    Fungal Genetics and Biology

    (2015)
  • L. Ghelardini et al.

    Drivers of emerging fungal diseases of forest trees

    Forest Ecol Manag

    (2016)
  • D.L. Hawksworth et al.

    Fungal diversity revisited: 2.2 to 3.8 million species

    Microbiol Spectr

    (2017)
  • The Fungal Kingdom

    (2018)
  • M.C. Fisher et al.

    Emerging fungal threats to animal, plant and ecosystem health

    Nature

    (2012)
  • H.N. Fones et al.

    Emerging fungal threats to plants and animals challenge agriculture and ecosystem resilience

    Microbiol Spectr

    (2017)
  • G.D. Brown et al.

    Hidden killers: human fungal infections

    Sci Transl Med

    (2012)
  • B.C. Monk et al.

    Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

    Proc Natl Acad Sci USA

    (2014)
  • P.L. Yeagle et al.

    Differential effects of cholesterol and lanosterol on artificial membranes

    Proc Natl Acad Sci USA

    (1977)
  • H. Vanden Bossche et al.

    Cytochrome P450: target for intraconazole

    Drug Devel Res

    (1986)
  • T.Y. Hargrove et al.

    Crystal structure of the new investigational drug candidate VT-1598 in complex with Aspergillus fumigatus sterol 14alpha-demethylase provides insights into its broad-spectrum antifungal activity

    Antimicrob Agents Chemother

    (2017)
  • A.A. Sagatova et al.

    Structural insights into binding of the antifungal drug fluconazole to Saccharomyces cerevisiae lanosterol 14alpha-demethylase

    Antimicrob Agents Chemother

    (2015)
  • E.L. Berkow et al.

    Fluconazole resistance in Candida species: a current perspective

    Infect Drug Resist

    (2017)
  • A. Resendiz Sharpe et al.

    I.E.A.R.S.w. group, Triazole resistance surveillance in Aspergillus fumigatus

    Med Mycol

    (2018)
  • D. Sanglard et al.

    The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents

    Antimicrob Agents Chemother

    (1999)
  • J.P. Vermitsky et al.

    Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor

    Antimicrob Agents Chemother

    (2004)
  • A.S. Chau et al.

    Molecular basis for enhanced activity of posaconazole against Absidia corymbifera and Rhizopus oryzae

    Antimicrob Agents Chemother

    (2006)
  • P.E. Russell

    A century of fungicide evolution

    J Agricultural Sci

    (2005)
  • E. Snelders et al.

    Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism

    PLoS Med

    (2008)
  • J.R. Lamichhane et al.

    Toward a reduced reliance on conventional pesticides in European agriculture

    Plant Dis

    (2016)
  • D.S. Perlin et al.

    The global problem of antifungal resistance: prevalence, mechanisms, and management

    Lancet Infect Dis

    (2017)
  • B. Havlickova et al.

    Epidemiological trends in skin mycoses worldwide

    Mycoses

    (2008)
  • K. Goralska et al.

    Neuroinfections caused by fungi

    Infection

    (2018)
  • D. Allen et al.

    Azole antifungals: 35 years of invasive fungal infection management

    Expert Rev Anti Infect Ther

    (2015)
  • A. Mourad et al.

    Present and future therapy of Cryptococcus infections

    J Fungi (Basel)

    (2018)
  • S.G. Whaley et al.

    Azole antifungal resistance in Candida albicans and emerging non-albicans Candida Species

    Front Microbiol

    (2016)
  • R. Becher et al.

    Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) and azole resistance in plant and human pathogens

    Appl Microbiol Biotechnol

    (2012)
  • S.A. Flowers et al.

    Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans

    Antimicrob Agents Chemother

    (2015)
  • A.A. Sagatova et al.

    The impact of homologous resistance mutations from pathogenic yeast on Saccharomyces cerevisiae lanosterol 14alpha-demethylase

    Antimicrob Agents Chemother

    (2017)
  • A.A. Sagatova et al.

    Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14alpha-demethylase

    Sci Rep

    (2016)
  • B.C. Monk et al.

    Outwitting multidrug resistance to antifungals

    Science

    (2008)
  • J. Berman

    Ploidy plasticity: a rapid and reversible strategy for adaptation to stress

    FEMS Yeast Res

    (2016)
  • A. Coste et al.

    Genotypic evolution of azole resistance mechanisms in sequential Candida albicans isolates

    Eukaryot Cell

    (2007)
  • A. Coste et al.

    A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans

    Genetics

    (2006)
  • C.B. Ford et al.

    The evolution of drug resistance in clinical isolates of Candida albicans

    Elife

    (2015)
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