Invited reviewFungal Lanosterol 14α-demethylase: A target for next-generation antifungal design
Graphical abstract
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)
- et al.
Structure-functional characterization of cytochrome P450 sterol 14alpha-demethylase (CYP51B) from Aspergillus fumigatus and molecular basis for the development of antifungal drugs
J Biol Chem
(2015) - et al.
Structural analyses of Candida albicans sterol 14alpha-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis
J Biol Chem
(2017) - et al.
Design and optimization of highly-selective fungal CYP51 inhibitors
Bioorg Med Chem Lett
(2014) - et al.
Genotype-phenotype complexity of the TR46/Y121F/T289A cyp51A azole resistance mechanism in Aspergillus fumigatus
Fungal Genet Biol
(2015) - et al.
The structure-function relationship of the Aspergillus fumigatus cyp51A L98H conversion by site-directed mutagenesis: the mechanism of L98H azole resistance
Fungal Genet Biol
(2011) Vulvovaginal candidosis
Lancet
(2007)- et al.
Molecular basis of antifungal drug resistance in yeasts
Int J Antimicrob Agents
(2017) - et al.
Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature
Diagn Microbiol Infect Dis
(2010) - et al.
Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol
Biochem Biophys Res Commun
(1995) - et al.
Candida auris for the clinical microbiology laboratory: Not your grandfather's Candida species
Clin Microbiol Newsl
(2017)