A First-in-Class Pyrazole-isoxazole Enhanced Antifungal Activity of Voriconazole: Synergy Studies in an Azole-Resistant Candida albicans Strain, Computational Investigation and in Vivo Validation in a Galleria mellonella Fungal Infection Model.

The widespread and irrational use of azole antifungal agents has led to an increase of azole-resistant Candida albicans strains with an urgent need for combination drug therapy, enhancing the treatment efficacy. Here, we report the discovery of a first-in-class pyrazole-isoxazole, namely, 5b, that showed remarkable growth inhibition against the C. albicans ATCC 10231 strain in combination with voriconazole, acting as a downregulator of ERG 11 (Cyp51) gene expression with a significant reduction of the yeast-to-hypha morphological transition. Furthermore, C. albicans CYP51 enzyme assay and in-depth molecular docking studies unveiled the unique ability of the combination of 5b and voriconazole to completely fill the CYP51 binding sites. In vivo studies using a Galleria mellonella model confirmed the previously in vitro observed synergistic effect of 5b with voriconazole. Also considering its biocompatibility in a cellular model of human keratinocytes, these results indicate that 5b represents a promising compound for a further optimization campaign.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

Oxygen-18 Labeling Reveals a Mixed Fe-O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α-Demethylation.

The 14α-demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three-step reaction terminates in a C-C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2 - ) or Compound I (perferryl oxygen, FeO3+ ) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18 O incorporation into formic acid from 18 O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18 O (one atom) into formic acid, consistent with a major but not exclusive FeO2 - mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X-ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Šaway from the heme iron atom. Experiments with human P450 51A1 and H2 18 O yielded primarily one 18 O atom but 14 % of the formic acid product with two 18 O atoms, indicative of a minor contribution of a Compound I mechanism. LC-MS evidence for a Compound 0-derived Baeyer-Villiger reaction product (a 14α-formyl ester) was also found.

Structural modeling of cytochrome P450 51 from a deep-sea fish points to a novel structural feature in other CYP51s.

Cytochromes P450 (CYP), enzymes involved in the metabolism of endogenous and xenobiotic substrates, provide an excellent model system to study how membrane proteins with unique functions have catalytically adapted through evolution. Molecular adaptation of deep-sea proteins to high hydrostatic pressure remains poorly understood. Herein, we have characterized recombinant cytochrome P450 sterol 14α-demethylase (CYP51), an essential enzyme of cholesterol biosynthesis, from an abyssal fish species, Coryphaenoides armatus. C. armatus CYP51 was heterologously expressed in Escherichia coli following N-terminal truncation and purified to homogeneity. Recombinant C. armatus CYP51 bound its sterol substrate lanosterol giving a Type I binding spectra (KD 15 µM) and catalyzed lanosterol 14α-demethylation turnover at 5.8 nmol/min/nmol P450. C. armatus CYP51 also bound the azole antifungals ketoconazole (KD 0.12 µM) and propiconazole (KD 0.54 µM) as determined by Type II absorbance spectra. Comparison of C. armatus CYP51 primary sequence and modeled structures with other CYP51s identified amino acid substitutions that may confer an ability to function under pressures of the deep sea and revealed heretofore undescribed internal cavities in human and other non-deep sea CYP51s. The functional significance of these cavities is not known. PROLOGUE: This paper is dedicated in memory of Michael Waterman and Tsuneo Omura, who as good friends and colleagues enriched our lives. They continue to inspire us.

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1.

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4ß,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, with koff rates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3ß-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14α C-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Unravelling the role of transient redox partner complexes in P450 electron transfer mechanics.

The molecular evolution of cytochromes P450 and associated redox-driven oxidative catalysis remains a mystery in biology. It is widely believed that sterol 14α-demethylase (CYP51), an essential enzyme of sterol biosynthesis, is the ancestor of the whole P450 superfamily given its conservation across species in different biological kingdoms. Herein we have utilized X-ray crystallography, molecular dynamics simulations, phylogenetics and electron transfer measurements to interrogate the nature of P450-redox partner binding using the naturally occurring fusion protein, CYP51-ferredoxin found in the sterol-producing bacterium Methylococcus capsulatus. Our data advocates that the electron transfer mechanics in the M. capsulatus CYP51-ferredoxin fusion protein involves an ensemble of ferredoxin molecules in various orientations and the interactions are transient. Close proximity of ferredoxin, however, is required to complete the substrate-induced large-scale structural switch in the P450 domain that enables proton-coupled electron transfer and subsequent oxygen scission and catalysis. These results have fundamental implications regarding the early evolution of electron transfer proteins and for the redox reactions in the early steps of sterol biosynthesis. They also shed new light on redox protein mechanics and the subsequent diversification of the P450 electron transfer machinery in nature.

Relaxed Substrate Requirements of Sterol 14α-Demethylase from Naegleria fowleri Are Accompanied by Resistance to Inhibition.

Naegleria fowleri is the protozoan pathogen that causes primary amoebic meningoencephalitis (PAM), with the death rate exceeding 97%. The amoeba makes sterols and can be targeted by sterol biosynthesis inhibitors. Here, we characterized N. fowleri sterol 14-demethylase, including catalytic properties and inhibition by clinical antifungal drugs and experimental substituted azoles with favorable pharmacokinetics and low toxicity. None of them inhibited the enzyme stoichiometrically. The highest potencies were displayed by posaconazole (IC50 = 0.69 µM) and two of our compounds (IC50 = 1.3 and 0.35 µM). Because both these compounds penetrate the brain with concentrations reaching minimal inhibitory concentration (MIC) values in an N. fowleri cellular assay, we report them as potential drug candidates for PAM. The 2.1 Å crystal structure, in complex with the strongest inhibitor, provides an explanation connecting the enzyme weaker substrate specificity with lower sensitivity to inhibition. It also provides insight into the enzyme/ligand molecular recognition process and suggests directions for the design of more potent inhibitors.

The antifungal drug isavuconazole inhibits the replication of human cytomegalovirus (HCMV) and acts synergistically with anti-HCMV drugs.

We recently reported that some clinically approved antifungal drugs are potent inhibitors of human cytomegalovirus (HCMV). Here, we report the broad-spectrum activity against HCMV of isavuconazole (ICZ), a new extended-spectrum triazolic antifungal drug. ICZ inhibited the replication of clinical isolates of HCMV as well as strains resistant to the currently available DNA polymerase inhibitors. The antiviral activity of ICZ against HCMV could be linked to the inhibition of human cytochrome P450 51 (hCYP51), an enzyme whose activity we previously demonstrated to be required for productive HCMV infection. Moreover, time-of-addition studies indicated that ICZ might have additional inhibitory effects during the first phase of HCMV replication. Importantly, ICZ showed synergistic antiviral activity in vitro when administered in combination with different approved anti-HCMV drugs at clinically relevant doses. Together, these results pave the way to possible future clinical studies aimed at evaluating the repurposing potential of ICZ in the treatment of HCMV-associated diseases.

Concerning P450 Evolution: Structural Analyses Support Bacterial Origin of Sterol 14α-Demethylases.

Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an "orphan" P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.

The Clinically Approved Antifungal Drug Posaconazole Inhibits Human Cytomegalovirus Replication.

Posaconazole (PCZ) is a clinically approved drug used predominantly for prophylaxis and salvage therapy of fungal infections. Here, we report its previously undescribed anti-human cytomegalovirus (HCMV) activity. By using antiviral assays, we demonstrated that PCZ, along with other azolic antifungals, has a broad anti-HCMV activity, being active against different strains, including low-passage-number clinical isolates and strains resistant to viral DNA polymerase inhibitors. Using a pharmacological approach, we identified the inhibition of human cytochrome P450 51 (hCYP51), or lanosterol 14α demethylase, a cellular target of posaconazole in infected cells, as a mechanism of anti-HCMV activity of the drug. Indeed, hCYP51 expression was stimulated upon HCMV infection, and the inhibition of its enzymatic activity by either the lanosterol analog VFV {(R)-N-(1-(3,4'-difluoro-[1,1'-biphenyl]-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide} or PCZ decreased HCMV yield and infectivity of released virus particles. Importantly, we observed that the activity of the first-line anti-HCMV drug ganciclovir was boosted tenfold by PCZ and that ganciclovir (GCV) and PCZ act synergistically in inhibiting HCMV replication. Taken together, these findings suggest that this clinically approved drug deserves further investigation in the development of host-directed antiviral strategies as a candidate anti-HCMV drug with a dual antimicrobial effect.

A requirement for an active proton delivery network supports a compound I-mediated C-C bond cleavage in CYP51 catalysis.

CYP51 enzymes (sterol 14α-demethylases) are cytochromes P450 that catalyze multistep reactions. The CYP51 reaction occurs in all biological kingdoms and is essential in sterol biosynthesis. It removes the 14α-methyl group from cyclized sterol precursors by first forming an alcohol, then an aldehyde, and finally eliminating formic acid with the introduction of a Δ14-15 double bond in the sterol core. The first two steps are typical hydroxylations, mediated by an electrophilic compound I mechanism. The third step, C-C bond cleavage, has been proposed to involve either compound I (i.e. FeO3+) or, alternatively, a proton transfer-independent nucleophilic ferric peroxo anion (compound 0, i.e. Fe3+O2-). Here, using comparative crystallographic and biochemical analyses of WT human CYP51 (CYP51A1) and its D231A/H314A mutant, whose proton delivery network is destroyed (as evidenced in a 1.98-Å X-ray structure in complex with lanosterol), we demonstrate that deformylation of the 14α-carboxaldehyde intermediate requires an active proton relay network to drive the catalysis. These results indicate a unified, compound I-based mechanism for all three steps of the CYP51 reaction, as previously established for CYP11A1 and CYP19A1. We anticipate that our approach can be applied to mechanistic studies of other P450s that catalyze multistep reactions, such as C-C bond cleavage.

A new chemotype with promise against Trypanosoma cruzi.

Pyridyl benzamide 2 is a potent inhibitor of Trypanosoma cruzi, but not other protozoan parasites, and had a selectivity-index of ≥10. The initial structure-activity relationship (SAR) indicates that benzamide and sulfonamide functional groups, and N-methylpiperazine and sterically unhindered 3-pyridyl substructures are required for high activity against T. cruzi. Compound 2 and its active analogs had low to moderate metabolic stabilities in human and mouse liver microsomes.

Validation of Human Sterol 14α-Demethylase (CYP51) Druggability: Structure-Guided Design, Synthesis, and Evaluation of Stoichiometric, Functionally Irreversible Inhibitors.

Sterol 14α-demethylases (CYP51) are the cytochrome P450 enzymes required for biosynthesis of sterols in eukaryotes, the major targets for antifungal agents and prospective targets for treatment of protozoan infections. Human CYP51 could be and, for a while, was considered as a potential target for cholesterol-lowering drugs (the role that is now played by statins, which are also in clinical trials for cancer) but revealed high intrinsic resistance to inhibition. While microbial CYP51 enzymes are often inhibited stoichiometrically and functionally irreversibly, no strong inhibitors have been identified for human CYP51. In this study, we used comparative structure/functional analysis of CYP51 orthologs from different biological kingdoms and employed site-directed mutagenesis to elucidate the molecular basis for the resistance of the human enzyme to inhibition and also designed, synthesized, and characterized new compounds. Two of them inhibit human CYP51 functionally irreversibly with their potency approaching the potencies of azole drugs currently used to inhibit microbial CYP51.

Successful Aspects of the Coadministration of Sterol 14α-Demethylase Inhibitor VFV and Benznidazole in Experimental Mouse Models of Chagas Disease Caused by the Drug-Resistant Strain of Trypanosoma cruzi.

Up to now, no vaccines are available for Chagas disease, and the current therapy is largely unsatisfactory. Novel imidazole-based scaffolds of protozoan sterol 14α-demethylase (CYP51) inhibitors have demonstrated potent antiparasitic activity with no acute toxicity. Presently our aim was to investigate the effectiveness of the experimental 14α-demethylase inhibitor VFV in the mouse models of Trypanosoma cruzi infection using a naturally drug-resistant Colombiana strain, under monotherapy and in association with the reference drug, benznidazole (Bz). The treatment with VFV resulted in complete parasitemia suppression and 100% animal survival when administered orally (given in 10% DMSO plus 5% Arabic gum) at 25 mg/kg (bid) for 60 days. However, as parasite relapse was found using VFV alone under this treatment scheme, the coadministration of VFV with Bz was assayed giving simultaneously (for 60 days, bid) by oral route, under two different drug vehicles (10% DMSO plus 5% Gum Arabic with or without 3% Tween 80). All tested mice groups resulted in >99.9% of parasitemia decrease and 100% animal survival. qPCR analysis performed on cyclophosphamide immunosuppressed mice revealed that, although presenting lack of cure, VFV given as monotherapy was 14-fold more active than Bz, and the coadministration of Bz plus VFV (given simultaneously, using 10% DMSO plus 5% Gum Arabic as vehicle) resulted in 106-fold lower blood parasitism as compared to the monotherapy of Bz. Another interesting finding was the parasitological cure in 70% of the animals treated with Bz and VFV when the coadministration was given using the VFV suspension in 10% DMSO + Arabic gum + Tween 80 (a formulation that we have found to provide a better pharmacokinetics), even after immunosuppression using cyclophosphamide cycles, supporting the promising aspect of the drug coadministration in improving the efficacy of therapeutic arsenal against T. cruzi.

Sterol 14α-Demethylase Structure-Based Optimization of Drug Candidates for Human Infections with the Protozoan Trypanosomatidae.

Sterol 14α-demethylases (CYP51) are cytochrome P450 enzymes essential for sterol biosynthesis in eukaryotes and therapeutic targets for antifungal azoles. Multiple attempts to repurpose antifungals for treatment of human infections with protozoa (Trypanosomatidae) have been undertaken, yet so far none of them have revealed sufficient efficacy. VNI and its derivative VFV are two potent experimental inhibitors of Trypanosomatidae CYP51, effective in vivo against Chagas disease, visceral leishmaniasis, and sleeping sickness and currently under consideration as antiprotozoal drug candidates. However, VNI is less potent against Leishmania and drug-resistant strains of Trypanosoma cruzi and VFV, while displaying a broader spectrum of antiprotozoal activity, and is metabolically less stable. In this work we have designed, synthesized, and characterized a set of close analogues and identified two new compounds (7 and 9) that exceed VNI/VFV in their spectra of antiprotozoal activity, microsomal stability, and pharmacokinetics (tissue distribution in particular) and, like VNI/VFV, reveal no acute toxicity.

Binding of a physiological substrate causes large-scale conformational reorganization in cytochrome P450 51.

Sterol 14α-demethylases (CYP51s) are phylogenetically the most conserved cytochromes P450, and their three-step reaction is crucial for biosynthesis of sterols and serves as a leading target for clinical and agricultural antifungal agents. The structures of several (bacterial, protozoan, fungal, and human) CYP51 orthologs, in both the ligand-free and inhibitor-bound forms, have been determined and have revealed striking similarity at the secondary and tertiary structural levels, despite having low sequence identity. Moreover, in contrast to many of the substrate-promiscuous, drug-metabolizing P450s, CYP51 structures do not display substantial rearrangements in their backbones upon binding of various inhibitory ligands, essentially representing a snapshot of the ligand-free sterol 14α-demethylase. Here, using the obtusifoliol-bound I105F variant of Trypanosoma cruzi CYP51, we report that formation of the catalytically competent complex with the physiological substrate triggers a large-scale conformational switch, dramatically reshaping the enzyme active site (3.5-6.0 Å movements in the FG arm, HI arm, and helix C) in the direction of catalysis. Notably, our X-ray structural analyses revealed that the substrate channel closes, the proton delivery route opens, and the topology and electrostatic potential of the proximal surface reorganize to favor interaction with the electron-donating flavoprotein partner, NADPH-cytochrome P450 reductase. Site-directed mutagenesis of the amino acid residues involved in these events revealed a key role of active-site salt bridges in contributing to the structural dynamics that accompanies CYP51 function. Comparative analysis of apo-CYP51 and its sterol-bound complex provided key conceptual insights into the molecular mechanisms of CYP51 catalysis, functional conservation, lineage-specific substrate complementarity, and druggability differences.

Sterol 14α-Demethylase Structure-Based Design of VNI (( R)- N-(1-(2,4-Dichlorophenyl)-2-(1 H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)) Derivatives To Target Fungal Infections: Synthesis, Biological Evaluation, and Crystallographic Analysis.

Because of the increase in the number of immunocompromised patients, the incidence of invasive fungal infections is growing, but the treatment efficiency remains unacceptably low. The most potent clinical systemic antifungals (azoles) are the derivatives of two scaffolds: ketoconazole and fluconazole. Being the safest antifungal drugs, they still have shortcomings, mainly because of pharmacokinetics and resistance. Here, we report the successful use of the target fungal enzyme, sterol 14α-demethylase (CYP51), for structure-based design of novel antifungal drug candidates by minor modifications of VNI [( R)- N-(1-(2,4-dichlorophenyl)-2-(1 H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)], an inhibitor of protozoan CYP51 that cures Chagas disease. The synthesis of fungi-oriented VNI derivatives, analysis of their potencies to inhibit CYP51s from two major fungal pathogens ( Aspergillus fumigatus and Candida albicans), microsomal stability, effects in fungal cells, and structural characterization of A. fumigatus CYP51 in complexes with the most potent compound are described, offering a new antifungal drug scaffold and outlining directions for its further optimization.

CYP51 as drug targets for fungi and protozoan parasites: past, present and future.

The efficiency of treatment of human infections with the unicellular eukaryotic pathogens such as fungi and protozoa remains deeply unsatisfactory. For example, the mortality rates from nosocomial fungemia in critically ill, immunosuppressed or post-cancer patients often exceed 50%. A set of six systemic clinical azoles [sterol 14α-demethylase (CYP51) inhibitors] represents the first-line antifungal treatment. All these drugs were discovered empirically, by monitoring their effects on fungal cell growth, though it had been proven that they kill fungal cells by blocking the biosynthesis of ergosterol in fungi at the stage of 14α-demethylation of the sterol nucleus. This review briefs the history of antifungal azoles, outlines the situation with the current clinical azole-based drugs, describes the attempts of their repurposing for treatment of human infections with the protozoan parasites that, similar to fungi, also produce endogenous sterols, and discusses the most recently acquired knowledge on the CYP51 structure/function and inhibition. It is our belief that this information should be helpful in shifting from the traditional phenotypic screening to the actual target-driven drug discovery paradigm, which will rationalize and substantially accelerate the development of new, more efficient and pathogen-oriented CYP51 inhibitors.

Antitrypanosomal and antileishmanial activity of prenyl-1,2,3-triazoles.

A series of prenyl 1,2,3-triazoles were prepared from isoprenyl azides and different alkynes. The dipolar cycloaddition reaction provided exclusively primary azide products as regioisomeric mixtures that were separated by column chromatography and fully characterized. Most of the compounds displayed antiparasitic activity against Trypanosoma cruzi and Leishmania donovani. The most active compounds were assayed as potential TcCYP51 inhibitors.

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana.

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme's active site, consistent with the fact that the resistant line continues to produce the enzyme's product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.