Elucidation of Antifungal Drug Action by High Resolution GC/MS

Importance of the Topic

Ergosterol is an essential component of fungal cell membranes and a prime target for antifungal drug development. Understanding how candidate compounds perturb sterol biosynthesis in model organisms like Saccharomyces cerevisiae is critical for elucidating mechanisms of action and for guiding the design of more effective therapeutics.

Objectives and Study Overview

The study aimed to combine a targeted analysis of known intermediates in the ergosterol pathway with an untargeted metabolic profiling approach, using high-resolution GC/Q-TOF technology, to:

• Validate the analytical workflow with established antifungal drugs (terbinafine and fluconazole).
• Identify enzymatic targets of novel compounds, including a new chemical entity (NCE) and natural product totarol.
• Employ MS/MS accurate mass data and structure‐correlation tools to confirm identities of unexpected sterol metabolites.

Methodology and Data Processing

Wild-type yeast cultures were exposed to drug concentrations inhibiting growth by ~10%. Lipids were extracted via a Folch protocol, derivatized (oximation and silylation), and analyzed in full-scan electron ionization mode on an Agilent 7890 GC coupled to a 7200 series quadrupole-time-of-flight MS. MS/MS product ion data were acquired for structure confirmation. Chromatographic deconvolution and compound detection were performed in MassHunter Unknowns Analysis. Putative identifications used NIST11 library matching and accurate mass filtering, while multivariate statistics in Mass Profiler Professional highlighted significant fold changes between treated and control samples. Quantitation of targeted sterols was carried out in MassHunter Quantitative software.

Used Instrumentation

• Agilent 7890 gas chromatograph with split/splitless inlet
• Agilent 7200 series GC/Q-TOF mass spectrometer
• Electron ionization source with full-scan MS and MS/MS acquisition
• Computational tools: MassHunter (Unknowns, Quantitative), Mass Profiler Professional, Molecular Structure Correlator, NIST11 spectral library

Primary Results and Discussion

Proof-of-concept experiments confirmed expected responses: terbinafine treatment led to squalene accumulation and depletion of downstream sterols, while fluconazole induced lanosterol build-up with reduced later pathway intermediates. Untargeted profiling in NCE‐treated yeast revealed a marked accumulation of 4,4-dimethyl-8,24-cholestadienol and downregulation of subsequent metabolites, implicating Erg25 as the specific enzymatic target. Similarly, totarol treatment resulted in increased levels of 4α-carboxy-4β-methyl-5α-cholesta-8,24-dienol, pointing to Erg26 inhibition. Structural assignments were supported by MS/MS accurate mass fragments and Molecular Structure Correlator scores.

Benefits and Practical Applications

Combining targeted sterol quantitation with untargeted, high-resolution MS profiling enables comprehensive mapping of pathway perturbations. This approach accelerates target identification for novel antifungal agents, aids in mechanism‐of‐action studies, and can be integrated into drug discovery pipelines for quality control and lead optimization.

Future Trends and Applications

• Integration of automated MS/MS fragmentation libraries and in silico prediction tools for rapid structural confirmation.
• Higher resolution and sensitivity mass spectrometers to detect trace sterol intermediates and novel metabolites.
• Application of similar workflows to other microbial models and alternative lipid pathways.
• Coupling metabolic profiling with genomics and transcriptomics to build multi-omic networks of drug response.

Conclusion

High-resolution GC/Q-TOF metabolic profiling, in tandem with genetic screening methods such as haploinsufficiency profiling, provides a powerful platform for elucidating the enzymatic targets of antifungal compounds. The combination of targeted quantitation, untargeted discovery, and accurate mass MS/MS confirmation yields detailed mechanistic insights that support the rational development of new therapeutics.

References

• Gushue J, Aronova S, Baumann S, Wu M, St. Onge B, Suresh S, Davis R, Peltz G. Elucidation of Antifungal Drug Action by High Resolution GC/MS. PITTCON 2012.