A Method for Quantitatively Differentiating Crude Natural Extracts Using High-Performance Liquid Chromatography−Electrospray Mass Spectrometry

Julian, Randall K.; Higgs, Richard E.; Gygi, Jeffrey D.; Hilton, Matthew D.

doi:10.1021/ac971055v

Cited by 39 publications

(27 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fermentation was carried out as described previously (22). The combined fermentation broth (26 liters) was centrifuged, and the cell mass was extracted twice with 9 VOL.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Cercosporamide, a Known Antifungal Natural Product, as a Selective Pkc1 Kinase Inhibitor through High-Throughput Screening

et al. 2004

View full text Add to dashboard Cite

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broadspectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a ␤-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and ␤-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

show abstract

“…The fermentation was carried out as described previously (22). The combined fermentation broth (26 liters) was centrifuged, and the cell mass was extracted twice with 9 VOL.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Cercosporamide, a Known Antifungal Natural Product, as a Selective Pkc1 Kinase Inhibitor through High-Throughput Screening

et al. 2004

View full text Add to dashboard Cite

show abstract

“…HPLC ES-MS. Chemical analysis of solid-phase extraction (SPE) eluates was performed by high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC ES-MS) as described by Julian et al (23). Methanolsolubilized analytes (50 l) were separated on a Novapak C 18 analytical column (3.9 by 100 mm) over a 30-min linear gradient from 2% methanol plus 6.5 mM ammonium acetate (pH 5.5) to 95% methanol plus 6.5 mM ammonium acetate (pH 5.5) at a flow rate of 1 ml min Ϫ1 .…”

Section: Methodsmentioning

confidence: 99%

Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from Methylococcus capsulatus Bath

et al. 2001

View full text Add to dashboard Cite

“…Chemical analyses of filtered ethanol extracts or of SPE eluates were performed by HPLC-ES-MS as described by Julian et al (23) or by ES-MS as described by Higgs et al (20). A standard mixture consisting of caffeine, m-cresol purple, Spinosad (a mixture of isomeric spinosyn factors A and D; Dow Agrosciences, Indianapolis, Ind.…”

Section: Methodsmentioning

confidence: 99%

“…Because of the many vagaries of biological assays (e.g., nonlinear responses, chemical interference, lack of correlation between bioassay data, and domination of activity outcomes by a few common metabolites), we favor more general chemical analyses to provide data to guide improved expression of secondary metabolites. At present, the best chemical analyses depend on initial separation of natural product extracts (by thin-layer chromatography or high-performance liquid chromatography [HPLC]) followed by detection of the resolved secondary metabolites by suitable detectors (15,23). However, the benefits of superior resolution resulting from chromatographic separation of natural product extracts are often offset by lower throughput because of the greater time, reagents, and labor necessary for chromatography.…”

mentioning

confidence: 99%

Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

Zahn

Higgs

Hilton

2001

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography-ES-MS-evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.Many traits of microbes, both positive and negative, are attributed to expression of biologically active secondary metabolites. These metabolites are typically produced only under specific physiological conditions, and many are not essential for growth (6,33). Although the physiological role of most secondary metabolites remains an area of speculation (25), these compounds have been ascribed apparent roles, including cell differentiation, cell signaling or communication, nutrient sequestering, and defense (6,7,12,21,26,33).The foremost value of secondary metabolites to humanity has been in providing the basis for new commercial drugs, both directly (e.g., penicillin) and indirectly (e.g., synthetic or semisynthetic compounds derived from secondary metabolites [8]). In recent years, several factors have increased the difficulty of discovering secondary metabolites with the potential to be drug leads. These factors include the difficulty of managing the compatibility of complex natural extracts with high-throughput and ultra-high-throughput screening methods, the reality that most discoveries are rediscoveries of known compounds, and competition from laboratory-synthesized chemical diversity, the supply of which has been dramatically increased by combinatorial methods (4,14). However, human awareness of microbial biodiversity has expanded tremendously in the past few years, so we now recognize that less than a few percent...

show abstract

A Method for Quantitatively Differentiating Crude Natural Extracts Using High-Performance Liquid Chromatography−Electrospray Mass Spectrometry

Cited by 39 publications

References 17 publications

Discovery of Cercosporamide, a Known Antifungal Natural Product, as a Selective Pkc1 Kinase Inhibitor through High-Throughput Screening

Discovery of Cercosporamide, a Known Antifungal Natural Product, as a Selective Pkc1 Kinase Inhibitor through High-Throughput Screening

Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from Methylococcus capsulatus Bath

Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

Contact Info

Product

Resources

About