Toward Global Metabolomics Analysis with Hydrophilic Interaction Liquid Chromatography–Mass Spectrometry: Improved Metabolite Identification by Retention Time Prediction

Creek, Darren J.; Jankevics, Andris; Breitling, Rainer; Watson, David; Barrett, Michael P.; Burgess, Karl

doi:10.1021/ac2021823

Cited by 317 publications

(304 citation statements)

References 44 publications

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“…LC-MS analysis utilized hydrophilic interaction chromatography with a ZIC-HILIC column (Sequant) and a formic acid mobile phase and was coupled to high-resolution MS with the Exactive Orbitrap (Thermo) operating in both positive and negative modes with all parameters as previously described (23).…”

Section: Methodsmentioning

confidence: 99%

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Creek

Nijagal

Kim

et al. 2013

Antimicrob Agents Chemother

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134

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dIn vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra-and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; ␣ ‫؍‬ 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.T rypanosoma brucei is the kinetoplastid parasite responsible for human African trypanosomiasis (HAT), a potentially fatal infection of sub-Saharan Africa. Current treatments for HAT are inadequate because of high toxicity and complex administration regimens, and new drugs are urgently required to ensure ongoing control of the disease in the face of emerging reports of drug resistance (1). Newly invigorated efforts to bring novel compounds forward as drugs have led to several promising candidates emerging through screening against parasites in culture (2-5). In vitro continuous culture methods enable the routine screening of compounds for trypanocidal activity against bloodstream form trypanosomes, for example, with resazurin fluorescence-based assays (5-7).Culture methods for bloodstream form T. brucei were first developed in the 1970s and required feeder layers to support continuous growth (8). This requirement could be overcome by regular supplementation with cysteine (9) and supplementation with mammalian serum (10). Continuous culture was successfully maintained by the addition of reducing and chelating agents (2-mercaptoethanol and bathocuproine sulfonate) to modified Iscove medium with 10% fetal bovine serum (HMI11) (11). HMI11 is now widely used for routine cell culture of laboratory strains of T. brucei and underpins many of the approaches employed in the search for new trypanocidal compounds. Nevertheless, HMI11 is a very rich medium compared to the natural in vivo environment of bloodstream form T. brucei, which may give...

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Section: Methodsmentioning

confidence: 99%

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Creek

Nijagal

Kim

et al. 2013

Antimicrob Agents Chemother

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134

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“…Liquid chromatography separation used a zwitterionic ZIC-hydrophilic interaction liquid chromatography column (Merck Sequant, Watford, UK) with a formic acid gradient, as previously described (Zhang et al, 2012). The method was performed on a Dionex U3000 RSLC (Thermo Fisher, Loughborough, UK) liquid chromatography system coupled to an Exactive Orbitrap (Thermo Fisher) operating at 50,000 resolution in positive and negative mode electrospray ionization (rapid switching), with MS parameters as described (Creek et al, 2011). Metabolomics data were analyzed using the IDEOM application (http:// mzmatch.sourceforge.net/ideom.php) with default parameters .…”

Section: Methodsmentioning

confidence: 99%

“…Metabolomics data were analyzed using the IDEOM application (http:// mzmatch.sourceforge.net/ideom.php) with default parameters . Metabolites were identified by accurate mass and retention time (or predicted retention time where standards were unavailable) according to the IDEOM metabolite database (Creek et al, 2011), with the addition of possible mBBr adducts for common cellular thiols. Molecular formulae for unidentified metabolites were determined based on accurate mass using the IDEOM application.…”

Section: Methodsmentioning

confidence: 99%

Potent Trypanocidal Curcumin Analogs Bearing a Monoenone Linker Motif Act on Trypanosoma brucei by Forming an Adduct with Trypanothione

et al. 2014

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We have previously reported that curcumin analogs with a C 7 linker bearing a C 4 -C 5 olefinic linker with a single keto group at C3 (enone linker) display midnanomolar activity against the bloodstream form of Trypanosoma brucei. However, no clear indication of their mechanism of action or superior antiparasitic activity relative to analogs with the original di-ketone curcumin linker was apparent. To further investigate their utility as antiparasitic agents, we compare the cellular effects of curcumin and the enone linker lead compound 1,7-bis(4-hydroxy-3-methoxyphenyl)hept-4-en-3-one (AS-HK014) here. An AS-HK014-resitant line, trypanosomes adapted to AS-HK014 (TA014), was developed by in vitro exposure to the drug. Metabolomic analysis revealed that exposure to AS-HK014, but not curcumin, rapidly depleted glutathione and trypanothione in the wild-type line, although almost all other metabolites were unchanged relative to control. In TA014 cells, thiol levels were similar to untreated wild-type cells and not significantly depleted by AS-HK014. Adducts of AS-HK014 with both glutathione and trypanothione were identified in AS-HK014-exposed wild-type cells and reproduced by chemical reaction. However, adduct accumulation in sensitive cells was much lower than in resistant cells. TA014 cells did not exhibit any changes in sequence or protein levels of glutathione synthetase and g-glutamylcysteine synthetase relative to wild-type cells. We conclude that monoenone curcuminoids have a different mode of action than curcumin, rapidly and specifically depleting thiol levels in trypanosomes by forming an adduct. This adduct may ultimately be responsible for the highly potent trypanocidal and antiparasitic activity of the monoenone curcuminoids.

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“…Other features were putatively annotated (MSI level 2) based on accurate mass and predicted retention time using the IDEOM database (35). The final data set consisted of 460 putatively identified metabolites, from a total of ϳ1,500 unique mass features.…”

Section: Cell Culture and Drug Incubations For Lc-ms Metabolomics Anamentioning

confidence: 99%

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action

Creek

Chua

Cobbold

et al. 2016

Antimicrob Agents Chemother

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130

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e High-throughput phenotypic screening of chemical libraries has resulted in the identification of thousands of compounds with potent antimalarial activity, although in most cases, the mechanism(s) of action of these compounds remains unknown. Here we have investigated the mode of action of 90 antimalarial compounds derived from the Malaria Box collection using high-coverage, untargeted metabolomics analysis. Approximately half of the tested compounds induced significant metabolic perturbations in in vitro cultures of Plasmodium falciparum. In most cases, the metabolic profiles were highly correlated with known antimalarials, in particular artemisinin, the 4-aminoquinolines, or atovaquone. Select Malaria Box compounds also induced changes in intermediates in essential metabolic pathways, such as isoprenoid biosynthesis (i.e., 2-C-methyl-D-erythritol 2,4-cyclodiphosphate) and linolenic acid metabolism (i.e., traumatic acid). This study provides a comprehensive database of the metabolic perturbations induced by chemically diverse inhibitors and highlights the utility of metabolomics for triaging new lead compounds and defining specific modes of action, which will assist with the development and optimization of new antimalarial drugs. Malaria remains a major global health problem, and there is a pressing need to discover and develop new antimalarials. Traditional antimalarial drugs have been severely undermined by the emergence of drug-resistant strains and current treatments rely heavily on artemisinin-based combination therapies. Alarmingly, artemisinin resistance has arisen in Southeast Asia during the last decade, highlighting the urgent need for new antimalarials (1). Extensive efforts to produce a malaria vaccine have met limited success and a pipeline of new antimalarial drugs will be required to mitigate extensive morbidity and mortality from malaria for the foreseeable future (2).High-throughput phenotypic screening of chemical libraries against the malaria parasite, Plasmodium falciparum, has provided a major step forward in the discovery of novel antimalarial compounds. Almost 30,000 compounds that selectively inhibit growth of cultured P. falciparum asexual red blood cell (RBC) stages have been identified in these screens, providing excellent starting points for the discovery of new antimalarial drugs (3-5). A prioritized collection of these "hit" compounds, known as the Malaria Box, has been assembled by the Medicines for Malaria Venture (MMV) and provided free to the research community to facilitate this drug development pipeline (6).A key limitation to the further optimization of many of these compounds is the lack of information on their mode of action. While not essential for registration, information on the mode of action of inhibitors discovered in phenotypic screens will significantly accelerate drug development by allowing structure-based drug design, monitoring of activity, toxicity and resistance, and facilitation of rational clinical usage in combination with other medicines. Furthermore, in...

show abstract

Toward Global Metabolomics Analysis with Hydrophilic Interaction Liquid Chromatography–Mass Spectrometry: Improved Metabolite Identification by Retention Time Prediction

Cited by 317 publications

References 44 publications

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Potent Trypanocidal Curcumin Analogs Bearing a Monoenone Linker Motif Act on Trypanosoma brucei by Forming an Adduct with Trypanothione

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action

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