Structural elucidation of novel biomarkers of known metabolic disorders based on multistage fragmentation mass spectra.

Specific diagnostic markers are the key to effective diagnosis and treatment of inborn errors of metabolism (IEM). Untargeted metabolomics allows for the identification of potential novel diagnostic biomarkers. Current separation techniques coupled to high-resolution mass spectrometry provide a powerful tool for structural elucidation of unknown compounds in complex biological matrices. This is a proof-of-concept study testing this methodology to determine the molecular structure of as yet uncharacterized m/z signals that were significantly increased in plasma samples from patients with phenylketonuria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. A hybrid linear ion trap-orbitrap high resolution mass spectrometer, capable of multistage fragmentation, was used to acquire accurate masses and product ion spectra of the uncharacterized m/z signals. In order to determine the molecular structures, spectral databases were searched and fragmentation prediction software was used. This approach enabled structural elucidation of novel compounds potentially useful as biomarkers in diagnostics and follow-up of IEM patients. Two new conjugates, glutamyl-glutamyl-phenylalanine and phenylalanine-hexose, were identified in plasma of phenylketonuria patients. These novel markers showed high inter-patient variation and did not correlate to phenylalanine levels, illustrating their potential added value for follow-up. As novel biomarkers for 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, three positional isomers of 3-methylglutaconyl carnitine could be detected in patient plasma. Our results highlight the applicability of current accurate mass multistage fragmentation techniques for structural elucidation of unknown metabolites in human biofluids, offering an unprecedented opportunity to gain further biochemical insights in known inborn errors of metabolism by enabling high confidence identification of novel biomarkers.

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.

Novel sulphur-containing imatinib metabolites found by untargeted LC-HRMS analysis.

Untargeted metabolite profiling using high-resolution mass spectrometry coupled with liquid chromatography (LC-HRMS), followed by data analysis with the Compound Discoverer 2.0™ software, was used to study the metabolism of imatinib in humans with chronic myeloid leukemia. Plasma samples from control (drug-free) and patient (treated with imatinib) groups were analyzed in full-scan mode and the unknown ions occurring only in the patient group were then, as potential imatinib metabolites, subjected to multi-stage fragmentation in order to elucidate their structure. The application of an untargeted approach, as described in this study, enabled the detection of 24 novel structurally unexpected metabolites. Several sulphur-containing compounds, probably originating after the reaction of reactive intermediates of imatinib with endogenous glutathione, were found and annotated as cysteine and cystine adducts. In the proposed mechanism, the cysteine adducts were formed after the rearrangement of piperazine moiety to imidazoline. On the contrary, in vivo S-N exchange occurred in the case of the cystine adducts. In addition, N-O exchange was observed in the collision cell in the course of the fragmentation of the cystine adducts. The presence of sulphur in the cysteine and cystine conjugates was proved by means of ultra-high resolution measurements using Orbitrap Elite. The detection of metabolites derived from glutathione might improve knowledge about the disposition of imatinib towards bioactivation and help to improve understanding of the mechanism of its hepatotoxicity or nephrotoxicity in humans.

Ultrafast Online SPE-MS/MS Method for Quantification of 3 Tyrosine Kinase Inhibitors in Human Plasma.

BACKGROUND: With an increasing number of cancer patients receiving tyrosine kinase inhibitors (TKIs), therapeutic drug monitoring of these molecules is becoming more widespread today. It is mainly based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) methods with typical run times of several minutes. In an online solid phase extraction-MS/MS (SPE-MS/MS) system, the chromatography column is replaced with a reusable solid phase extraction (SPE) cartridge and the analysis time is shortened to less than half a minute. The aim of this study was to develop such a method and test the performance of this high-throughput system in the analysis of imatinib (IMA), nilotinib (NIL), and lapatinib (LAP) in human plasma. METHODS: Samples were prepared by simple protein precipitation with methanol containing deuterated internal standards. After centrifugation, the supernatant was diluted 10 fold with a mixture of methanol and water (1:1). A C4 cartridge was used for SPE and the analytes were eluted by acetonitrile. All the analytes were measured within a wide calibration range (50-5000 ng/mL for nilotinib and imatinib, 100-10,000 ng/mL for lapatinib). The method was compared with the LC-MS/MS method by the analysis of 176 clinical samples. RESULTS: Intraday and interday inaccuracies within 15% and a coefficient of variation less than 15% were achieved for all the TKIs that were measured. Even though the matrix effects were higher in comparison with LC-MS/MS methods, their effect on the performance of the method was eliminated by the usage of deuterated internal standards. The total run time of the new method was 29 seconds for one analysis and the results were fully comparable with LC-MS/MS. CONCLUSIONS: Routine clinical practice requiring high-throughput methods for therapeutic drug monitoring of TKIs may benefit from the online SPE-MS/MS method that provides fast, low-cost analysis, and results that are comparable with conventional methods.

Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients.

The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liquid chromatography mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metabolism. We observed differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an additional tool for the assessment of response/resistance to imatinib.