The role of pharmacometabonomics in predicting drug pharmacokinetics

Individual variability in drug response is a key challenge in current clinical practice and in drug discovery and development. Pharmacological response is closely associated with drug concentration at the site of action and therefore knowledge of drug pharmacokinetics is vital to delivering effective therapy. In addition to genetic polymorphisms, environmental factors also play an important role in determining drug efficacy, safety, metabolism and pharmacokinetics. The newly emerging field of pharmacometabonomics uses information from pre-dose metabolite profiles to predict individual drug responses, can be sensitive to both genetic and environmental factors and thus has great promise to help the future delivery of personalized medicine. This article introduces pharmacometabonomics and covers its application to the prediction of pharmacokinetics

Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines

Introduction: KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) mutations occur in approximately one-third of colorectal (CRC) tumours and have been associated with poor prognosis and resistance to some therapeutics. In addition to the well-documented pro-tumorigenic role of mutant Ras alleles, there is some evidence suggesting that not all KRAS mutations are equal and the position and type of amino acid substitutions regulate biochemical activity and transforming capacity of KRAS mutations. Objectives: To investigate the metabolic signatures associated with different KRAS mutations in codons 12, 13, 61 and 146 and to determine what metabolic pathways are affected by different KRAS mutations. Methods: We applied an NMR-based metabonomics approach to compare the metabolic profiles of the intracellular extracts and the extracellular media from isogenic human SW48 CRC cell lines with different KRAS mutations in codons 12 (G12D, G12A, G12C, G12S, G12R, G12V), 13 (G13D), 61 (Q61H) and 146 (A146T) with their wild-type counterpart. We used false discovery rate (FDR)-corrected analysis of variance (ANOVA) to determine metabolites that were statistically significantly different in concentration between the different mutants. Results: CRC cells carrying distinct KRAS mutations exhibited differential metabolic remodelling, including differences in glycolysis, glutamine utilization and in amino acid, nucleotide and hexosamine metabolism. Conclusions: Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS

Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines.

INTRODUCTION: KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. OBJECTIVES: To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. METHODS: Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRAS G13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS +/- and KRAS G13D/-. RESULTS: Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/- cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. CONCLUSIONS: Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS

Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of fmo5-/- mice, decreases plasma cholesterol and epididymal fat deposition

We previously showed that Fmo5−/− mice exhibit a lean phenotype and slower metabolic ageing. Their characteristics include lower plasma glucose and cholesterol, greater glucose tolerance and insulin sensitivity, and a reduction in age-related weight gain and whole-body fat deposition. In this paper, nuclear magnetic resonance (NMR) spectroscopy-based metabolite analyses of the urine of Fmo5−/− and wild-type mice identified two isomers of 2,3-butanediol as discriminating urinary biomarkers of Fmo5−/− mice. Antibiotic-treatment of Fmo5−/− mice increased plasma cholesterol concentration and substantially reduced urinary excretion of 2,3-butanediol isomers, indicating that the gut microbiome contributed to the lower plasma cholesterol of Fmo5−/− mice, and that 2,3-butanediol is microbially derived. Short- and long-term treatment of wild-type mice with a 2,3-butanediol isomer mix decreased plasma cholesterol and epididymal fat deposition but had no effect on plasma concentrations of glucose or insulin, or on body weight. In the case of long-term treatment, the effects were maintained after withdrawal of 2,3-butanediol. Short-, but not long-term treatment, also decreased plasma concentrations of triglycerides and non-esterified fatty acids. Fecal transplant from Fmo5−/− to wild-type mice had no effect on plasma cholesterol, and 2,3-butanediol was not detected in the urine of recipient mice, suggesting that the microbiota of the large intestine was not the source of 2,3-butanediol. However, 2,3-butanediol was detected in the stomach of Fmo5−/− mice, which was enriched for Lactobacillus genera, known to produce 2,3-butanediol. Our results indicate a microbial contribution to the phenotypic characteristic of Fmo5−/− mice of decreased plasma cholesterol and identify 2,3-butanediol as a potential agent for lowering plasma cholesterol

Flavin-Containing Monooxygenase 1 Catalyzes the Production of Taurine from Hypotaurine

Taurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and by de novo synthesis, from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part this is due to the incorrect assignment, in 1962, of the enzyme as an NAD-dependent hypotaurine dehydrogenase. For more than 55 years the literature has continued to refer to this enzyme as such. Here we show, both in vivo and in vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase 1 (FMO1). Metabolite analysis of the urine of Fmo1-null mice by 1H NMR spectroscopy revealed a build-up of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice. In vitro assays confirmed that human FMO1 catalyzes the conversion of hypotaurine to taurine utilizing either NADPH or NADH as co-factor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biological processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, anti-oxidant and anti-inflammatory functions, and the pathogenesis of obesity and skeletal muscle disorders

Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of Fmo5-/- mice, decreases plasma cholesterol and epididymal fat deposition

We previously showed that Fmo5-/- mice exhibit a lean phenotype and slower metabolic ageing. Their characteristics include lower plasma glucose and cholesterol, greater glucose tolerance and insulin sensitivity, and a reduction in age-related weight gain and whole-body fat deposition. In this paper, nuclear magnetic resonance (NMR) spectroscopy-based metabolite analyses of the urine of Fmo5-/- and wild-type mice identified two isomers of 2,3-butanediol as discriminating urinary biomarkers of Fmo5-/- mice. Antibiotic-treatment of Fmo5-/- mice increased plasma cholesterol concentration and substantially reduced urinary excretion of 2,3-butanediol isomers, indicating that the gut microbiome contributed to the lower plasma cholesterol of Fmo5-/- mice, and that 2,3-butanediol is microbially derived. Short- and long-term treatment of wild-type mice with a 2,3-butanediol isomer mix decreased plasma cholesterol and epididymal fat deposition but had no effect on plasma concentrations of glucose or insulin, or on body weight. In the case of long-term treatment, the effects were maintained after withdrawal of 2,3-butanediol. Short-, but not long-term treatment, also decreased plasma concentrations of triglycerides and non-esterified fatty acids. Fecal transplant from Fmo5-/- to wild-type mice had no effect on plasma cholesterol, and 2,3-butanediol was not detected in the urine of recipient mice, suggesting that the microbiota of the large intestine was not the source of 2,3-butanediol. However, 2,3-butanediol was detected in the stomach of Fmo5-/- mice, which was enriched for Lactobacillus genera, known to produce 2,3-butanediol. Our results indicate a microbial contribution to the phenotypic characteristic of Fmo5-/- mice of decreased plasma cholesterol and identify 2,3-butanediol as a potential agent for lowering plasma cholesterol

NMR-based pharmacometabonomics: A new paradigm for personalised or precision medicine

Metabolic profiling by NMR spectroscopy or hyphenated mass spectrometry, known as metabonomics or metabolomics, is an important tool for systems-based approaches in biology and medicine. The experiments are typically done in a diagnostic fashion where changes in metabolite profiles are interpreted as a consequence of an intervention or event; be that a change in diet, the administration of a drug, physical exertion or the onset of a disease. By contrast, pharmacometabonomics takes a prognostic approach to metabolic profiling, in order to predict the effects of drug dosing before it occurs. Differences in pre-dose metabolite profiles between groups of subjects are used to predict post-dose differences in response to drug administration. Thus the paradigm is inverted and pharmacometabonomics is the metabolic equivalent of pharmacogenomics. Although the field is still in its infancy, it is expected that pharmacometabonomics, alongside pharmacogenomics, will assist with the delivery of personalised or precision medicine to patients, which is a critical goal of 21st century healthcare

Species-Specific Variations in the Metabolomic Profiles of Acropora hyacinthus and Acropora millepora Mask Acute Temperature Stress Effects in Adult Coral Colonies

Coral reefs are suffering unprecedented declines in health state on a global scale. Some have suggested that human assisted evolution or assisted gene flow may now be necessary to effectively restore reefs and pre-condition them for future climate change. An understanding of the key metabolic processes in corals, including under stressed conditions, would greatly facilitate the effective application of such interventions. To date, however, there has been little research on corals at this level, particularly regarding studies of the metabolome of Scleractinian corals. Here, the metabolomic profiles [measured using 1H nuclear magnetic resonance spectroscopy (1H NMR) and ultra-high-performance liquid chromatography-mass spectrometry (LC-MS)] of two dominant reef building corals, Acropora hyacinthus and A. millepora, from two distinct geographical locations (Australia and Singapore) were characterized. We assessed how an acute temperature stress (an increase of 3.25°C ± 0.28 from ambient control levels over 8 days), shifted the corals’ baseline metabolomic profiles. Regardless of the profiling method utilized, metabolomic signatures of coral colonies were significantly distinct between coral species, a result supporting previous work. However, this strong species-specific metabolomic signature appeared to mask any changes resulting from the acute heat stress. On closer examination, we were able to discriminate between control and temperature stressed groups using a partial least squares discriminant analysis classification model (PLSDA). However, in all cases “late” components needed to be selected (i.e., 7 and 8 instead of 1 and 2), suggesting any treatment effect was small, relative to other sources of variation. This highlights the importance of pre-characterizing the coral colony metabolomes, and of factoring that knowledge into any experimental design that seeks to understand the apparently subtle metabolic effects of acute heat stress on adult corals. Further research is therefore needed to decouple these apparent individual and species-level metabolomic responses to climate change in corals.NER

HMDB 5.0: the Human Metabolome Database for 2022

The Human Metabolome Database or HMDB (https://hmdb.ca) has been providing comprehensive reference information about human metabolites and their associated biological, physiological and chemical properties since 2007. Over the past 15 years, the HMDB has grown and evolved significantly to meet the needs of the metabolomics community and respond to continuing changes in internet and computing technology. This year's update, HMDB 5.0, brings a number of important improvements and upgrades to the database. These should make the HMDB more useful and more appealing to a larger cross-section of users. In particular, these improvements include: (i) a significant increase in the number of metabolite entries (from 114 100 to 217 920 compounds); (ii) enhancements to the quality and depth of metabolite descriptions; (iii) the addition of new structure, spectral and pathway visualization tools; (iv) the inclusion of many new and much more accurately predicted spectral data sets, including predicted NMR spectra, more accurately predicted MS spectra, predicted retention indices and predicted collision cross section data and (v) enhancements to the HMDB's search functions to facilitate better compound identification. Many other minor improvements and updates to the content, the interface, and general performance of the HMDB website have also been made. Overall, we believe these upgrades and updates should greatly enhance the HMDB's ease of use and its potential applications not only in human metabolomics but also in exposomics, lipidomics, nutritional science, biochemistry and clinical chemistry.Analytical BioScience

Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines

AbstractIntroductionKirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) mutations occur in approximately one-third of colorectal (CRC) tumours and have been associated with poor prognosis and resistance to some therapeutics. In addition to the well-documented pro-tumorigenic role of mutant Ras alleles, there is some evidence suggesting that not allKRASmutations are equal and the position and type of amino acid substitutions regulate biochemical activity and transforming capacity ofKRASmutations.ObjectivesTo investigate the metabolic signatures associated with differentKRASmutations in codons 12, 13, 61 and 146 and to determine what metabolic pathways are affected by differentKRASmutations.MethodsWe applied an NMR-based metabonomics approach to compare the metabolic profiles of the intracellular extracts and the extracellular media from isogenic human SW48 CRC cell lines with differentKRASmutations in codons 12 (G12D, G12A, G12C, G12S, G12R, G12V), 13 (G13D), 61 (Q61H) and 146 (A146T) with their wild-type counterpart. We used false discovery rate (FDR)-corrected analysis of variance (ANOVA) to determine metabolites that were statistically significantly different in concentration between the different mutants.ResultsCRC cells carrying distinctKRASmutations exhibited differential metabolic remodelling, including differences in glycolysis, glutamine utilization and in amino acid, nucleotide and hexosamine metabolism.ConclusionsMetabolic differences among differentKRASmutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenicKRAS.</jats:sec