Applications of LC-MS based metabolomics in pharmaceutical science
Creators: name: Mary Peter, Rebecca
Publisher: name: No Publisher Supplied
Year: 2025

description: This dissertation is to support the Degree of Doctor of Philosophy in Pharmaceutical Science at Rutgers, The State University of New Jersey. It documents my research work conducted under the guidance of Dr. Ah-Ng Kong in the Department of Pharmaceutics. The work presented here is original, except for where suitable references are made to previous work. This dissertation consists of five chapters and one appendix chapter. They are either published manuscripts or intended to be submitted for publication to journals indexed by PubMed Central. Chapter 1 provides the background for my research. It discusses the importance of metabolomic studies in biomedical research. Metabolomics describes the end products of biochemical processes which are greatly influenced by genetic and environmental factors. Metabolic alterations can be linked to potential biochemical reactions/enzymes and their corresponding genes. Thus, these results can be further validated via a multi-omics approach including genomics, transcriptomics, and proteomics. Metabolomic analysis of biological samples exhibits the possibility to determine the mechanism of action of anti-cancer agents, biomarker discovery, and impact of genetic alterations. Specific emphasis is on evolution of metabolomics in cancer research, metabolomics workflow, general understanding of LC-MS based platform for quantitation of metabolites and their biological interpretation. Chapter 2 discusses the first application of metabolomics in pharmaceutical science and has been published. Herein, we investigated the effect of HDAC inhibitor (HDACi) belinostat at clinically relevant concentration on nuclear factor erythroid 2-related factor 2 (NRF2) and mitochondrial metabolism for the treatment of KRAS-mutant human lung cancer. LC-MS metabolomic study of belinostat on mitochondrial metabolism was performed in G12C KRAS-mutant H358 non-small cell lung cancer cells. Furthermore, L-methionine (methyl-13C) isotope tracer was used to explore the effect of the belinostat in one carbon metabolism. Bioinformatic analyses of metabolomic data were performed to identify the pattern of the significantly regulated metabolites. To study the effect of belinostat on redox signaling Are-NRF2 pathway, luciferase reporter activity assay was done in stably transfected HepG2-C8 cells (containing pARE-TI-luciferase construct), followed by qPCR analysis of NRF2 and its target gene in H358 and A549 cells. Metabolomic study reveals significantly altered metabolic levels related to redox homeostasis including tricarboxylic acid (TCA) cycle metabolites (citrate, aconitate, fumarate, malate and α-ketoglutarate); urea cycle metabolites (Arginine, ornithine, arginino-succinate, aspartate and fumarate); and glutathione metabolism pathway (GSH/GSSG and NAD/NADH ratio) after belinostat treatment. 13C stable isotope labeling data indicates potential role of belinostat in creatine biosynthesis via methylation of guanidinoacetate. Moreover, belinostat downregulated the expression of NRF2 and its target gene NAD(P)H:quinone oxidoreductase 1 (NQO1), indicating potential anticancer effect of belinostat is mediated, potentially via Nrf2-regulated glutathione pathway. Another HDACi, panobinostat showed potential anticancer effect in both H358 and A549 cells.Chapter 3 discusses the second application of metabolomics in pharmaceutical science. Herein, we utilized Pten knockout (KO)-induced prostate tumorigenesis mouse model to examine mechanism of action of phytochemical, indole-3-carbinol (I3C) via metabolic rewiring. I3C is a heterocyclic bioactive chemical found in cruciferous vegetables, possessing multiple biological activities such as antimicrobial, antioxidant, antiviral, anti-inflammatory, and anti-cancer activities. Untargeted metabolomics was performed using LC-MS based platform to investigate Pten-dependent and Pten-independent metabolic targets of I3C. The most impacted pathways by I3C included pyrimidine metabolism, arginine and proline metabolism, porphyrin metabolism, citrate cycle and lipoic acid metabolism. Chapter 4 discusses the third application of metabolomics in pharmaceutical science. Herein, we established and verified an Nrf2 KO transgenic mouse model and studied resultant metabolic reprogramming in the gut and vital organs of mouse to understand the significant role of Nrf2 at metabolic level. To validate the Nrf2 KO mouse model, gene expression of Nrf2 and its target genes including Nqo1, Ho1 and Gclm were examined and were found to be significantly downregulated compared to wildtype. Additionally, regulation of antioxidant defense mechanism in the gut based on glutathione metabolism in small &amp; large intestines of Nrf2 KO mice confirmed genetic knockout of Nrf2. Based on bioinformatic analysis of metabolomic information derived from small intestine, large intestine, liver, lung and brain of Nrf2 KO vs wild type mice, most regulated metabolites and metabolic pathways are identified and biologically interpreted. The most regulated metabolic pathways/metabolites in small intestine are citrate cycle and purine metabolism; in large intestine are pyrimidine metabolism; in liver are valine, leucine &amp; isoleucine biosynthesis/degradation, phenylalanine biosynthesis/metabolism, fructose &amp; mannose metabolism, taurine &amp; hypotaurine metabolism, arginine &amp; proline metabolism; in lung is lysine degradation; in brain are arginino succinate, cystine and xanthine. This has provided great starting point to further investigate correlation between metabolite concentrations and altered biological states mediated by Nrf2 in different mice tissues. Chapter 5 provides conclusion for my research and discusses future perspectives including improved experimental design, bioinformatic and modelling approaches to further advance our understanding of metabolomic studies in biomedical research. Appendix chapter 1 is a published review article that provides comprehensive review that identifies molecular biomarkers of pre-malignant and cancerous lung cells providing a strong basis to develop early cancer detection and interception methods. We discussed molecular changes, including genetic alterations, inflammation, signal transduction pathways, redox imbalance, epigenetic and proteomic signatures associated with initiation and progression of lung carcinoma. We also highlighted molecular targets of phytochemicals during lung cancer development. These targets mainly consist of cellular signaling pathways, epigenetic regulators and metabolic reprogramming. With growing interest in natural products research, translation of these compounds into new treatments or approaches to medical care has gained high importance. In this context, we discussed their overall disposition with pharmacokinetics. Lastly, we discussed clinical trials of phytochemicals in lung cancer patients.