title: Development of cellular metabolite-based MALDI mass spectrometry assays for drug discovery
creator: Weigt, David
description: Due to their speed and lack of photonic interferences MALDI MS assays have a great potential to support drug discovery. Previous work demonstrated the feasibility of whole cell-based MALDI MS assays for abundant pharmacodynamics protein markers. Analogous assays for metabolites, which are of interest for phenotypic and mechanistic drug profiling studies due to their potentially high dynamic range, have been missing. In this thesis a score-based method development approach was implemented to develop a metabolite-based whole cell MALDI MS fingerprinting workflow. The final workflow enabled optimized discrimination of three cancer cell lines by principal component analysis. This MS fingerprinting workflow was used for development of the first metabolite-based phenotypic MALDI MS assay, in this case to monitor BCR-Abl tyrosine kinase inhibition in K562 leukemia cells. To add to the strength of this workflow, a data processing pipeline was developed in R programming language for the automated extraction of drug-sensitive m/z features. This data processing pipeline enabled the extraction of metabolite concentration-response markers that were structurally characterized by ultra-high resolution MALDI-FTICR MS/MS. To this end, heme B, a known marker for erythropoiesis in K562 cells, was identified as a suitable phenotypic marker of redifferentiation of these leukemia cells. The potassium adduct of the membrane lipid phosphatidylcholine (36:1) enabled to compare potencies of different tyrosine kinase inhibitors. In the second part of this thesis, the first MALDI MS-based mechanistic cell assay was developed, using inhibitors of the fatty acid synthase (FASN) as an example. Here, the employment of a 13C-labelled internal isotope standard enabled standardized monitoring of cellular malonyl-CoA accumulation upon FASN inhibition. Automation enabled assays in 96-well format. The dynamic range of the signal readout and Z-factors suggested excellent assay performance. Exploratory, unbiased data analysis followed by MS/MS structural characterization revealed CDP-choline as a generalized marker for lipid pathway inhibition, which not only responds to confirmed FASN inhibitors but also to the acetyl-CoA carboxylase inhibitor TOFA. The assay confirmed publications highlighting BI 99179 and GSK2194069 as potent FASN inhibitors but also gave evidence that a third inhibitor, triclosan, which also was published as a FASN inhibitor, indeed affects the lipid synthesis pathway at one synthetic step, but most likely not FASN activity. In conclusion, this thesis demonstrated that metabolite-based MALDI MS cell assays – both phenotypic and mechanistic - are feasible, standardizable, and automatable, and that they generate robust data useful for chemical biology work or compound profiling in drug discovery. Future work is likely going to focus on scaling up the throughput of MALDI MS cell assays by miniaturization and automation as well as expanding the repertoire of metabolic enzymes targetable by MALDI MS cell assays.
date: 2019
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/26246/1/Doktorarbeit.pdf
identifier: DOI:10.11588/heidok.00026246
identifier: urn:nbn:de:bsz:16-heidok-262465
identifier:   Weigt, David  (2019) Development of cellular metabolite-based MALDI mass spectrometry assays for drug discovery.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/26246/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng