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Dissertation Submitted to the Combined Dissertation submitted to the Combined Faculty of Natural Sciences and Mathematics of the Ruperto Carola University Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Luuk Nico van Oosten, M. Sc. born in: Woerden, The Netherlands Oral examination: June 3rd 2020 A novel high-throughput and label-free phenotypic drug screening approach: MALDI-TOF mass spectrometry combined with machine learning strategies Referees: Prof. Dr. Christian D. Klein Prof. Dr. Jeroen Krijgsveld Notice Parts of the work described in this thesis have been published as follows: 1) An international PCT patent application by Heidelberg University, filed under reference number PCT/EP2018/079221, with the author and first referee of this thesis listed as inventors. This patent application is under review at the time of publishing this thesis (April 2020). 2) A submission to the bioRxive preprint server, which can be cited as follows: Luuk N. van Oosten and Christian D. Klein, 2019, PhenoMS-ML: Phenotypic Screening by Mass Spectrometry and Machine Learning, DOI: https://doi.org/10.1101/593244 3) A conference poster publication, which can be cited as follows: Luuk N. van Oosten and Christian D. Klein, 2019, MALDI-TOF Mass Spectrometry Profiling of Bacteria Coupled with Machine Learning Techniques Identifies Mode of Action of Antibacterial Drugs, Poster Presentation, Annual Meeting of the German Pharmaceutical Society, September 1-4, 2019 4) A peer-reviewed publication in the Journal of Medicinal Chemistry, which can be cited as follows: Luuk N. van Oosten and Christian D. Klein, 2020, Machine learning in mass spectrometry: A MALDI-TOF MS approach to phenotypic antibacterial screening, Journal of Medicinal Chemistry, DOI: https://doi.org/10.1021/acs.jmedchem.0c00040 Abstract A renewed and growing interest in phenotypic drug screening approaches in the field of drug discovery is observed, as it has become apparent that target-oriented drug discovery assays have inherent limitations and cannot fulfil the urgent unmet medical need for novel drugs. The shortcomings of target-oriented drug screening assays are especially apparent in the field of antibiotic drug discovery, where target-based approaches largely failed to translate screening hits to clinically relevant drugs. In this thesis, a proteomics-based phenotypic drug screening approach using MALDI-TOF mass spectrometry was developed, which is able to detect sub-lethal stress in bacterial cells provoked by antibiotics. To achieve this, mass spectra of whole-cells exposed to known antibiotics at concentrations below the minimal inhibitory concentration (MIC) were used to extract relevant mass spectral peaks with a data-dependent and automated computational pipeline created in the MATLAB environment. Using the selected subset of mass spectral peaks, classification models were trained to recognize general mass spectral responses provoked by unknown drugs in the cellular proteome. Additionally, the classification models proved capable of identifying the mechanisms of action of unknown drugs. To establish and validate the best performing classification modeling procedure, four different feature selection algorithms and nine classification models were analyzed in detail using an Escherichia coli data set composed of over 900 spectra, involving 17 antibiotics with 1 four different mechanisms of action, at concentrations ranging 1×MIC down to /32×MIC in a two-fold dilution series. Four different feature selection approaches were investigated to ensure the extraction of relevant mass spectral data in response to the different antibiotics for classification modeling. The selection approaches included (1) a random forest of decision trees, (2) sequential forward feature selection, and (3) sequential backward feature selection. Mass spectral peaks selected by two or all three of these feature selection approaches were combined into (4) an aggregated feature set. Classification models were trained for all combinations of nine model types and the four feature sets. In this thesis two classification problems were investigated. First, a binary classification problem, to differentiate between affected cells, and non-affected cells based on selected mass spectral peaks. Second, a multi-class model was trained to detect and distinguish between the different antibiotic mechanisms of action, a highly desired drug screening assay characteristic. The combination of these elements yielded 72 models, which were evaluated based on their overall classification accuracy. The overall classification accuracy was determined using internal 10-fold cross-validation and external validation, which was performed with a blind set of 20 drugs. The internal and external validation studies showed that the aggregated feature set in combination with a quadratic support vector machine- based model (Q-SVM) resulted in the best classification performance. For the E. coli data set, this was represented by an overall accuracy of 0.92 for internal validation and an i accuracy of 0.95 for the external validation of the Q-SVM model. Classifying based on the mechanism of action of the antibiotics resulted in a classification accuracy of 0.67 for internal validation and 0.80 for external validation. Furthermore, it was shown that the peak selection method was able to identify relevant, known stress associated proteins within the aggregated feature sets of both the binary and the mechanism of action model. After the experimental workflow and the computational pipeline were established based on E. coli data, the method was applied to four different organisms (the Gram-positive bacterium Staphylococcus aureus, the fungi Saccharomyces cerevisiae and Candida albicans, and human HeLa cancer cell line) and different proteomic responses, to explore the versatility and transferability of the developed screening assay. The applicability of the method was demonstrated by the consistent performance of the classification models generated with the experimental and computational pipeline. This resulted in binary model accuracies between 0.92 and 0.97 for internal and 0.77 and 0.95 for external validation, depending on the assayed organism and data set complexity. For mechanism of action models, model accuracies ranged between 0.73 and 0.96 for internal and 0.66 and 0.93 for external validation. The application of the developed assay on different organisms with different drug stressors highlighted several advantageous characteristics of the developed MALDI-TOF MS screening approach. Both the binary and mechanism of action classification models of S. aureus correctly identified an antibiotic drug (fusidic acid) in the blind test set, which had a target binding activity that was not present in the training data set. This implicates the ability of the method to detect novel drugs within known global mechanism of action for which the model was trained. Moreover, external validation of S. cerevisiae showed that the binary classification model is able to detect antifungal drugs (tavaborole, an antifungal protein synthesis inhibitor) with a mechanism of action which was not present in the training data set. This is a highly desirable property of any phenotypic screening assay, as it shows that the assay allows for the identification of drugs with novel mechanisms of action. Lastly, the proteomic effect of different types of drugs on mammalian cells was explored by using the HeLa cancer cell line. It was shown that the presented proteomic profiling approach can easily detect several types of drug-induced stresses in HeLa cells, in particular corticosteroids and tubulin (de)polymerization inhibitors, but is less suitable for distinguishing other types of drug classes (neurotransmitter antagonists, statins, opioids). Additionally, the application of the assay on HeLa cells demonstrated the ability to detect different types of stresses, such as the cells’ proteomic response to UV exposure or heat shocks. These results pave the way for possible distinction between apoptosis and necrosis pathways in HeLa cells using the presented MALDI-TOF MS based method. To conclude, a high-throughput compatible, label free, MALDI-TOF mass spectrometry- based screening assay is described in this thesis, which measures sub-lethal drug effects on ii the cellular proteome in a phenotypic and pharmacological relevant setting. The method was found suitable for whole-cell screening of small libraries of drugs, and showed the ability to distinguish different types of stresses elicited on multiple types of cell cultures. The potential to find new, weakly active drugs within a known mechanism of action, as well as the ability to detect sub-lethal drug responses with new mechanisms of action for which the model was not trained, was demonstrated. The characteristic to identify novel mechanisms of action in a cell-based screen can be exploited to solve the most pressing issues in drug discovery today. In addition, mechanistic information of the drugs activity can be used as a starting point for further target elucidation or to prioritize drug screening hits. The studies performed in this thesis have resulted in a solid foundation for further research that expand the capabilities of the MALDI-TOF MS-based assay in a broad range of phenotypic profiling applications in the drug discovery field. iii iv Zusammenfassung Aktuell ist ein erneutes und wachsendes Interesse an phänotypischen Screening-Ansätzen in der Medikamenten-Entdeckung zu beobachten, da deutlich wurde, dass Target-orientierte
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