Gregg Morin

High-throughput phenotype-based screening of large libraries of compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Through such a screen, the novel compound LCS3 was previously identified that selectively kills lung adenocarcinoma (LUAD) cells, but its mechanism of action remained unknown. This thesis used gene expression profiling to elucidate the cellular responses of LUAD cells to LCS3. I demonstrated that LCS3 induces NRF2 pathway activation and oxidative stress through the generation of reactive oxygen species in sensitive LUAD cell lines. I then developed and applied a thermal proteome profiling (TPP) approach and identified the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, I confirmed that LCS3 inhibits disulfide reductase activity through a reversible and uncompetitive mechanism. The results demonstrated that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways, suggesting a mechanistic overlap in cell death induced by LCS3 and lethality arising from disulfide reductase inhibition. I established that challenging resistant cells with oxidative stress increases reliance on the glutathione and thioredoxin pathways and sensitizes cells to LCS3 and dual disulfide reductase inhibition. Finally, a genome-wide CRISPR-Cas9 knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. Together, this work shines light on the mechanism of action of LCS3 and demonstrates the potential utility of disulfide reductase inhibition in lung cancer. This work also highlights the ability of TPP to uncover novel targets of novel small molecules identified by high-throughput screens.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Capillary electrophoresis (CE) is known for its low sample consumption, high-resolution efficiency as a separation technique, while mass spectrometry (MS) provides unprecedented selectivity and sensitivity as a detector, while adding another dimension in separation. The development of a robust CE-MS interface makes it possible to combine these two technologies for the analysis of many types of compounds. However, the coupling of CE to MS reduces its usable electrolyte ingredients substantially, and many of the popular buffer systems based on phosphate or borate, the MEKC methods dependent on the use of non-volatile surfactants, have to be excluded. As a result, formic acid and acetate buffers are the most widely used background electrolytes (BGEs) in CE-MS. In this work, we used mainly organic solvent systems to expand the choices of BGEs for CE-MS. In chapters 2 and 3, we presented two quantitative 100 % nonaqueous CE-MS methods for the separation and determination of small hydrophobic molecules. Two different BGEs were developed in this part: a basic buffer system for the analysis of negatively charged compounds and an acidic buffer system for the analysis of positively charged compounds. The compounds studied are three anthraquinones extracted from traditional Chinese medicine (TCM), Rhubarb, and six synthesized hydrophobic peptides, respectively. A high-organic-content CE-MS (HOCE) method was developed for the proteomics analysis of envelope proteins. A field amplified sample stacking technique was optimized to improve the concentration sensitivity of CE-MS for samples containing a large number of different analytes. The introduction of methanol into the buffer increased the performance of CE-MS for the detection of hydrophobic peptides in complex proteins digest. A half organic CE-MS method was used for the sequencing of novel mAbs. It was demonstrated that CE-MS/MS provided highly complementary information to LC-MS/MS with much less sample consumption. The last part of this thesis describes the application of a new generation mass spectrometry, timsTOF Pro connected with LC for the site-specific O-glycomics. In TIMS, charged compounds were separated based on their differential sizes and charges, similar to CE. The results suggest that combining CE-MS for PTMs analysis can be even more productive in the future.

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No abstract available.