Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells
department or school: Medicine, Faculty of
data provider: DSpace
degree campus: UBCV
creator: Johnson, Fraser
issued: 2022-04-25T19:09:26Z
issued: 2022
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: 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.
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/81356
note: CHARACTERIZATION OF A SMALL MOLECULE INHIBITOR OF DISULFIDE REDUCTASES THAT INDUCES OXIDATIVE STRESS AND LETHALITY IN LUNG CANCER CELLS by Fraser Johnson B.Sc., The University of British Columbia, 2015 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Interdisciplinary Oncology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2022 © Fraser Johnson, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells submitted by Fraser Johnson in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Interdisciplinary Oncology Examining Committee: Dr. William W. Lockwood, Associate Professor, Pathology and Laboratory Medicine, UBC Co-supervisor Dr. Gregg B. Morin, Associate Professor, Medical Genetics, UBC Co-supervisor Dr. Peter Stirling, Associate Professor, Medical Genetics, UBC Supervisory Committee Member Dr. Christopher Ong, Associate Professor, Urologic Sciences, UBC University Examiner Dr. Christopher A. Maxwell, Associate Professor, Pediatrics, UBC University Examiner Additional Supervisory Committee Member: Dr. Poul H. Sorensen, Professor, Pathology and Laboratory Medicine, UBC Supervisory Committee Member iii Abstract 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 expr…
has type: Thesis/Dissertation
date issued: 2022-05
is shown at: 10.14288/1.0413031
language: eng
degree discipline: Interdisciplinary Oncology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Lockwood, William W.
contributor: Morin, Gregg B.
type: Text
identifier URI: http://hdl.handle.net/2429/81356