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dc.contributor.advisorSamali, Afshin
dc.contributor.advisorGorman, Adrienne
dc.contributor.authorNandy, Argha
dc.description.abstractThe RNA ligase RtcB has been shown to regulate RNA function by direct exon ligation of spliced XBP1 messenger RNA cleaved by the endoplasmic reticulum (ER) stress sensor IRE1α. Ligation by RtcB leads to the expression of the transcription factor XBP1s that controls ER homeostasis and is surprisingly also utilized for oncogenic transformation, survival, and progress. Through elevated levels of XBP1s, tumor cells acquire a pro-survival advantage and promote tumor aggressiveness by an increase in tumor immune infiltration, angiogenesis, and cell invasive properties. Using RNA interference (RNAi), depleting human RtcB (hRtcB) in MDA-MB-231 triple-negative breast cancer (TNBC) cells significantly reduced basal XBP1s expression both at the mRNA and protein level. As the crystal structure of hRtcB is yet to be resolved, in silico techniques were therefore employed to develop a 3-dimensional predicted model of the hRtcB protein for the first time. Built from the Pyrococcus horikoshii RtcB (bacterial RtcB) crystal structure, PDB ID 4DWQA, the hRtcB homology model is in complex with manganese and covalently bound ligand GMP. The developed model clearly showed that the GTP binding site of the enzyme is a well-defined pocket that can be utilized as a target site for in silico drug discovery. A virtual high throughput screening (VHTS) campaign was thereafter executed to develop drugs that would halt or modulate the hRtcB-mediated XBP1s formation in TNBC. ~Eighteen million small molecule compounds were screened against the ligand binding site/active site of the hRtcB homology model. From the 3129 hits generated, the top 200 highest scoring binders were subjected to the structural hit fingerprint analysis. The top 100 highest scoring binders that exhibited similar interactions to the natural ligand GMP in the active site of the hRtcB homology model were further narrowed down to the top 20 and finally to the top 8 hit candidates based on criteria including high, medium, and poor scoring binders and purchasability. Through establishing an in vitro screening platform (MDA-MB-231/XBP1-Luciferase cell line), the top 8 hit candidates were tested in the luciferase assay from which compound-001 yielded a significant reduction (~50%) in XBP1s-luciferase under ER stress. Additionally, compound-007 showed a trend in the reduction of XBP1s-luciferase under stress in the assay. Testing these 2 compounds further in MDA-MB-231 TNBC cells didn’t yield any reduction in XBP1s under ER stress. Based on the luciferase assay results, these two hit compounds can become potential leads once tested in a biochemical assay setting and if successful, further optimized towards structure-activity relationships.en_IE
dc.publisherNUI Galway
dc.subjectHuman RtcBen_IE
dc.subjecttRNA ligaseen_IE
dc.subjectHomology modelingen_IE
dc.subjectHuman RtcB active siteen_IE
dc.subjectVirtual high-throughput screeningen_IE
dc.subjectSmall-molecule screeningen_IE
dc.subjectCell-based assaysen_IE
dc.subjectXBP1 mRNA splicingen_IE
dc.subjectRNA ligationen_IE
dc.subjectTriple-negative breast canceren_IE
dc.subjectNatural Sciencesen_IE
dc.titleDevelopment of drugs targeting the human tRNA ligase RtcB in breast canceren_IE
dc.contributor.funderHardiman Research Scholarship, NUI Galwayen_IE

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