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Discovery of a Small Molecule TUBB3/Βiii-Tubulin Modulator in Lung Cancer

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Discovery of a Small Molecule TUBB3/Βiii-Tubulin Modulator in Lung Cancer Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer Felicity Chao Lin Kao A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Women’s and Children’s Health Faculty of Medicine The University of New South Wales March 2016 TH E UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Kao First name: Felicity Other name/s: Chao Lin Abbreviation for degree as g iven in the University calendar: PhD School: School of Women's and Children's Health Faculty: Faculty of Medicine Title: Discovery of a small molecule TU883/I311 1·tubulin modulator in lung cancer Abstract Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant clinical problem. 13111-tubulin (encoded by TUBB3 gene) is aberranlly expressed and is associated with chemoresistance and tumour aggressiveness in NSCLC, where it has been identified as a bona fide target for chemosensitisation. Currently, there is no commercially available TUB83J13111-tubulin inhibitor. Regulation of 13111 -tubulin is poorly understood, making it difficult to target this protein. We sought to identify a chemical modulator of TU883J1311Hubulin expression, as it will be a valuable research tool to probe TU883J!3111-tubulin regulation . A novel small molecule TU883/13111-tubulin enhancer, WECC0017371 , was identified in our high throughput screen, based on its ability to modulate TUBB3 promoter activity. WECC001 7371 demonstrated the ability to significantly enhance TUBB3 mRNA and 13111-tubulin protein expression in a time· and dose-dependent manner. Additionally, WECC0017371 did not alter microtubule morphology but enhanced 13111-tubuli n immunostaining in two independent NSCLC cell lines, H460 and H1299, compared to control. Importantly, WECC0017371 enhanced 13111 -tubulin aberrant expression was functional and led to a significant decrease in in vitro sensitivity to DNA-damaging and tubulin-binding agents. Using chemical modifications, the efficacy, potency and selectivity of WECC0017371 was further improved for the study of TU883/13 111-tubulin regulation. The superior WECC0017371 analogue, ENTD014 was developed into an affinity chromatography probe for future studies to identify intracellular proteins that it binds to enhance TU883/1311Hubulin expression. Negative control probes were also developed to identify and eliminate proteins that bind non-specifically to the affinity probe. To understand how WECC001 7371 is exerting its effects on TUBB3 expression. NSCLC cells were treated, mRNA isolated and a microarray and bioinformatics approach was used. Coordinated gene expression changes in multiple members of the mitotic and p53 signalling pathways were detected in WECC0017371-treated cells. In conclusion, a novel chemical research tool has been developed in this thesis to better understand the molecular mechanisms underlying TUBB3/13111-tubulin regulation in NSCLC and 13111-tubulin-mediated pathobiology. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise U n iv~ i c rofilms to use the 350 word abstract of my thesis in Dissertation Abstracts International.-------- / ../1 ~ The University recognises that there may be exceptional circumstances requ1nng restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requi rements for Award: COPYRIGHT STATEMENT 'I hereby grant the University of New South Wales or its agents the right to archive and to 111,ake available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis--or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed Date ............ 4.. 1.. ~.':?. .(. ~ .~······················· ···· ··········· ····· AUTHENTICITY STATEMENT 'I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.' Signed Date ..............~ .f. .l..~!.! .. l::? .............................. :...... ..... Table of Contents Table of Contents .......................................................................................................... 1 Publications arising from this thesis .............................................................................. 6 Awards ......................................................................................................................... 7 Acknowledgements ....................................................................................................... 8 List of Abbreviations .................................................................................................. 10 Chapter 1 Introduction ............................................................................................... 12 1.1 Thesis overview ............................................................................................ 12 1.2 Lung cancer .................................................................................................. 15 1.2.1 Staging of lung cancer ........................................................................... 16 1.2.2 Treatment strategies for NSCLC ............................................................ 16 1.3 Tubulin/ microtubule system ......................................................................... 22 1.3.1 Structure and function of microtubules .................................................. 22 1.3.2 The β-tubulin family .............................................................................. 26 1.3.3 Microtubule interacting proteins ............................................................ 30 1.3.4 Posttranslational modification ................................................................ 31 1.3.5 Tubulin binding agents for NSCLC treatment ........................................ 34 1.3.6 Drug resistance in NSCLC and βIII-tubulin ........................................... 37 1.4 βIII-tubulin: expression and regulation .......................................................... 42 1.4.1 Expression and function of βIII-tubulin .................................................. 42 1.4.2 Gene structure of human TUBB3 gene ................................................... 49 1 1.4.3 Regulation of TUBB3 expression in non-neoplastic cells ........................ 52 1.4.4 Regulation of TUBB3 expression in cancer cells .................................... 57 1.5 High throughput drug discovery .................................................................... 70 1.5.1 βIII-tubulin as a drug target.................................................................... 70 1.5.2 High throughput screening ..................................................................... 71 1.5.3 Assay types ............................................................................................ 72 1.5.4 Experimental design and planning ......................................................... 74 1.5.5 Assay development and optimisation for HTS ....................................... 75 1.5.6 Execution of HTS .................................................................................. 77 1.5.7 Validation of drug candidate and lead optimisation ................................ 78 1.6 Thesis perspective and significance ............................................................... 79 1.7 Thesis aims ................................................................................................... 80 Chapter 2 Materials and Methods ............................................................................... 81 2.1 Materials ....................................................................................................... 81 2.1.1 Cytotoxic drugs and other chemicals ...................................................... 81 2.1.2 Tissue culture ........................................................................................ 81 2.1.3 Plasmid sequencing and restriction digest .............................................. 82 2.1.4 Plasmid transfection and single clone selection .....................................
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