Splice switching small molecules as inducers of apoptosis

One of the most important post-transcriptional processes that occurs during the transcription of DNA to RNA is alternative splicing. Alternative splicing is a highly regulated process that occurs during gene expression and is the basis for the large protein diversity that is achieved in eukaryotes. It allows multiple protein isoforms to be formed from one pre-mRNA sequence.;Although alternative splicing is necessary to provide protein diversity, aberrant splicing can also lead to diseases such as cancer. Recently, there has been an interest in the ability of small molecules to exogenously modulate alternative splicing as therapeutics for aberrant splicing diseases. The ellipticine analogue GQC-05 has been found to have splice switching capabilities towards the apoptotic oncogene Bcl-x, however the mode of action of GQC-05 is not understood.;This thesis describes efforts made to elucidate the mode of action of GQC-05 by synthesising a library of ellipticine derivatives for testing in a range of biochemical assays to evaluate their ability to bind and stabilise Bcl-x.;The background to alternative RNA splicing and the aberrant splicing process that can lead to diseases such as cancer is introduced in Chapter 1. The importance of the oncogene Bcl-x and the current limitations in targeting it therapeutically are also presented.;Chapter 2 investigates current literature methods to prepare 9-methoxyellipticine. However it is found that these methods do not provide a modular framework in which to systematically alter the ellipticine structure.;Chapter 3 describes the development of a one-pot, sequential, palladium cross-coupling methodology to construct the backbone of the natural product ellipticine. This methodology has been further extended to synthesise a range of ellipticine analogues with modular changes throughout the structure.;Chapter 4 examines the ability of the ellipticine derivatives synthesised to stabilise a putative Gquadruplex structure within Bcl-x through the use of UV melting assays. The ligands have also been investigated for their ability to stabilise a range of competitor DNA structures with the help of FRET melting assays. The interaction of GQC-05 with the putative Bcl-x G-quadruplex has also initially been examined using NMR spectroscopy. And finally, the ellipticine derivatives have been tested in Bcl-x in vitro splicing assays to determine whether they are capable of exerting the same splicing modulation on Bcl-x as their parent compound GQC-05.One of the most important post-transcriptional processes that occurs during the transcription of DNA to RNA is alternative splicing. Alternative splicing is a highly regulated process that occurs during gene expression and is the basis for the large protein diversity that is achieved in eukaryotes. It allows multiple protein isoforms to be formed from one pre-mRNA sequence.;Although alternative splicing is necessary to provide protein diversity, aberrant splicing can also lead to diseases such as cancer. Recently, there has been an interest in the ability of small molecules to exogenously modulate alternative splicing as therapeutics for aberrant splicing diseases. The ellipticine analogue GQC-05 has been found to have splice switching capabilities towards the apoptotic oncogene Bcl-x, however the mode of action of GQC-05 is not understood.;This thesis describes efforts made to elucidate the mode of action of GQC-05 by synthesising a library of ellipticine derivatives for testing in a range of biochemical assays to evaluate their ability to bind and stabilise Bcl-x.;The background to alternative RNA splicing and the aberrant splicing process that can lead to diseases such as cancer is introduced in Chapter 1. The importance of the oncogene Bcl-x and the current limitations in targeting it therapeutically are also presented.;Chapter 2 investigates current literature methods to prepare 9-methoxyellipticine. However it is found that these methods do not provide a modular framework in which to systematically alter the ellipticine structure.;Chapter 3 describes the development of a one-pot, sequential, palladium cross-coupling methodology to construct the backbone of the natural product ellipticine. This methodology has been further extended to synthesise a range of ellipticine analogues with modular changes throughout the structure.;Chapter 4 examines the ability of the ellipticine derivatives synthesised to stabilise a putative Gquadruplex structure within Bcl-x through the use of UV melting assays. The ligands have also been investigated for their ability to stabilise a range of competitor DNA structures with the help of FRET melting assays. The interaction of GQC-05 with the putative Bcl-x G-quadruplex has also initially been examined using NMR spectroscopy. And finally, the ellipticine derivatives have been tested in Bcl-x in vitro splicing assays to determine whether they are capable of exerting the same splicing modulation on Bcl-x as their parent compound GQC-05