Award details

G-Quadruplex-Based Chemical Genetics

Reference	BB/K018043/1
Principal Investigator / Supervisor	Professor Sir Shankar Balasubramanian
Co-Investigators / Co-Supervisors	Dr Marco Di Antonio
Institution	University of Cambridge
Department	Chemistry
Funding type	Research
Value (£)	343,893
Status	Completed
Type	Research Grant
Start date	05/08/2013
End date	04/08/2016
Duration	36 months

Abstract

In situ "click chemistry" has been developed in the past to identify and screen novel enzyme inhibitors. This approach relies on the use of alkynes and azides that can bind protein active sites and by doing so they are allowed to react, as the protein acts as a catalyst. However, this methodology has never been extended to nucleic acids, mainly due to the less structured and more dynamic folding properties of the latter. We recently demonstrated that in situ "click chemistry" could be successfully applied for screening novel ligands for nucleic acids folded into G-quadruplex structures. G-quadruplexes are non-canonical DNA and RNA secondary structures that can be generated in G-rich sequences. Their formation has been detected in promoter regions of oncogenes (i.e SRC, c-MYC and c-KIT) and their stabilization by means of small molecules has been associated with down-regulation of those genes and DNA damage response activation. The development of this method has already provided the first small molecule capable of strongly discriminating between RNA and DNA G-quadruplexes. Such a molecule could potentially provide the chemical means to decipher and distinguish between the biological function(s) of RNA and DNA G-quadruplexes. In this proposal we aim to extend the in situ "click chemistry" methodology by increasing the chemical complexity of the libraries of compounds used to generate the first generation of ligands possessing intra-G-quadruplex selectivity. This will enable an understanding of the function(s) of different G-quadruplexes formed in very different genomic regions. Our strategy will generate the tools to interrogate cells about the function(s) associated with a given G-quadruplex forming sequence. The goal is to generate molecules capable of selectively targeting G-quadruplexes associated with a given gene, enabling the specific alteration of gene expression by small-molecule targeting (Chemical Genetics).

Summary

Targeting DNA and RNA by means of small molecules is one the most successful strategies to interfere with and study cancer related biology. Nucleic acids can adopt non-canonical structures that have been suggested to regulate biological processes crucial for cellular stability, such as gene regulation and protein expression. Therefore many studies have focussed on the development of molecules with selective recognition properties towards DNA and RNA secondary structures. Nucleic acid secondary structures named G-quadruplexes have emerged as candidates for gene and protein regulation. Targeting and stabilising the G-quadruplexes present in some genes with small molecules leads to an alteration in the gene expression. A large number of G-quadruplexes have been found in the whole genome, suggesting that these structures can be involved in a myriad of biological processes. Despite the large number of disclosed ligands that selectively recognize G-quadruplexes over the canonical double stranded DNA, none of them is able to selectively recognize one particular G-quadruplex over the others present in the genome. Therefore, having a ligand selective for one particular structure will provide the means to interfere only with the biological functions associated with the targeted structure. We recently developed a new method to generate molecules "in situ" in the presence of the targeted G-quadruplex, generating ligands specific for the targeted structure. When running a chemical ligation in the presence of a targeted nucleic acid (i.e a specific DNA or RNA G-quadruplex) only those functionalities that specifically interact with the target will give rise to the ligation and generate an adduct. The molecules generated by this method will be extremely selective for the target, as they will posses the perfect geometry to interact with it. By developing this method we have already demonstrated that is possible to generate molecules that are selective towards RNA over DNA G-quadruplexes. We now want to further develop and extend this method increasing the complexity and the variability of the possible adducts to enable the formation of highly specific ligands. The chemical tools generated by this method will provide further insights into the mechanism behind G-quadruplex formation in different genomic locations. Our final goal is to achieve selective gene regulation by using small molecules that target only the G-quadruplexes present in a given genomic region.

Impact Summary

Developing small molecules that selectively interact with a given genomic sequence altering a specific gene expression will enable the study of the effect of these changes in vivo with temporal resolution. This approach represents a valid alternative to the classic genetics approach of studying gene dysfunction and will be of huge impact for genetic research, chemical biology and molecular recognition development. The SRC gene in particular has been extensively studied because of its importance in cell cycle regulation and cell migration. Our study will provide the chemical tools to interfere selectively with the expression of this gene, having a large impact on the many molecular biology and genetic studies associated with cells that are SRC dependent (mainly cancer cells). Moreover the selective targeting of G-quadruplex RNA over DNA will have a major impact on the further advancement of the understanding of G-quadruplex functions in cells. In fact, generating a novel class of ligands capable of strongly discriminating between different G-quadruplexes represents one of the major challenges in this field. Indeed, this will lead to a better understanding of the different functions associated with the single structure and, moreover, will allow the targeting of a single structure/sequence without interfering with the phenotypes associated with all the others. The study of these novel ligands is crucial for progressing G-quadruplex binding small molecules into clinical applications because: i) ligands will be more effective and lower concentrations will be required, thereby reducing possible side effects ii) targeting a single structure will reduce the intrinsic toxicity of the ligand which could result from indiscriminate stabilization of all G-quadruplexes present in cells. We believe that such a study is crucial for the development of novel cancer therapeutics and will provide further insights for the generation of novel chemotherapeutics. Exploring novel chemical tools that can regulate gene expression selectively is crucial especially in the light of the high impact that cancer has as disease worldwide.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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