Department of Chemistry: Part III Project 2011/2012 Prof. S. Balasubramanian Embedding Crosslinking Agents within G-4 Ligands: “An Old Strategy for a New Purpose” EMAIL firstname.lastname@example.org Group web site http://www-shankar.ch.cam.ac.uk/ Contact details: Dr Marco di Antonio, email@example.com
Guanine rich oligonucleotides are capable of self-organization into secondary structures also known as G-quadruplexes (G-4).1 Besides the pure supramolecular aspects, what really makes these structures interesting is their potential as cancer selective targets. Indeed, it has been shown that guanine rich oligonucleotides capable of forming G-quadruplex structures in vitro are present in crucial genomic regions, such as in the telomeric DNA and at the transcriptional level of several oncogenes.2 Moreover, it is widely recognized that the G-quadruplex folding equilibrium could be implied in the transcriptional regulation of oncogenes as well as in the inhibition of the cancer related enzyme Telomerase.3 For these reasons several efforts have been made in the last few years in order to engineer small molecules capable of selective recognition and stabilization of G-4 structures, with the aim to eventually develop new selective anticancer drugs. On the other hand, the most successful cancer treatments are still based on the use of crosslinking agents, such as Cisplatin and Chlorambucil. The aim of this project is to link the selective recognition properties of an already described G-4 ligand with the alkylating properties of reactive electrophilic species.4 The final goal is to transfer the crosslinking approach used for double stranded DNA to the G-folded DNA or RNA. This should lead to ligands capable of specifically delivering the alkylating agent to a desired biological target. This concept is schematically depicted in Figure 1.
Figure 1. General illustration of a G-4 structure cross-linked by using a hybrid ligand/alkylating molecule.
Such a strategy allows, in principle, an irreversible stabilization of the biological target, which can be useful to further unravel the biological functions of G-quadruplexes. In order to control the alkylation process, electrophilic species generated from stable silent precursors will be used. Indeed, the generation of transient alkylating species from stable precursors through mild chemical generation is a key aspect, which can lead to a temporal and spatial control of the alkylation process. Nevertheless, such an approach should avoid alkylation of undesired sites, one of the main
1 Davis, J.T. Angew. Chem. Int. Ed. Engl. 2004; 43(6): 668-98. 2 Balasubramanian, S.; Hurley, L.; Neidle, S. Nat. Rev. Drug Discov.2011; 10(4): 261-75. 3 Neidle, S. Curr. Opin. Struct. Biol.2009; 19 (3): 239-50. 4 Di Antonio, M. et al. J. Am. Chem. Soc.2009; 131(36): 13132-41.
causes of side-effects from the use of chemotherapeutics. The release of the alkylating agent will be achieved by using mild chemical triggers, such as reduction, pH modification or light absorption, as schematically depicted in Figure 2.
Figure 2. Reactivity and generation of an alkylating agent from a stable precursor tethered to a recognition moiety.
The hybrid ligand/alkylating agents proposed in this project are pyridostatin (PDS) based molecules, which are well known G-4 selective ligands and are depicted in Figure 3. One of the synthetic procedures proposed to achieve the synthesis of the above agents is based on a Cu(I) catalysed 1,3 dipolar cycloaddition, also known as a “click” reaction. Such an approach should afford a small library of PDS based hybrid ligand/alkylating agents.
The ability of the synthetized molecules to covalently interact with G-quadruplex folded oligonucleotides will be tested by means of FRET melting experiments, circular dichroism and gel electrophoresis. Moreover, the best ligands will be further assessed in cellular assays as well as gene down-regulation.
Figure 3. One of the possible PDS based molecules proposed as object of this project.
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