Final Activity Report Summary - SMRPQ (Small molecule regulators of promoter quadruplexes) Targeting G-quadruplex nucleic acids with small molecules is emerging as a potential strategy for anti-cancer drug design. G-quadruplex sequence motifs are widespread in genomic region such as telomeres, and in gene promoters. Small molecules that selectively bind and stabilize the telomeric quadruplex can inhibit telomerase, an enzyme up-regulated in cancer cells. There is evidence that the binding of small molecules to the G-quadruplex associated with proto-oncogenes including c-myc, KRAS, PDGF-A and c-kit, can modulate transcription. Recent data suggests that RNA G-quadruplex in the 5' un-translated regions may also offer another target for small molecule intervention. We have developed bis-indole carboxamides and bis-phenylethynyl amide derivatives as G-quadruplex binding small molecule ligands. The amide derivatives were efficiently prepared using 3-4 steps by employing Sonogashira coupling, 5-endo cyclization, ester hydrolysis and a chemoselective amide coupling. Ligand-quadruplex recognition has been evaluated using a fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. The bis-indole carboxamides were designed and synthesised, which are a novel class small molecule scaffold that exhibit highest stabilization potential for DNA G-quadruplex sequences associated with the promoters of c-kit2 and c-myc (G-quadruplex recognition by bis-indole carboxamides; Dash, J.; Shirude, P. S.; Balasubramanian, S. Chem. Commun. 2008, 3055 - 3057). While most of the G-quadruplex ligands reported so far comprise a planar, aromatic core designed to stack on the terminal tetrads of a G-quadruplex, the second class of compounds we have designed; such as bis-phenylethynyl amides are neither polycyclic, nor macrocyclic and have free rotation around the triple bond enabling conformational flexibility. Such molecules show very good binding affinity, excellent quadruplex:duplex selectivity and also promising discrimination between intramolecular promoter quadruplexes. Our results indicate that the recognition of the c-kit2 quadruplex by these ligands is achieved through groove binding, which favours the formation of a parallel conformation (Diarylethynyl Amides That Recognize the Parallel Conformation of Genomic Promoter DNA G-Quadruplexes; Dash, J. Shirude, P. S.; Hsu, S-T D; Balasubramanian, S. J. Am. Chem. Soc., 130, 15950-15956, 2008.) The properties of these ligands make them attractive probes to explore hypotheses for the biological function of G-quadruplexes and such investigations are currently underway.
