A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with Gquadruplex DNA within the c-myc gene promoter were evaluated. Complex 1, which has a flat planar 2,6-bis(benzimidazol-2-yl)pyridine (bzimpy) scaffold, was found to stabilize the c-myc G-quadruplex structure in a cellfree system. An in silico G-quadruplex DNA model has been constructed for structure-based virtual screening to develop new Pt-based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit-to-lead optimization, bzimpy and related 2,6-bis(pyrazol-3-yl) pyridine (dPzPy) scaffolds containing amine side-chains emerge as the top candidates. Six of the top-scoring complexes were synthesized and their interactions with c-myc G-quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c-myc G-quadruplex. Complex 3a ([PtL2R]; L2 = 2,6bis[1-(3-piperidinepropyl)-1H-enzo[d]imidazol-2-yl]pyridine, R = Cl) displayed the strongest inhibition in a cell-free system (IC = 2.2 μM) and was 3.3-fold more potent than that of 1. Complexes 3a and 4a ([PtL3R]; L3 = 2,6-bis[1-(3-morpholinopropyl)1H- pyrazol-3-yl]pyridine, R = Cl) were found to effectively inhibit c-myc gene expression in human hepatocarcinoma cells with IC values of 17 μM, whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 μM. Complexes 3a and 4a have a strong preference for Gquadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3a and 4 a bind G-quadruplex DNA with binding constants (K) of approximately 10-10dm mol, which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c-myc G-quadruplex DNA through an external end-stacking mode at the 3'terminal face of the G-quadruplex. Intriguingly, binding of c-myc G-quadruplex DNA by 3b is accompanied by an increase of up to 38-fold in photoluminescence intensity at λmax = 622 nm. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.