Platinum-based DNA binding agents as telomerase inhibitors and cancer therapeutics

The targeting of key processes encountered in cancer may rest in the telomeres found at the end of eukaryotic chromosome. The major function of telomeric DNA is to protect the loss of coding DNA with each round of replication, thereby defining a finite lifetime for the cell. In somatic cells, with each cellular division the length of the telomere decreases, eventually reaching a critical length that signals apoptosis. Conversely in cancerous cells, the length of the telomere is maintained by the reverse transcriptase enzyme telomerase. This enzyme operates on the long single stranded extreme 3'-terminus of the telomere that consists mostly of guanine bases. There is evidence that telomerase can be inhibited by the folding of this G-rich portion of the telomere into a quadruplex structure, consisting of a planar arrangement of four guanine bases held together by a Hoogsteen hydrogen bonded array. Therefore, it is of great interest to create small molecules that bind strongly and preferentially to the G-quadruplex DNA motif for telomerase inhibition as a cancer therapy.In Chapter 2, a series of first generation phenanthroimidazole platinum(II) complexes were synthesized, containing large pi-surfaces that can bias their binding to G-quadruplex DNA. In comparison to a previously reported smaller pi-surface bipyridine platinum(II) complex, binding affinity and selectivity for the intermolecular G-quadruplex, (T4G 4T4)4 is compared through UV-Vis titrations, thermal denaturation, competitive dialysis, circular dichroism and molecular modeling.In an attempt to place our studies in a greater biological context, Chapter 3 examines the interactions of these phenanthroimidazole platinum(II) complexes with the G-quadruplex forming human telomeric repeat, (GGGTTA) n. Their ability to bind and stabilize this intramolecular G-quadruplex is examined through a fluorescence resonance energy transfer (FRET) melting assay and molecular modeling. In addition, the ability of these large pi-surface complexes to template G-quadruplex folding is explored through circular dichroism. Lastly, their ability to inhibit telomerase is evaluated through a modified version of the telomerase repeat amplification protocol assay (TRAP-LIG).In Chapter 4, a second generation of phenanthroimidazole platinum(II) complexes were designed containing substituents (indolyl, quinolinyl) that promote hydrogen bonding within the ligand to achieve increased binding affinity and selectivity. The synthesis of the quinolinylphenanthroimidazole ligand and the indolylphenanthroimidazole platinum(II) complex is reported. A FRET melting assay, molecular modeling and templation experiments monitored by circular dichroism are used to assay the interactions of this complex with the intramolecular G-quadruplex based on the human telomeric repeat, (GGGTTA) n.Thus far, there have been many elegant studies on organic G-quadruplex binders that have appeared, but examples involving inorganic complexes are rare. Implementation of transition metals in these scaffolds provides the opportunity to generate G-quadruplex binders of multiple geometries beyond those that carbon can access. Comparatively speaking, introduction of metals into these scaffolds can lead to simplified syntheses of these binders over their organic counterparts with generation of compound libraries able to selectively target G-quadruplex DNA.In Chapter 5, the importance of pi-surface in binding to the G-quadruplex is examined through the synthesis of a metallosupramolecular platinum(II) square, [Pt(en)(4,4'-bpy)]48+. Molecular modeling and a FRET melting assay were performed to evaluate the interactions, binding affinity and selectivity of this complex for a G-quadruplex based on the human telomeric repeat, (GGGTTA)n. In addition, the ability of these compounds to inhibit telomerase by the TRAP and TRAP-LIG assay are discussed.In Chapter 6, the ability of DNA intercalating drugs to self-assemble was also explored. A terpyridine platinum(II) complex containing an alkyl dendritic substituent was synthesized and self-assembled. The self-assembly was characterized using atomic force microscopy (AFM), transmission electron microscopy (TEM), Optical Microscopy and dynamic light scattering (DLS). The ability of these assemblies to interact with DNA was observed by UV-Vis, circular dichroism, thermal denaturation and AFM studies.