DNA and its Secondary Structures as Targets for Small Molecule Cancer Therapeutics - An NMR Structural Study

Abstract

DNA serves as a major target for mainstream drugs used in the treatment of cancer, but current DNA-targeted drugs have significant issues due to their poor selectivity giving rise to adverse effects. Recent research on targeting DNA has focused on DNA interactive compounds with novel mechanisms of action and new cancer-related DNA molecular targets. An understanding of molecular level details of small molecule interactions with their DNA targets is critical for understanding the molecular mechanisms of action and for structure-based rational drug design. This dissertation presents two studies focused on gaining a structural understanding of DNA-targeted small molecules, one with a novel mechanism of action and the second with a cancer-specific DNA molecular target. XR5944 is potent anticancer drug and a novel mechanism of action, DNA bis-intercalation with a major groove binding. It is able to recognize and bind the estrogen response element (ERE) sequence via the major groove to inhibit estrogen receptor-α activity. This mechanism of action may be useful for overcoming drug resistance to currently available antiestrogen treatments for breast cancer, all of which target the hormone-receptor complex. We determined the nuclear magnetic resonance solution structure of the 2:1 complex of XR5944 with the naturally occurring TFF1-ERE, which exhibits important and unexpected features. In the determined structure, each bis-intercalating XR5944 molecule is strongly bound at one of its intercalating site, but weakly at the other. Our results show the sites of intercalation within a native promoter sequence appear to be context and sequence dependent. The binding of one drug molecule influences the binding site of the second. The structure underscores the fact that the DNA binding of a bis-intercalator is directional and differs from the simple addition of two single intercalation sites. Our results provide insights toward future structure-based rational drug design of DNA bis-intercalators to modulate ERα-induced transcriptional activity, as well as for designing bis-intercalators with major groove binding modes in general. Human telomeric DNA G-quadruplex secondary structures have emerged as an attractive molecular target for anticancer drugs. G-quadruplex formation in human telomeres inhibits telomerase, which plays a key role in maintaining the malignant phenotype by stabilizing telomere length and integrity. Under physiologically relevant conditions, human telomeric DNA sequences form two equilibrating G-quadruplex structures, with the hybrid-2 structure being the predominant in an extended sequence Thus, the hybrid-2 human telomeric G-quadruplex is considered to be a potential target for anticancer drugs targeting telomere biology and telomerase. We discovered that epiberberine, a naturally occurring isoquinoline alkaloid, can specifically bind the hybrid-2 telomeric G-quadruplex and induce the conversion of hybrid-1 telomeric G-quadruplex to the hybrid-2 structure. We determined the structure of the hybrid-2 G-quadruplex in complex with epiberberine by NMR in K⁺ solution. This NMR solution structure shows an unexpected, large, drug-induced conformational change in the flanking and loop regions, creating a very well-defined “induced intercalated quasi-triad pocket” with an extensive capping structure. Our result demonstrates the importance of ligand shape as well as the G-quadruplex folding topology and flanking and loop sequences in small molecule targeting the intramolecular hybrid-2 human telomeric G-quadruplex. Our result also indicates that asymmetric compounds containing a crescent-shaped moiety are more likely to bind in a specific manner to an intramolecular G-quadruplex.