Nucleic acid triggered catalytic drug release: A new concept for the design of disease-specific chemotherapeutic agents

Chemotherapeutic approaches to cure infectious diseases or cancer depend on drugs that are selectively toxic to the disease-causing organism or the diseased cell. Viral infections and cancer pose the greatest challenge for chemotherapy because there is little biochemically to distinguish an infected or cancerous cell from a normal cell, and as a result many currently used drugs show little selectivity. In this dissertation we propose a new concept for the rational design and synthesis of highly selective chemotherapeutic agents that makes direct use of genetic information about the disease state. The key idea is to use the mRNA or DNA specific to the disease state to trigger the catalytic release of a cytotoxic drug by promoting the association of a prodrug with a catalyst capable of releasing the drug.; We demonstrate the feasibility of such an approach in vitro first with a model system in which a imidazole was attached to 5′ end of an oligodeoxynucleotide (ODN) hairpin and dipivaloyl fluorescein was linked to an ODN complementary to the single strand stem of the hairpin. It was found that only in the presence of 5′ imidazole linked hairpin was the pivaloyl group of fluorescein efficiently hydrolyzed. Then we developed a three-component system in which the catalytic component consists of an imidazole group linked to the 5′-end of a 15-mer that is complementary to the 5′-end of the triggering oligonucleotide. In this system the prodrug component consists of a p-nitrophenol ester linked to the 3′-end of an 8-mer oligonucleotide that is complementary to 3′-end of the triggering sequence. We demonstrate that p-nitrophenol is only released efficiently in the presence of all three components, and that the reaction is catalytic and undergoes multiple turnovers. We also show that the complex between the catalytic component and the triggering ODN behaves like an enzyme and follows Michaelis Menten kinetics. Most importantly, we show that catalytic release of p-nitrophenol is sensitive to the presence of a single base pair mismatch.; We then developed a peptide nucleic acid (PNA) based system suitable for directing the assembly of the prodrug and the prodrug activation component. We show that PNA components could bind simultaneously to specific sequences on folded E. Coli 5S rRNA structure to release the drug. To determine whether or not prodrugs based on all kinds of ester linkages would be suitable for use in humans, we also investigated the stability of esters of 7-hydroxycoumarin in human serum. Finally we describe how the ester hydrolysis efficiency of imidazole can be enhanced by using phenyl-pentafluorophenyl stacking interactions to preorganize the imidazole and the ester group.