Towards novel cancer therapeutics : development of programmable, synthetic DNA polymers to inhibit tubulin polymerization

Abstract

Paclitaxel (Taxol®) is a microtubule stabilizer and an effective chemotherapeutic. Yet, it suffers from limitations, which oligonucleotide therapeutics might be able to address. Tubulin is a dimeric protein comprising α/β subunits which assemble into microtubules. It is a prominent cancer drug target and essential for to cell growth, division and stability, intracellular transport and cell cycle regulation. Paclitaxel's potent mechanism of action leads to hyper-stabilization of microtubules and ultimately, apoptosis. But emerging resistance, undesirable properties as well as its environmentally destructive extraction, scarcity and astronomical price pose severe risks to it’s therapeutic success and highlight the pressing need for alternatives. Aptamers are versatile biological polymers made from nucleic acids, which fold into three-dimensional structures and can bind a broad range of targets, with affinity and specificity that rivals antibodies, while offering compelling advantages. Aptamers are versatile ‘chemical antibodies’ which can be synthesized and modified cost-effectively and rapidly in large quantities, are chemically benign and non-immunogenic. They are typically generated from a random pool of sequences by an iterative enrichment, called in vitro selection (SELEX) and have already found clinical success. In the motivation to overcome the under-addressed hurdles of chemotherapeutics, aptamers are explored as novel anti-mitotic agents. In this work, a population of previously published tubulin-binding aptamers with affinities in the low micromolar range are evaluated in in vitro drug discovery screening for the first time and a comprehensive analysis framework is developed for the rapid and robust characterization of nucleic acids as drug-like molecules. In vitro selection of more tightly-binding aptamers and a range of post-selection modification strategies are pursued. This involves development of a rapid prototyping template and a ligand-responsive anti-tubulin chimera. Furthermore, a novel polymeric miniature microtubule target is fabricated to facilitate epitope-specific selection of Paclitaxel-like binders.