Development of a Small Molecule Tubulysin B Conjugate for Treatment of Carbonic Anhydrase IX Receptor Expressing Cancers.

Carbonic anhydrase IX (CAIX) is a membrane-spanning zinc metalloenzyme that catalyzes the reversible consumption of CO2 and water to form H+ + HCO3-. Many human cancers upregulate CAIX to help control the pH in their hypoxic microenvironments. The consequent overexpression of CAIX on malignant cells and low expression on normal tissues render CAIX a particularly attractive target for small molecule inhibitors, antibody-drug conjugates, and ligand-targeted drugs. In this study, CAIX-targeted fluorescent reporter molecules were initially exploited to investigate CAIX-specific binding to multiple cancer cell lines, where they were shown to display potent and selective binding to CAIX positive cells. A small molecule CAIX-targeted tubulysin B conjugate was then synthesized and examined for its ability to kill CAIX-expressing tumor cells in vitro. Potent therapeutic conjugates were subsequently tested in vivo and demonstrated to eliminate solid human tumor xenografts in murine tumor models without exhibiting overt signs of toxicity. Because most solid tumors contain hypoxic regions where CAIX is overexpressed, development of a method to selectively deliver drugs to these hypoxic regions could aid in the therapy of otherwise difficult to treat tumors.

Strain-promoted "click" chemistry for terminal labeling of DNA.

1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.