Cyclooxygenases (COXs) play a vital role in the inflammatory cascade through the generation of prostaglandins. Their over-expression is implicated in numerous diseases. In particular, over-expression of COX-2 is a predictive biomarker for progression of pre-malignant lesions towards invasive cancer. This makes the early detection of COX-2 expressing lesions of high clinical relevance.Current fluorescent probes for COX-2 imaging mostly employ a ligand-linker-fluorophore design. This has proved successful but retains limitations, including background signal from non-bound probes. The development of a self-immolating trigger specific for COX-2 would facilitate the generation of activatable probes, reducing background signal, as well as COX-2 targeted pro-drugs. This presentation describes our first steps towards such a trigger.Figure 1: Mechanism of Probe Activation by COXs.Our approach (figure 1) is based upon aspirin, which acetylates a serine in the COX active site. We installed a self-immolating linker and latent fluorophore parato aspirin's phenolic acetyl and sought to introduce selectivity for COX-2 over COX-1 by replacing the carboxyl moiety with either an ester or an alkyl thioether ('R'). When deacetylated by COXs the revealed phenol participates in a 1,6-quinone-methide elimination, releasing the activated fluorophore.We synthesized two probes and showed that they were specifically activated by COXs in vitro. We then demonstrated activation by COX-2 in two separate cellular models, including COX-2 over-expressing human mammary epithelial cells. Our results validate this mechanistic approach to targeting COXs.

Development of cyclooxygenase specific self-immolating triggers and their use in activatable fluorescent probes to detect cyclooxygenase-2 in cells

248th National Meeting (2014)

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