Exploring Fundamental Strategies For Imaging Hydrolytic Enzymes

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The focus of my research program is to apply fundamental bio-organic chemistry approaches, enzymology and rationale probe design to develop innovative chemical strategies for imaging lysosomal enzymes: most notably (CTB) and glucocerebrosidase (GCase). To improve MI of lysosomal proteases, in vitro and in vivo, we have prepared a series of novel prodrug inspired peptide derivatives designed to be substrate-based probes for CTB. However, a substrate-based approach requires the accumulation of the reporter signal, fluorescent or radioactive, within the local area of enzyme activity to improve detection. To test this approach, we prepared substrates bearing various aminoquinoline (AMQ) reporters that would become ionized and accumulate into the acidic environment of the lysosomes after CTB release. Weakly basic, lipophilic amines, like AMQs, are well known lysosomotropic molecules that spontaneously accumulate to high levels inside of cells. Indeed, peptides derivatized with PABA, a common prodrug linker and AMQs proved to be excellent substrates of CTB. However, most AMQs have no inherent fluorescent properties and cannot be easily labeled with 18F for positron emission tomography (PET) imaging studies in vivo. To address these limitations and explore lysosomal trapping to improve imaging of protease enzymes, we synthesized a series of AMQ derivatives designed to be highly fluorescent and lysosomotropic to enable cell imaging studies and to guide PET tracer discovery. We discovered novel AMQ derivatives that are highly fluorescent, have tunable lipophilicity, pKa, and accumulate inside the lysosomes of living cells through the lysosomotropic effect. Surprisingly, one derivative had near-infrared fluorescence emission and accumulated in the lysosomes of breast cancer cells, generating high resolution images using confocal microscopy. Further, several derivatives were fluorinated, using approaches compatible with 18F radiochemistry while also maintaining strong fluorescence. We will continue to investigate the photochemical properties of these novel AMQs (relative quantum yields, modes of fluorescence), physiochemical properties (pKa, logD) and biological properties (lysosomal trapping, toxicity). This will guide the development of new derivatives with improved imaging performance. To demonstrate the utility of these novel AMQs as powerful new reporter groups, we will conjugate the best derivatives onto irreversible inhibitors of GCase and into peptide conjugates to generate fluorogenic substrate-based probes for detecting CTB. In parallel, we will use the most efficient and selective substrates, bearing fluorinated AMQs, as potential PET tracers for imaging CTB in vivo. Importantly, the same prodrug strategies and lysosomotropic reporter groups developed for CTB can be easily adapted for imaging other proteases as well as other enzyme classes including glucosidases such as GCase thus having broad long term impact.