Specific Detection and Imaging of Enzyme Activity by Signal‐Amplifiable Self‐Assembling ¹⁹F MRI Probes

Abstract

Specific turn-on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal-amplifiable self-assembling (19) F NMR/MRI probe for turn-on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are "silent" when aggregated, but exhibit a disassembly driven turn-on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these (19) F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by (19) F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by (19) F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn-on (19) F probes may serve as a screening platform for the activity of MMPs.

Affinity purification in target identification: the specificity challenge

“…Parallel labeling experiments against the target protein carbonic anhydrase have shown that the moderately reactive epoxide crosslinker exhibits the optimal balance of selectivity and reactivity (Chen et al 2003). Hamachi, Fenical and their respective co-workers developed a series of novel traceless affinity labeling probes containing electrophilic organocatalysts such as acyl phenolate, (Hughes et al 2009) 4-dimethylaminopyridine (DMAP) (Koshi et al 2008;Sun et al 2011;Wang et al 2011), tosylate, (Tsukiji et al 2009a, b;Tamura et al 2012), and acyl imidazole (Fujishima et al 2012;Matsuo et al 2013;Miki et al 2014). By targeting specific nucleophilic groups in the ligand-binding pocket of the protein, these probes exhibited high target specificity (Fig.…”

Rotaxane probes for protease detection by 129Xe hyperCEST NMR

“…[1][2][3] These methods rely on suppressing background signals until a chemical or biological event occurs that activates the probe, at which point a detectable signal is generated. This strategy has been used to detect enzymes, [2][3][4] signaling molecules, 3 and redox conditions, 5 among other targets. Proteases, in particular, are widely used markers for disease detection, as certain proteases are overexpressed in diseased tissues and cells.…”

“…Rotaxane 2 was synthesized with pyrene and (5,6)-carboxytetramethylrhodamine (TAMRA) stoppers, and an axle containing the PLG-LAG recognition sequence of matrix metalloprotease-2 (MMP-2). MMPs are overexpressed in a wide range of metastatic tumors, 4,24,25 and are present in heart tissue after myocardial infarction. 26 MMP-2 has been used successfully as a target for a variety of drug delivery and imaging applications.…”

“…26 MMP-2 has been used successfully as a target for a variety of drug delivery and imaging applications. 4,7,24,25 Previously, a xenon sensor for MMP was developed with a peptide recognition sequence for MMP-7 covalently attached to CryA. 27 Upon cleavage by MMP-7, a change of approximately 0.5 ppm in the 129 Xe@CryA chemical shift was observed.…”

Disassembly-driven Turn-on Sensing of Enzyme Activity by Substrate-based Fluorescent Nanoprobe

“…Our switching strategy for an turn-on response (based on self-assembly and enzymatic reaction-driven disassembly of the nanoprobe) could be applied not only in test tube but also in conditioned media of tumor cells containing a high concentration of various proteins and metabolites. 16 The probe design is modular and thus applicable for specific detection of various proteins, including enzymes and nonenzymatic proteins. 4 We believe that the present progress in supramolecular approach may facilitate the development of protein or enzyme-specific switchable fluorescent probes that are useful for screening of cancer diagnosis, development of the inhibitors for enzyme and/or molecular imaging of the biomarkers in cells or in vivo.…”