Abstract 2621: Development of Fluoro-UK-101, an immunoproteasome-targeting activity-based fluorescent probe

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: This study was conducted to develop the first activity-based fluorescent probe targeting the immunoproteasome subunit LMP2. The immunoproteasome is an alternate form of the constitutive proteasome which is composed of unique catalytic subunits. While immunoproteasomes were believed to primarily function in generating peptides for antigen presentation on MHC class I molecules, antibody-based studies have revealed the expression of immunoproteasome catalytic subunits in a variety of cancers. However, antibodies are incapable of distinguishing between active and inactive forms of these proteins, a limitation that can be overcome using activity-based probes. Experimental Procedures: Fluorescent compounds were synthesized by derivatization of the LMP2-specific inhibitor UK-101 at the P2 position, where lysine was substituted for alanine. The free amino group on the side chain of lysine was coupled to linkers of various lengths. Fluorescein was then coupled to each linker to yield the fluorescent probes. These compounds were characterized in prostate cancer cell lines via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescence (IF) microscopy. One analog was selected for further characterization. Prostate cancer cells were treated with an LMP2-selective dose of this analog, Fluoro-UK-101, and the co-localization of this fluorescent probe with antibodies specific for proteasome subunits and intracellular organelles was examined via IF. Data Summary: The structures of the synthesized fluorescent derivatives of UK-101 were confirmed via mass spectrometry and nuclear magnetic resonance spectroscopy. Treatment of prostate cancer cells with these compounds gave punctate staining in a dose-dependent manner, indicating the cell permeability and activity of these probes. The impact of the linker length on LMP2 binding specificity and fluorescent signal strength was determined via SDS-PAGE and IF microscopy. Doses at which these fluorescent analogs specifically target LMP2, but not other proteasome subunits, were identified via SDS-PAGE. One analog, Fluoro-UK-101, was selected based on binding specificity and IF intensity. Additionally, IF microscopy techniques using the selected fluorescent analog, Fluoro-UK-101, revealed differences in LMP2 localization patterns among prostate cancer cell lines. Conclusions: We have successfully developed Fluoro-UK-101, an activity-based fluorescent probe that selectively binds the immunoproteasome catalytic subunit LMP2. Fluoro-UK-101 allows the visualization of catalytically active immunoproteasomes in intact cells. Modification of the fluorophore will facilitate its application in a rapid readout assay to detect tumors in animal models using LMP2 as a biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2621. doi:10.1158/1538-7445.AM2011-2621