Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

<div><p>Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker^® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.</p></div

Permeabilizing Groups are Required for Efficient Entry of Substrates into Cells.

<p>Healthy donor fibroblasts were incubated for 16 hours with 5(6)-(2-dimethylaminoethyl)carboxamido)-2’7’-dichlorofluorescein-3’,6’-di-O-β-D-glucopyranoside (3) (B,D) or 5(6)-(2-dimethylaminoethyl)carboxamido-2',7'-dichlorofluorescein-3’,6’-di-O-β-D-glucopyranoside octaacetate(4) (A,C), at 12.5uM (A,B) and 25uM (C,D). Prior to imaging staining media was removed and cells washed 3 times with PBS. Cells were then bathed in Opti-Klear^™ Live Cell Imaging Buffer containing 10ug/ml Hoechst 33342 and images were captured using an AMG EVOS Auto FL with a 40X objective and TRANS, GFP and DAPI lightcubes. Only the targeted substrate with the peracetate permeabilizing groups exhibited significant staining.</p

Localization of Targeted Lysosomal Substrate and anti-LAMP1 Antibody.

<p>Colocalization of the targeted lysosomal substrate <b>(5)</b> with anti-LAMP1 antibody staining was measured in AG06173 live human fibroblasts by incubation with 5uM 5(6)-(2-dimethylaminoethyl)carboxamido-2',7'-dichlorofluorescein diacetate (<b>5</b>) for 1 hour, followed by fixing the cells, permeabilization and staining with a primary rabbit anti-LAMP1 pAb antibody at 1.6ug/mL and incubation at room temperature for 1 hour. After washing, a secondary Alexa Fluor^® 555 conjugated Goat Anti-Rabbit antibody was added and incubated for 1 hour at room temperature. Cells were washed 3 times in PBS containing Hoechst 33342 added to the second wash and imaged. Fluorescence staining of nuclei (blue) and lysosomes with both anti-LAMP1 (red) and <b>(5)</b> (green) was captured using fluorescence microscopy. Merging of green and red images demonstrated >99% degree of colocalization between the two stains.</p

Inhibition of Lysosomal Metabolism by Chloroquine Leads to Reduction in Lysosomal Staining.

<p>Healthy donor fibroblasts were incubated for 16 hours with or without 300uM chloroquine at 37°C/5% CO₂. The media was removed and replaced with serum free media containing 10uM 5(6)-dimethylaminoethylcarboxamido-2',7'-dichlorofluorescein diacetate (5) and cells were incubated for 2 hours. Staining media was removed and cells were washed 3 times with PBS prior to the addition of Opti-Klear^™ Live Cell Imaging Buffer. Images were then captured on a Zeiss Axio Observer A1 inverted microscope fitted with a 40X lens and FITC filter set. After treatment with chloroquine, staining in lysosomes is significantly reduced (right panel).</p

Analysis of Lysosomal Enzyme Activity Using Alternative Fluorescent Substrates.

<p>AG06173 human fibroblast cells were stained with 10 uM of an esterase substrate with an alternative morpholino based targeting group (6) (C), 10uM of the resorufin based β-glucosidase substrate (9) (B), or 10uM of the resorufin based esterase substrate (10) (A) followed by incubation at 37°C in 5% CO₂ atmosphere for 16 hours. The cells were then washed with pre-warmed PBS followed by addition of Opti-Klear^™ Live Cell Imaging Buffer containing 10ug/ul Hoechst 33342). Cells were imaged using an AMG EVOS Auto FL Fluorescence Microscope equipped with appropriate lightcubes and 40X air objective.</p

Inhibition of Lysosomal Glucocerebrosidase by CBE Leads to Reduction in Lysosomal Staining.

<p>Healthy donor fibroblasts AG06173 were incubated for 16 hours with 0(A), 5(B), 10(C) and 50uM (D) of Conduritol β-Epoxide (CBE), then treated with 5uM 5(6)-(2-dimethylaminoethyl)carboxamido-2',7'-dichlorofluorescein-3’,6’-di-O-β-D-glucopyranoside, octaacetate (4) at 37°C/5% CO₂ for 16 hours. The media containing the β-glucosidase substrate and inhibitor was removed and the cells washed 3 times with PBS prior to the addition of Opti-Klear^™ Live Cell Imaging Buffer containing 10ug/ml Hoechst 33342. Images were captured using an AMG EVOS Auto FL with a 40X objective and GFP and DAPI lightcubes. Intensity of green fluorescent signal was quantified using Cell Profiler (E). As the inhibitor concentration increases, a relativel reduction in staining is observed.</p

Localization of Targeted Lysosomal Substrate and LysoTracker^® Red.

<p>Colocalization (C) of the targeted lysosomal substrate <b>(4)</b> (B) and LysoTracker^® Red (A) was measured by incubation with 5uM <b>(4)</b> for 16 hours followed by incubation with 75nM LysoTracker^® Red DND for 30minutes. Cells were washed with PBS and bathed in Opti-Klear^™ Live Cell Imaging buffer containing 10ug/mL Hoechst 33342 prior to imaging using an AMG EVOS Auto FL with appropriate lightcubes. Fluorescence analysis of LysoTracker^® Red (red) and <b>(4)</b> (green) exhibited a high degree of co-localization between the two stains in nearly all cases, with LysoTracker^® occasionally staining some additional structures indicated by arrows in panel D.</p

Lysosomal beta-Glucocerebrosidase Activity Measured by Multiple Methods.

<p>Glucocerebrosidase activity in healthy (GM14643) and two Gaucher I cell lines (GM10870,GM10874 containing the N370S mutation) was measured by staining with (<b>4</b>) and image analysis by flow cytometry as well as by using a standard 4-methylumbelliferyl β-D-glucopyranoside (MUG) lysis assay. Enzyme activity measurements are shown to be consistent when measured using (<b>4</b>) in two different formats when compared to the standard diagnostic assay using MUG.</p

Lysosomal Galactosylceramide Activity Measured by Multiple Methods.

<p>Galactosylceramide activity in Normal and Krabbe cells was measured using the standard MUG assay and using staining with (<b>2</b>) in both imaging and flow cytometry formats.</p

Image Analysis of Stained Cells using Cell Profiler.

<p>AG06173 human fibroblast cells were stained with the targeted lysosomal substrate <b>(4)</b> (green) and Hoechst 33342 nuclear stain (blue). Captured images (A) were then quantified using Cell Profiler image analysis. Objects in the blue and green channel images were identified using Global Otsu Two-Class thresholding method (B,C) and the intensity of the pixels contained within those objects measured for the green channel and the number of objects measured for the blue channel. The average green intensity per cell was calculated for each image and the average over all images. The intensity of the selected objects was measured in relative intensity units (RIU) and is represented as a heat map (D).</p