Concentration responses of three GCase inhibitors determined using recombinant WT GCase, recombinant N370S mutant enzyme, WT GCase from normal spleen extract and N370S mutant enzyme from a spleen of a patient with GD (genotype N370S/N370S).

<p>The three inhibitors were identified from the previous HTS using recombinant WT GCase.</p

pH profiles of recombinant N370S and WT GCase.

<p>The assay was performed in the presence of 10 mM NaTC. While the WT enzyme has a pH optimum of 5.9, the N370S enzyme has a broad optimum of around pH 7.2.</p

Classification of active compounds identified from the screen assay using the N370S mutant enzyme in spleen tissue.

<p>*Criteria for classification of compounds are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029861#pone.0029861.s003" target="_blank">table S2</a>.</p

Characterization of spleen GCase activity.

<p>A. pH optimum, B. NaTC dependency, C. Inhibition of spleen GCase activity by CBE.</p

Compounds discovered in the spleen qHTS tested for modulation of GCase activity and GCase translocation in cell assays.

<p>*As measured on spleen enzyme preparation.</p><p>**Translocation experiments performed on WT, N370S/N370S, L444P/L444P and L444P/Rec<i>Nci1</i> fibroblasts.</p

Effect of adding the spleen preparation on the activity of WT recombinant GCase.

<p>pH dependency and effect of sodium NaTC on the recombinant enzyme activity were assayed. A. activity of WT recombinant enzyme with and without NaTC. B and C. Different dilutions of N370S spleen in the presence (B) or absence (C) of NaTC. D. and E. WT recombinant enzyme mixed with increasing concentrations of spleen extract with (D) and without (E) NaTC.</p

Effect of SapC versus NaTC on the activity of WT (A) and N370S recombinant (B) GCase.

<p>In the presence of SapC, the activity of both WT (A), N370S (B) enzyme gave a sharp peak near pH 5, compared to the broader peak and higher pH seen using NaTC.</p

Comparison of the active compounds identified in the WT and N370S HTS experiments.

<p>A. Distribution of active compounds in the WT (blue) and N370S/N370S spleen (red) HTS experiments. B. Overlap between active compounds tested in both experiments by type of activity.</p

Dose response increase in lysosomal content of GCase in fibroblasts treated with selected identified compounds.

<p>Cells were treated with six drug concentrations (1 nM to 50 µM) for 5 days. The intensity of GCase staining in the lysosomes was measured using an automatic fluorescent microscope. The slope of dose dependent increase in fluorescence was plotted and statistically significant slopes are labeled with (*). Some compounds, including the activator NCGC00182510, were able to increase the lysosomal content of both N370S and L444P mutant enzymes. Some compounds were found to be toxic at the highest concentration and that point was removed from calculations.</p

Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids

<p>Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.</p