Tool Compounds Robustly Increase Turnover of an Artificial Substrate by Glucocerebrosidase in Human Brain Lysates

<div><p>Mutations in glucocerebrosidase (GBA1) cause Gaucher disease and also represent a common risk factor for Parkinson’s disease and Dementia with Lewy bodies. Recently, new tool molecules were described which can increase turnover of an artificial substrate 4MUG when incubated with mutant N370S GBA1 from human spleen. Here we show that these compounds exert a similar effect on the wild-type enzyme in a cell-free system. In addition, these tool compounds robustly increase turnover of 4MUG by GBA1 derived from human cortex, despite substantially lower glycosylation of GBA1 in human brain, suggesting that the degree of glycosylation is not important for compound binding. Surprisingly, these tool compounds failed to robustly alter GBA1 turnover of 4MUG in the mouse brain homogenate. Our data raise the possibility that <i>in vivo</i> models with humanized glucocerebrosidase may be needed for efficacy assessments of such small molecules.</p></div

CBE and isofagomine effectively inhibit purified GBA1 at 1 mM and 1 μM, respectively.

<p>A) Dose response of GBA1 inhibition in a cell free system by CBE and isofagomine. Similar dose response curves are seen with and without sodium taurocholate (NaTc). A representative experiment is shown. B) IC₅₀ values for isofagomine and CBE and % inhibition at 1 mM CBE and 1 μM isofagomine, respectively (mean ± SEM). Summary of data from three independent experiments is shown. C) CBE and isofagomine are not potent inhibitors of recombinant GBA3. A representative experiment is shown. D) CBE (1 mM) and isofagomine (1 μM) effectively inhibits GBA1 but not GBA3, irrespective of whether sodium taurocholate is present. The graph shows a summary of three independent experiments. E) Addition of sodium taurocholate (NaTc) increases activity of GBA1 ~ 5-fold but decreases activity of GBA3 by ~ 50%. Graph depicts a summary of three independent experiments.</p

Tool compounds increase activity of human brain-derived glucocerebrosidase.

<p>A) Compounds 40 and 43 increase GBA1 activity in lysates from human cortex. In the graphs depicting % signal, vehicle treated samples were set as 100%. Representative experiments are shown. B) Table depicting average EC₅₀ values and % activation from three independent experiments. Compound 43 activates GBA1 to a greater extent than compound 40 (90% vs 55%, respectively), based on activation at 31.6 μM, the highest concentration, at which both compounds were soluble.</p

Tool compounds increase activity of human GBA1 when added into mouse brain lysates.

<p>A) Compounds 40 & 43 robustly increase activity of human purified GBA1 when added to mouse brain lysates (Mouse+rhGBA) while they do not substantially affect mouse GBA1 (Mouse), as measured by turnover of 4MUG substrate. Vehicle treated samples were set as 100%. A representative experiment is shown. B) Greater activation of human recombinant protein in mouse lysates is seen without sodium taurocholate. Activity of human recombinant protein in mouse lysates was obtained by subtracting values from a parallel experiment with mouse brain lysates but without added human recombinant protein. C) Table depicting EC₅₀ values and % signal for purified human protein added to mouse brain lysates (three independent experiments). Mean ± SEM is shown.</p

Tool compounds do not robustly increase activity of mouse brain derived GBA1.

<p>A) Compounds 40 and 43 do not robustly increase GBA1 activity in lysates from mouse brain. Subtle increases in activity were seen with sodium taurocholate (+NaTc) whereas subtle inhibition was observed without sodium taurocholate (-NaTc) upon compound addition. The inhibition was not dose dependent. Vehicle treated samples were set as 100% in the graph depicting % signal. Representative experiments are shown. B) Table depicting average EC₅₀ values and % activation at the highest soluble concentration (31.6 μM) from three independent experiments.</p

Turnover of 4MUG substrate detects GBA1 activity in mouse brain lysates in the presence of sodium taurocholate.

<p>A) Increasing amount of protein lysates in the activity assay leads to increased signal (RFU—relative fluorescence unit). When converted to specific activity, similar values are obtained when using 10–60 μg of mouse brain lysates, as expected. A representative experiment is shown. B) Turnover of 4MUG substrate in lysates from mouse brain is mostly due to GBA1 activity when using sodium taurocholate (NaTc). 1 mM CBE and 1 μM isofagomine (Iso) decrease the signal by ~ 80% in the presence of sodium taurocholate (+ NaTc) whereas substantially smaller reduction is seen without sodium taurocholate (- NaTc) in the assay buffer. 10 μg of protein lysate was used in the reaction. A representative experiment is shown in the graphs, a summary of three experiments is depicted in the table (mean ± SEM). In the graph, control for each condition (with and without NaTc) was set as 100%.</p

Turnover of 4MUG substrate reads out GBA1 activity in human brain lysates in the presence of sodium taurocholate.

<p>A) Glucocerebrosidase in human brain migrates at lower molecular weight compared to purified protein or GBA from Hela cells, likely due to lower glycosylation. This difference in migration is removed upon deglycosylation with PNGase. Asterisks denote migration of glycosylated glucocerebrosidase (in Hela cells and brain), arrows point to deglycosylated glucocerebrosidase protein. GBA KD – glucocerebrosidase knock-down with shRNA. B) Increasing amount of protein lysates in the activity assay leads to increased signal (RFU — relative fluorescence unit). When converted to specific activity, similar values are obtained when using 10–60 μg of human cortex lysates, as expected. A representative experiment is shown. C) Turnover of 4MUG substrate in lysates from human cortex is mostly due to GBA1 activity when using sodium taurocholate (NaTc). 1 mM CBE and 1 μM isofagomine (Iso) decrease the signal by ~ 90% in the presence of sodium taurocholate whereas substantially smaller reduction (~ 30%) is seen without sodium taurocholate in the assay buffer. 10 μg of protein lysate was used. A representative experiment is shown in the graphs, a summary of three experiments is depicted in the table (mean ± SEM). In the graph, control for each condition (with and without sodium taurocholate, NaTc) was set as 100%.</p

Norsk

<p>(A) Rat muscle extracts were separated by SDS PAGE and probed by immunoblotting as described in the Methods. The analysis was limited to 15 animals (n = 8 biome enriched; lanes E1 through E8, and n = 7 biome depleted; lanes D1 through D7) due to size constraints of the gel. A control strip with no serum is labeled “C”, and indicates reactivity of the anti-IgM conjugate with muscle-derived antigens. (B) The number of bands recognized by natural IgM in individual sera (p = 0.0089) and the total reactivity of natural IgM from each serum sample (p = 0.0093) are shown, with the bars indicating the mean and standard error. (C) The distribution of bands as a function of band size is shown. For this analysis, the average number of bands in biome depleted and biome enriched rats (Y-axis) was plotted on linear and log scales (main figure and figure inset, respectively) versus different band sizes (X-axis).</p

Relative concentration of DNP-specific antibody in the serum of biome depleted (n = 20) and biome enriched (n = 15) rats.

<p>The relative concentration of antibody was determined by ELISA as described in the Methods. Relative levels of (A) IgM, (B) IgG, and (C) subclasses of IgG are shown. The means and standard errors are shown. The <i>p</i>-values associated with comparing data from biome depleted and biome enriched animals using a t-test are shown. (NS = not significant)</p

Natural anti-human serum albumin antibody levels in the serum of biome depleted (n = 20) and biome enriched (n = 15) rats.

<p>The relative concentration of antibody was determined by ELISA as described in the Methods. Relative levels of (A) IgM and (B) IgG are shown. Binding to human serum albumin (HSA) was used as a measure of reactivity toward a xenogeneic antigen for which the animals lacked previous exposure. The means, standard errors, and the <i>p</i>-values associated with comparing data from biome depleted and biome enriched animals using a t-test are shown. (NS = not significant)</p