human full-length wild-type gcase protein (cerezyme)  (Genzyme)

Journal: Scientific Reports

Article Title: A novel allosteric GCase modulator prevents Tau accumulation in GBA1 WT and GBA1 L444P/L444P cellular models

doi: 10.1038/s41598-025-02346-8

Figure Lengend Snippet: ( A ) Molecular structure of the GCase pharmacological chaperone GT-02216. ( B ) SPR dose-response for GT-02216 binding to immobilized human GCase protein monitored at acidic (pH 5.0) and neutral (pH 7.4) conditions. ( C ) SPR binding properties determined at the indicated pH values.

Article Snippet: Human full-length wild-type GCase protein (Cerezyme, Genzyme, Naarden, NL) was immobilized on the SPR CM5 sensor (GE Healthcare, #29149603,) by standard amino coupling using relatively high protein concentration of 100 μg/mL.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: A novel allosteric GCase modulator prevents Tau accumulation in GBA1 WT and GBA1 L444P/L444P cellular models

doi: 10.1038/s41598-025-02346-8

Figure Lengend Snippet: ( A ) Basal GCase activity in wild-type and mutant GBA1 fibroblasts (mean ± SD normalized on GBA1 WT(XY) , n = 6–48). Ordinary 1way ANOVA ( p < .0001) and Dunnet’s multiple tests against GBA1 WT(XY) , **** p < .0001. ( B ) Effect of 4-days treatment with 12.5 µmol/L GT-02216 on GCase activity on fibroblast lines (mean ± SD normalized on the respective basal activity, n = 5–42). Ordinary 1way ANOVA ( p < .0001) and Šidák’s multiple tests against the respective basal activity, **** p < .0001. (C) GT-02216 dose-response on GBA1 L444P/L444P(I) α fibroblasts (mean ± sem normalized on vehicle, n = 14) or (D) on GBA1 WT (XY) fibroblasts (mean ± sem normalized on vehicle, n = 8) treated for 4 days. Non-linear fit with four parameters log(agonist); EC 50 = 2.4 µmol/L on GBA1 L444P/L444P(I) α (1.3–10.5 µmol/L 95% confidentiality interval, top 1.8–2.4 µmol/L, R 2 = 0.71) and 1.5 µmol/L on GBA1 WT(XY) (1.3–1.8 µmol/L 95% confidentiality interval, top 1.9-2.0 µmol/L, R 2 = 0.95).

Article Snippet: Human full-length wild-type GCase protein (Cerezyme, Genzyme, Naarden, NL) was immobilized on the SPR CM5 sensor (GE Healthcare, #29149603,) by standard amino coupling using relatively high protein concentration of 100 μg/mL.

Techniques: Activity Assay, Mutagenesis

Journal: Scientific Reports

Article Title: A novel allosteric GCase modulator prevents Tau accumulation in GBA1 WT and GBA1 L444P/L444P cellular models

doi: 10.1038/s41598-025-02346-8

Figure Lengend Snippet: ( A ) GCase activity in doxycycline-inducible Tau-mCherry human fibroblasts with the genotype GBA WT(XX) or GBA1 L444P/L444P(I) α (mean ± SD normalized on GBA WT(XX) , n = 7). Mann-Whitney test, *** p 0.0006. ( B ) GT-02216 dose-response on Tau- GBA1 L444P/L444P(I) α fibroblasts (mean ± sem normalized on vehicle, n = 8) treated for 4 days. Non-linear fit with four parameters log(agonist); EC 50 = 1.1 µmol/L (0.7–1.6 µmol/L 95% confidentiality interval, top 1.5–1.6 µmol/L, R 2 = 0.79). ( C ) Basal and GT-02166-rescued GCase activity is not affected by the induction of Tau-mCherry expression with doxycycline in Tau-GBA1 WT(XX) fibroblasts (mean ± SD normalized on basal ctrl, n = 3). 2way ANOVA ( p < .0001 for treatment, ns for Tau expression) and Šidák’s multiple tests against the respective ctrl, **** p < .0001. ( D ) GT-02216 dose-response on Tau-GBA1 WT(XX) fibroblasts (mean ± sem normalized on vehicle, n = 8) treated for 4 days. Non-linear fit with four parameters log(agonist); EC 50 = 1.0 µmol/L (0.7–2.3 µmol/L 95% confidentiality interval, top 1.4–1.6 µmol/L, R 2 = 0.78).

Article Snippet: Human full-length wild-type GCase protein (Cerezyme, Genzyme, Naarden, NL) was immobilized on the SPR CM5 sensor (GE Healthcare, #29149603,) by standard amino coupling using relatively high protein concentration of 100 μg/mL.

Techniques: Activity Assay, MANN-WHITNEY, Expressing

Journal: Scientific Reports

Article Title: A novel allosteric GCase modulator prevents Tau accumulation in GBA1 WT and GBA1 L444P/L444P cellular models

doi: 10.1038/s41598-025-02346-8

Figure Lengend Snippet: ( A ) Quantification of Tau Puncta (TP) in Tau-GBA1 WT(XX) and Tau-GBA1 L444P/L444P(I) α fibroblasts at basal conditions (mean ± sem normalized on Tau-GBA WT(XX) , n = 55–60). Mann-Whitney test, *** p 0.0004. ( B ) Quantification of Tau puncta in Tau-GBA1 L444P/L444P(I) α fibroblasts treated overnight in the absence (ctrl) or presence (seeds) of Alzheimer’s brain-derived Tau seeds (mean ± sem normalized on ctrl, n = 45). Mann-Whitney test, **** p 4 × 10 − 11 . A representative image of ctrl or Tau seeds conditions are shown (Tau in magenta, nuclei stained with DAPI in blue). ( C ) GCase activity in Tau-GBA1 L444P/L444P(I) α fibroblasts treated overnight in the absence or presence of Tau seeds (mean ± sem normalized on ctrl, n = 3). Mann-Whitney test, not significant. ( D ) as in B. for Tau-GBA1 WT(XX) fibroblasts ( n = 45). Mann-Whitney test, **** p 0.00001. ( E ) as in C. for Tau-GBA1 WT(XX) fibroblasts ( n = 6–9). Mann-Whitney test, not significant. ( F ) Dose-dependent reduction of Tau Puncta in Tau- GBA1 L444P/L444P(I) α fibroblasts or ( G ) in GBA1 WT(XX) fibroblasts treated for 4 days with the indicated amount of GT-02216 (mean ± sem normalized on vehicle, no seed ctrl, n = 15–45). 2way ANOVA ( p < .0001 for GT-02216 concentration and seeds) and Šídák’s multiple tests against vehicle, **** p < .0001.

Article Snippet: Human full-length wild-type GCase protein (Cerezyme, Genzyme, Naarden, NL) was immobilized on the SPR CM5 sensor (GE Healthcare, #29149603,) by standard amino coupling using relatively high protein concentration of 100 μg/mL.

Techniques: MANN-WHITNEY, Derivative Assay, Staining, Activity Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Developing Allosteric Chaperones for GBA1 -Associated Disorders—An Integrated Computational and Experimental Approach

doi: 10.3390/ijms26010009

Figure Lengend Snippet: The SEE-Tx ® approach to discover non-competitive, pharmacological allosteric regulators for the GCase protein: ( A ) Schematic workflow of the procedure used to discover new allosteric regulators; ( B ) Docking-based high-throughput virtual screening of small molecules. Abbreviation: GCase; Glucocerebrosidase; VS, virtual screening; DSF, differential scanning fluorometry.

Article Snippet: The human full-length GCase protein (Cerezyme, wild type, not tagged; Genzyme, Naarden, The Netherlands) was immobilized on an SPR CM5 sensor (29149603, GE Healthcare, Chicago, IL, USA) using standard amino coupling with a high protein concentration of 100 µg/mL.

Techniques: High Throughput Screening Assay

Journal: International Journal of Molecular Sciences

Article Title: Developing Allosteric Chaperones for GBA1 -Associated Disorders—An Integrated Computational and Experimental Approach

doi: 10.3390/ijms26010009

Figure Lengend Snippet: Binding of small molecule hit compounds to GCase, as determined by DSF: ( A ) Difference in melting temperature (ΔTm) relative to recombinant human GCase in the presence of hit #1 to #28 at 30 μM and pH 7.2. The mean ΔTm values ± SD are from 2 independent experiments ( n = 2). The dotted line shows the threshold value for the DSF screening, which was ΔTm ≥ 0.5 °C; ( B ) Compound 1 (hit #22) structure; and ( C ) Dose-dependent effect on the thermal stability of GCase in the presence of hit #22 (compound 1 ). The mean ΔTm values ± SD are from 2 independent experiments ( n = 2). DSF, differential scanning fluorimetry; GCase; glucocerebrosidase; SD, standard deviation; Tm, melting temperature.

Article Snippet: The human full-length GCase protein (Cerezyme, wild type, not tagged; Genzyme, Naarden, The Netherlands) was immobilized on an SPR CM5 sensor (29149603, GE Healthcare, Chicago, IL, USA) using standard amino coupling with a high protein concentration of 100 µg/mL.

Techniques: Binding Assay, Recombinant, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Developing Allosteric Chaperones for GBA1 -Associated Disorders—An Integrated Computational and Experimental Approach

doi: 10.3390/ijms26010009

Figure Lengend Snippet: Validation of STAR compounds: ( A ) Structures of compound 2 ; ( B ) structure of compound 3 ; and measurement of GCase activity in WT fibroblasts treated for four days with ( C ) compound 2 and ( D ) compound 3 ; and GBA1 -associated patient-derived fibroblasts treated for four days with compound 2 and 3 , respectively: ( E , F ) p.L444P/p.L444P; ( G , H ) p.N370S/84gg; ( I , J ) p.N188S/p.S107L; and ( K , L ) p.L444P/p. WT. Mean values from at least three replicates of three independent experiments are represented by bars. Results are normalized to the untreated and presented as mean ± SD values of three experiments after one-way ANOVA with Dunnett’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s.—no significant; WT, wild-type.

Article Snippet: The human full-length GCase protein (Cerezyme, wild type, not tagged; Genzyme, Naarden, The Netherlands) was immobilized on an SPR CM5 sensor (29149603, GE Healthcare, Chicago, IL, USA) using standard amino coupling with a high protein concentration of 100 µg/mL.

Techniques: Activity Assay, Derivative Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Developing Allosteric Chaperones for GBA1 -Associated Disorders—An Integrated Computational and Experimental Approach

doi: 10.3390/ijms26010009

Figure Lengend Snippet: Dose-response for: ( A ) compound 2 and ( B ) compound 3 binding to immobilized human recombinant GCase monitored at neutral pH (7.4) by SPR; and GCase activity assay in wild-type lysates after treatment with ( C ) compound 2 , ( D ) compound 3, and ( E ) isofagomine (IFG) at acidic pH (5.6). Dose-response curves are plotted with mean values based on two independent assays with three replicates each. Error bars represent the standard deviation of the means.

Article Snippet: The human full-length GCase protein (Cerezyme, wild type, not tagged; Genzyme, Naarden, The Netherlands) was immobilized on an SPR CM5 sensor (29149603, GE Healthcare, Chicago, IL, USA) using standard amino coupling with a high protein concentration of 100 µg/mL.

Techniques: Binding Assay, Recombinant, Activity Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Developing Allosteric Chaperones for GBA1 -Associated Disorders—An Integrated Computational and Experimental Approach

doi: 10.3390/ijms26010009

Figure Lengend Snippet: Measurement of GCase activity in WT cell lines and fibroblasts derived from GD patients treated for four days. Mean values were derived from ( A ) one independent experiment with two replicates for compound 2 and ( B ) three independent experiments with two replicates each for compound 3 . HexCer substrate quantification to evaluate the substrate depletion in GD patient-derived fibroblasts (L444P/L444P) by LC/MS-MS for ( C ) compound 2 and ( D ) compound 3 . Results are presented as mean values from three replicates of two independent experiments after one-way ANOVA with Dunnett’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, no significant; WT, wild-type.

Article Snippet: The human full-length GCase protein (Cerezyme, wild type, not tagged; Genzyme, Naarden, The Netherlands) was immobilized on an SPR CM5 sensor (29149603, GE Healthcare, Chicago, IL, USA) using standard amino coupling with a high protein concentration of 100 µg/mL.

Techniques: Activity Assay, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Developing Allosteric Chaperones for GBA1 -Associated Disorders—An Integrated Computational and Experimental Approach

doi: 10.3390/ijms26010009

Figure Lengend Snippet: ( A ) Basal GCase activity of WT, p.N370S and p.L444P GBA1 mutant BE(2)M17 cell lines, results are presented as mean ± SD values after two-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.001, **** p < 0.0001; ( B ) GCase activity assay of differentiated WT, N370S and L444P neuronal cell lines treated for four days with 25 μM compound 3 . Activity is expressed as fold activity versus vehicle in each cell line (dashed line); ( C ) Representative images of GCase immunodetection by western blot and ( D ) quantification of GCase protein levels (GCase protein levels in vehicle-treated cells are represented as a dashed line) of the three differentiated neuronal cell lines (WT, N370S, and L444P) treated with 25 µM compound 3 for ten days; ( E ) GCase activity assay of three differentiated neuronal cell lines (WT, N370S and L444P) treated for 10 days with 25 μM of the selected compound 3 . Activity is expressed as fold activity versus vehicle in each cell line (dashed line); ( F ) Quantification of GlcSph levels, the substrate of GCase, following treatment of 10 days with compound 3 at 25 µM. Lipid levels are expressed in pmol/mg of tissue; and ( G ) Viability assays for the three differentiated neuronal cell lines treated for 10 with compound 3 at 25 µM. Viability is expressed as a fold percentage of live cells versus vehicles in each cell line. Results in panel ( B , D – G ) are presented as mean ± standard deviation values; significance is shown within each cell line compared to their vehicle after two-way ANOVA followed by Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. GCase, glucocerebrosidase; GlcSph, glucosylsphingosine; WT, wild-type.

Article Snippet: The human full-length GCase protein (Cerezyme, wild type, not tagged; Genzyme, Naarden, The Netherlands) was immobilized on an SPR CM5 sensor (29149603, GE Healthcare, Chicago, IL, USA) using standard amino coupling with a high protein concentration of 100 µg/mL.

Techniques: Activity Assay, Mutagenesis, Immunodetection, Western Blot, Standard Deviation