Allosteric Modulation of GCase Enhances Lysosomal Activity and Reduces ER Stress in GCase-Related Disorders

Abstract

Variants in the GBA1 gene, encoding the lysosomal enzyme glucosylceramidase beta 1 (GCase), are linked to Parkinson's disease (PD) and Gaucher disease (GD). Heterozygous variants increase PD risk, while homozygous variants lead to GD, a lysosomal storage disorder. Some GBA1 variants impair GCase maturation in the endoplasmic reticulum, blocking lysosomal transport and causing glucosylceramide accumulation, which disrupts lysosomal function. This study explores therapeutic strategies to address these dysfunctions. Using Site-directed Enzyme Enhancement Therapy (SEE-Tx^®), two structurally targeted allosteric regulators (STARs), GT-02287 and GT-02329, were developed and tested in GD patient-derived fibroblasts with relevant GCase variants. Treatment with GT-02287 and GT-02329 improved the folding of mutant GCase, protected the GCase_Leu444Pro variant from degradation, and facilitated the delivery of active GCase to lysosomes. This enhanced lysosomal function and reduced cellular stress. These findings validate the STARs' mechanism of action and highlight their therapeutic potential for GCase-related disorders, including GD, PD, and Dementia with Lewy Bodies.

Keywords: Gaucher disease; Parkinson’s disease; allosteric modulation; glucosylceramidase beta 1 (GCase); lysosomal dysfunction; lysosomal storage disorders; pharmacological chaperones; protein misfolding; site-directed enzyme enhancement therapy (SEE-Tx); structurally targeted allosteric regulators (STARs).

Figure A1

Structural localization of L444 and N370 amino acid residues in GCase. The molecular model shows the positions of L444 (orange sticks, Domain III) and N370 (yellow sticks, Domain II) relative to the enzyme’s active site (grey spheres, BTB substrate). Both residues are located distal to the catalytic pocket, supporting their involvement in allosteric regulation. Domain I (magenta), Domain II (blue), and Domain III (green) are depicted in ribbon representation. The structure was modeled using wild-type GCase (PDB 2V3F).

Figure A2

Surface plasmon resonance dose–response for GT compounds binding to immobilized recombinant human glucocerebrosidase (rhGCase) monitored at neutral (7.4) and acidic (5.0) pH with and without the presence of the inhibitory chaperone isofagomine (IFG).

Figure 1

Targeting GCase offers a promising approach for disease-modifying treatment in Parkinson’s Disease. Gain Therapeutic’s structurally targeted allosteric regulators (STARs) address GCase misfolding, enhancing its enzymatic activity and promoting its transport to lysosomes. By restoring GCase function, these regulators provide neuroprotective effects, improve lysosomal health, and reduce toxic substrate buildup, thereby mitigating neurodegenerative processes. GCase, glucocerebrosidase. For a detailed view of the L444P and N370S variant locations relative to the active site, see Appendix A Figure A1.

Figure 2

GCase binding confirmation: Surface plasmon resonance confirmed the direct binding of compounds GT-02287 and GT-02329 to recombinant human GCase protein (Cerezyme^®) at (a) pH 7.4 and (b) pH 5.0. All values are presented as the mean ± SD from duplicate measurements. GCase, glucocerebrosidase; SD, standard deviation.

Figure 3

Competitive inhibition assay with CBE in lysates. Wild-type fibroblast lysates were incubated with (a) CBE (black); (b) GT-02287 (green); or (c) GT-02329 (purple) for 15 min at the indicated concentrations. GCase activity was measured using the 4-methylumbelliferyl β-D-glucopyranoside substrate. In (b) and (c), where indicated, an additional preincubation with 40 µM CBE (grey) was performed to inhibit 50% of GCase activity. All values represent the mean ± SD from three wells per condition. CBE, conduritol-β-epoxide; GCase, glucosylceramidase beta 1; SD, standard deviation.

Figure 4

Cellular Gcase enhancing activity of GT-02287. Patient-derived fibroblasts (Coriell GM00372, Coriell GM08760, Telethon 20526, Telethon 21142, Telethon 20624, and Telethon 20843) were treated with GT-02287 (a–g) at the specified concentrations for 4 days. Gcase activity was measured using the fluorogenic substrate 4-methylumbelliferyl β-D-glucopyranoside. The dose–response effect is presented as the percentage increase in activity relative to untreated cells. Data represent the mean ± SD from at least three wells per condition. Gcase, glucocerebrosidase; SD, standard deviation.

Figure 5

Cellular GCase enhancing activity of GT-02329. Patient-derived fibroblasts (Coriell GM00372, Coriell GM08760, Telethon 20526, Telethon 21142, Telethon 20624, and Telethon 20843) were treated with GT-02329 (a–g) at the specified concentrations for 4 days. GCase activity was measured using the fluorogenic substrate 4-methylumbelliferyl β-D-glucopyranoside. The dose–response effect is presented as the percentage increase in activity relative to untreated cells. Data represent the mean ± SD from at least three wells per condition. GCase, glucocerebrosidase; SD, standard deviation.

Figure 6

GlcCer substrate depletion following treatment with GT-02287 and GT-02329. p.L444P/p.L444P fibroblasts (Coriell GM08760) were treated with (a) GT-02287 or (b) GT-02329 at the specified concentrations for 10 days. Data represent the mean ± SD from three wells per condition. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s Multiple Comparison Test. Asterisks indicate statistically significant differences compared to untreated GCase_Leu444Pro fibroblasts: *** p ≤ 0.001, **** p ≤ 0.0001. ANOVA, analysis of variance; GCase, glucocerebrosidase; GlcCer, glucosylceramide; SD, standard deviation.

Figure 7

Lysosomal delivery of wild-type and disease-linked GCase variants: (a) sequence of GCase showing the signal sequence (underlined) and the disease-causing variants Asn370Ser and Leu444Pro (bold italics); (b) lysosomal delivery of HA-tagged GCase variants. Representative CLSM images of MEF cells expressing GCase variants for 48 h and treated with 50 nM BafA1 for 17 h. Immunostaining was performed with anti-HA (red) and anti-LAMP1 (green) antibodies, with nuclei stained using DAPI (blue). Scale bars: 10 µm (merged images) and 1 µm (insets); (c) quantification of (b) showing the percentage of LAMP1-positive lysosomes containing GCase variants. Images were analyzed using LysoQuant. Data are presented as mean ± SEM from three independent experiments. Statistical analysis: One-way ANOVA with Dunnett’s multiple comparison test; non-significant (ns) p > 0.05, **** p < 0.0001; (d) same as (b) for HT GCase variants. MEF cells were treated with 50 nM BafA1 and 100 nM TMR for 17 h. GCase variants were visualized with TMR (red), and lysosomes were stained with anti-LAMP1 (green). Scale bars: 10 µm (merged images) and 1 µm (insets); (e) quantification of (d) showing the percentage of LAMP1-positive lysosomes containing the TMR signal. Images were analyzed using LysoQuant. Data are presented as mean ± SEM from two independent experiments; (f) co-immunoprecipitation of GCase-HA variants in HEK293 cells. WB analysis shows CNX (upper panel), BiP (middle panel), and GCase-HA (lower panel); (g) quantification of CNX association with the three GCase variants; (h) quantification of BiP association with the three GCase variants. Data in (g) and (h) are presented as mean from two independent experiments; (i) fluorescent gel showing the TMR signal of GCase-HT variants expressed in HEK293 cells for 4 days, with or without 50 nM BafA1 treatment for the last 17 h; and (j) quantification of (i). Data are presented as mean ± SEM from five independent experiments. Statistical analysis: One-way ANOVA with Dunnett’s multiple comparison test; * p < 0.05, **** p < 0.0001. Bafilomycin A1, BafA1; binding immunoglobulin protein, BiP; calnexin, CNX; confocal laser scanning microscopy, CLSM; DAPI, 4′,6-diamidino-2-phenylindole; EL, endolysosomes; FL, full-length; GCase, glucocerebrosidase; HT, HaloTag; HA, hemagglutinin tag; HEK293, human embryonic kidney 293 cells; LAMP1, lysosomal-associated membrane protein 1; MEF, mouse embryonic fibroblasts; NT, non-transfected; SEM, standard error of the mean; TMR, tetramethylrhodamine; WB, Western blot.

Figure 8

STAR compounds promote lysosomal delivery of the ER-retained variant GCase_Leu444Pro: (a) TMR fluorescence of GCase_Leu444Pro-HT was analyzed in HEK293 cells treated for 4 days with either DMSO or GT compounds (GT-02287 or GT-02329) at concentrations ranging from 3.1 µM to 25 µM. During the last 17 h of treatment, cells were incubated with 100 nM TMR. Control cells were treated with 50 nM BafA1 for 17 h (lane 2). After treatment, proteins were transferred onto a PVDF membrane and immunoblotted against GAPDH; (b) the Halo fragment was quantified, corrected for the GAPDH signal, and normalized to the DMSO control. The mean ± SEM is shown, with four independent experiments for 25 µM concentrations, two for 6.25 µM and 12.5 µM, and three for BafA1. Statistical analysis was performed using an unpaired t-test, with *** p < 0.001 and **** p < 0.0001. BafA1, bafilomycin A1; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK293, human embryonic kidney 293 cells; PVDF, polyvinylidene fluoride; SEM, standard error of the mean; TMR, tetramethylrhodamine.

Figure 9

STAR compounds promote CNX release and prevent proteasomal degradation of GCase_Leu444Pro: (a) co-immunoprecipitation of endogenous GCase_Leu444Pro (lower panel) with CNX (upper panel) was analyzed in patient fibroblasts (Coriell GM08760) treated for 4 days with DMSO or the compound GT-02329 at concentrations ranging from 3.1 µM to 25 µM. Protein content in the total cell extracts (TCEs) is shown. (*) indicates an unspecific band; (b) quantification of (a). The mean is shown, based on two independent experiments; (c) immunoisolated GCase_Leu444Pro from patient fibroblasts (Coriell GM10915) treated for 4 days with DMSO or compounds GT-02287; (d) quantification of (c). The mean ± SEM is shown, based on four independent experiments, using an unpaired t-test with ** p < 0.01 and **** p < 0.0001; (e) radiolabeled protein content in the TCE is shown; (f) radiolabeled GCase_Leu444Pro immunoisolated from patient fibroblasts (Coriell GM10915); (g) quantification of (f). The mean is shown, based on two independent experiments; (h) GCase_Leu444Pro levels in patient fibroblasts (Coriell GM08760) treated for 4 days with DMSO or compounds GT-02287 or GT-02329 and, for the last 3 h, with the proteasome inhibitor PS341. (*) indicates an unspecified band. CNX, calnexin; DMSO, dimethyl sulfoxide; TCE, total cell extracts; SEM, standard error of the mean; PS341, proteasome inhibitor (bortezomib).

Figure 10

STAR compounds alleviate UPR in p.N370S/ins and p.L444P/p.L444P patient fibroblasts: (a) BiP mRNA levels determined by RT-PCR in fibroblasts from healthy donors (lane 1) and patient-derived fibroblasts (lanes 2 and 3); (b) BiP mRNA levels monitored by qPCR in GCase_Leu444Pro patient fibroblasts (Coriell GM08760) treated for 4 days with DMSO or the STAR compound GT-02287 at concentrations ranging from 1.5 µM to 25 µM; (c) same as (b), but for patient fibroblasts treated with GT-02329; the mean ± SEM is shown, based on two independent experiments; (d) mRNA levels of selected UPR markers monitored by qPCR in GCase_Leu444Pro patient fibroblasts (Coriell GM10915) treated for 10 days with DMSO or 12.5 µM GT-02287; the mean ± SEM is shown, based on two independent experiments for GRP170 and CRELD2, three for the other UPR markers; (e) same as (d), but for cells treated with 6.25 µM GT-02329; the mean ± SEM is shown, based on three independent experiments for BiP, two for the other UPR markers; (f) BiP and GRP94 protein levels monitored by WB analysis in GCase_Leu444Pro patient fibroblasts (Coriell GM08760) treated for 4 days with 25 µM GT-02287 and 12.5 µM GT-02329; (g,h) quantification of (f). The mean ± SEM is shown based on four independent experiments for BiP 25 μM 2287, three for GRP94 25 μM 2287, and two for 12.5 μM 2329. UPR, unfolded protein response; BiP, binding immunoglobulin protein; mRNA, messenger ribonucleic acid; RT-PCR, reverse transcription polymerase chain reaction; qPCR, quantitative polymerase chain reaction; DMSO, dimethyl sulfoxide; STAR, structurally targeted allosteric regulators; GRP94, glucose-regulated protein 94; WB, Western blot; SEM, standard error of the mean.