rhgba1 cerezyme  (Sanquin)

Journal: Chemical Science

Article Title: Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases †

doi: 10.1039/d3sc01021j

Figure Lengend Snippet: Fluorescence polarisation activity-based protein profiling (FluoPol-ABPP) screening of our 358-compound iminosugar library on rhGBA1 (Cerezyme®) and rhGAA (Myozyme®) subject of the here presented studies, and structures of the TAMRA-ABPs I and II used in the FluoPol-ABPP screenings.

Article Snippet: Recombinant human rhGBA1 (Cerezyme® from Genzyme) was used during FluoPol-ABPP assays.

Techniques: Fluorescence, Activity Assay

Journal: Chemical Science

Article Title: Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases †

doi: 10.1039/d3sc01021j

Figure Lengend Snippet: (a) Synthesis of GBA1 ABP I. Reagents and conditions: (i) mixture of 5′-and 6′-TAMRA-alkyne (2), sodium ascorbate, CuSO 4 , t BuOH/toluene/H 2 O (1 : 1 : 1 v : v : v), 18 h at ambient temperature, 20% yield. (b) Chemical structure of ABP III. (c) Competitive inhibitors used for validation of the FluoPol ABPP assay on GBA1. Apparent inhibitory potency (IC 50 ) values given are derived from competitive ABPP on rhGBA1 using ABP I as the readout. (d) In-gel fluorescence reveals GBA1 selectivity of ABP I compared to broad-spectrum retaining β-glucosidase ABP III in a 1 : 1 mixture of HEK293T GBA2 overexpressing and HEK293T GBA3 overexpressing cell lysates (labeling with 500 nM probe). (e) and (f) Optimization of ABP I concentration (e) and pH (f). (g) Inhibition curves of 3 (blue line), 4 (green line) and 5 (pink line) as determined by FluoPol-ABPP.

Article Snippet: Recombinant human rhGBA1 (Cerezyme® from Genzyme) was used during FluoPol-ABPP assays.

Techniques: Biomarker Discovery, Derivative Assay, Fluorescence, Labeling, Concentration Assay, Inhibition

Journal: Chemical Science

Article Title: Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases †

doi: 10.1039/d3sc01021j

Figure Lengend Snippet: Screening of our in-house iminosugar library containing 358 entries at a concentration of 5 μM in the FluoPol-ABPP assay on rhGBA1. 38 Compounds showed more than 50% reduction in ABP I FP-signal. Representative inhibitors include compounds 6, 7 and 8.

Article Snippet: Recombinant human rhGBA1 (Cerezyme® from Genzyme) was used during FluoPol-ABPP assays.

Techniques: Concentration Assay

Journal: Chemical Science

Article Title: Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases †

doi: 10.1039/d3sc01021j

Figure Lengend Snippet: IC 50 values (μM) for in vitro inhibition of human recombinant lysosomal α-glucosidase GAA (Myozyme), ER α-glucosidase GANAB (from Pompe disease Fibroblast lysates), β-glucosidases rhGBA1 (Cerezyme) and GBA2 (GBA2-overexpressing HEK293T lysate), and in situ cell inhibition of glucosylceramide synthase (GCS) (RAW 264.7 cells). Reported values are mean ± standard deviation from 3 technical replicates (Fig. S8)

Article Snippet: Recombinant human rhGBA1 (Cerezyme® from Genzyme) was used during FluoPol-ABPP assays.

Techniques: In Vitro, Inhibition, Recombinant, In Situ, Standard Deviation