recombinant human β-gal protein  (Lonza)

Journal: International Journal of Molecular Sciences

Article Title: Development of Small-Molecule Allosteric Modulators of Beta-Galactosidase (β-Gal) for the Treatment of GM1 Gangliosidosis and Morquio B

doi: 10.3390/ijms27083631

Figure Lengend Snippet: Magellan platform-driven virtual screening for non-competitive, allosteric pharmacological regulators of β-Gal. ( a ) Ribbon diagram of β-Gal monomer used for VS (PDB ID: 3THC), with domains individually colored: β-domain 1 (red), TIM barrel (blue), TIM-β1 loop (yellow), and β-domain 2 (green). ( b ) High-throughput, docking-based virtual screening workflow for identifying small molecules. β-Gal, β-galactosidase; DSF, differential scanning fluorimetry; PDB, Protein Data Bank; TIM, triosephosphate isomerase; VS, virtual screening.

Article Snippet: Each 25 μL reaction contained 12.5 μL of 1.5 μM recombinant human β-Gal protein (rhGLB1; Novoprotein, Shanghai, China) in PBS (pH 7.4), resulting in a final protein concentration of 1 μM, and 12.5 μL of compound solution dissolved in 100% DMSO and diluted in protein buffer to achieve a final DMSO concentration of 2%.

Techniques: High Throughput Screening Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of Small-Molecule Allosteric Modulators of Beta-Galactosidase (β-Gal) for the Treatment of GM1 Gangliosidosis and Morquio B

doi: 10.3390/ijms27083631

Figure Lengend Snippet: Binding of small-molecule hit compounds to β-Gal protein as measured by DSF. ( a ) Change in melting temperature (ΔTm) of recombinant human β-Gal in the presence of hit compounds #1–3 at 100 μM and #1–9 at 30 μM, measured at pH 7.4. Mean ΔTm ± SD values are shown. The dotted line indicates the DSF screening threshold (ΔTm ≥ 0.5 °C). ( b ) Dose-dependent effect of Hit 3 on β-Gal thermal stability, with mean ΔTm ± SD. ( c ) Chemical structure of Hit 3 and Hit 5. DSF, differential scanning fluorimetry; SD, standard deviation; Tm, melting temperature.

Article Snippet: Each 25 μL reaction contained 12.5 μL of 1.5 μM recombinant human β-Gal protein (rhGLB1; Novoprotein, Shanghai, China) in PBS (pH 7.4), resulting in a final protein concentration of 1 μM, and 12.5 μL of compound solution dissolved in 100% DMSO and diluted in protein buffer to achieve a final DMSO concentration of 2%.

Techniques: Binding Assay, Recombinant, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Development of Small-Molecule Allosteric Modulators of Beta-Galactosidase (β-Gal) for the Treatment of GM1 Gangliosidosis and Morquio B

doi: 10.3390/ijms27083631

Figure Lengend Snippet: ( a ) Hit 3 (left panel) and Hit 5 (right panel) reduction in GM1 ganglioside accumulation in canine fibroblasts. Representative immunofluorescence images of WT and R60H β-Gal canine fibroblasts (untreated or treated with indicated compounds). Images show GM1 ganglioside antibody staining (first column), merge of GM1 ganglioside staining and DAPI (second column), inset of a region (third column), and CellMask staining (fourth column). Scale bar: 100 μM; inset scale 10 μM. ( b , c ) Dose–response of Hit 3 and Hit 5, respectively. Results are presented as mean ± SD. Data represent the percentage of GM1 ganglioside area per cell area relative to untreated R60H canine fibroblasts. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s Multiple Comparison Test. Asterisks indicate statistically significant differences compared to untreated R60H canine fibroblasts: *** p < 0.001, **** p < 0.0001; WT, wild type.

Article Snippet: Each 25 μL reaction contained 12.5 μL of 1.5 μM recombinant human β-Gal protein (rhGLB1; Novoprotein, Shanghai, China) in PBS (pH 7.4), resulting in a final protein concentration of 1 μM, and 12.5 μL of compound solution dissolved in 100% DMSO and diluted in protein buffer to achieve a final DMSO concentration of 2%.

Techniques: Immunofluorescence, Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Development of Small-Molecule Allosteric Modulators of Beta-Galactosidase (β-Gal) for the Treatment of GM1 Gangliosidosis and Morquio B

doi: 10.3390/ijms27083631

Figure Lengend Snippet: ( a ) Representative immunofluorescence images of WT or R60H β-Gal canine fibroblasts (untreated or treated with indicated compounds). Images show GM1 ganglioside antibody staining (first column), merge of GM1 ganglioside staining and DAPI (second column), inset of a region (third column), and CellMask staining (fourth column). Scale bar: 100 μm; inset scale 10 μm. ( b ) Quantification of GM1 ganglioside area per cell area at 3.13 μM and 12.5 μM concentrations. Results are expressed as a percentage relative to untreated cells and are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s Multiple Comparison Test. Asterisks indicate statistically significant differences compared to untreated R60H canine fibroblasts: ns, not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Each 25 μL reaction contained 12.5 μL of 1.5 μM recombinant human β-Gal protein (rhGLB1; Novoprotein, Shanghai, China) in PBS (pH 7.4), resulting in a final protein concentration of 1 μM, and 12.5 μL of compound solution dissolved in 100% DMSO and diluted in protein buffer to achieve a final DMSO concentration of 2%.

Techniques: Immunofluorescence, Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Development of Small-Molecule Allosteric Modulators of Beta-Galactosidase (β-Gal) for the Treatment of GM1 Gangliosidosis and Morquio B

doi: 10.3390/ijms27083631

Figure Lengend Snippet: Surface plasmon resonance (SPR) dose–response for compounds ( a ) Cpd 6, ( b ) Cpd 13, and ( c ) Cpd 18, showing their binding to immobilized β-Gal at neutral pH (7.4).

Article Snippet: Each 25 μL reaction contained 12.5 μL of 1.5 μM recombinant human β-Gal protein (rhGLB1; Novoprotein, Shanghai, China) in PBS (pH 7.4), resulting in a final protein concentration of 1 μM, and 12.5 μL of compound solution dissolved in 100% DMSO and diluted in protein buffer to achieve a final DMSO concentration of 2%.

Techniques: SPR Assay, Binding Assay