Validation of a highly sensitive HaloTag-based assay to evaluate the potency of a novel class of allosteric β-Galactosidase correctors

by Selleckchem | Jan 11 2024

Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology is a disease-agnostic drug discovery tool that can be applied to any protein target of interest with a known three-dimensional structure. We used this proprietary technology to identify and characterize the therapeutic potential of structurally targeted allosteric regulators (STARs) of the lysosomal hydrolase β-galactosidase (β-Gal), which is deficient due to gene mutations in galactosidase beta 1 (GLB1)-related lysosomal storage disorders (LSDs). The biochemical HaloTag cleavage assay was used to monitor the delivery of wildtype (WT) β-Gal and four disease-related β-Gal variants (p.Ile51Thr, p.Arg59His, p.Arg201Cys and p.Trp273Leu) in the presence and absence of two identified STAR compounds. In addition, the ability of STARs to reduce toxic substrate was assessed in a canine fibroblast cell model. In contrast to the competitive pharmacological chaperone N-nonyl-deoxygalactonojirimycin (NN-DGJ), the two identified STAR compounds stabilized and substantially enhanced the lysosomal transport of wildtype enzyme and disease-causing β-Gal variants. In addition, the two STAR compounds reduced the intracellular accumulation of exogenous GM1 ganglioside, an effect not observed with the competitive chaperone NN-DGJ. This proof-of-concept study demonstrates that the SEE-Tx® platform is a rapid and cost-effective drug discovery tool for identifying STARs for the treatment of LSDs. In addition, the HaloTag assay developed in our lab has proved valuable in investigating the effect of STARs in promoting enzyme transport and lysosomal delivery. Automatization and upscaling of this assay would be beneficial for screening STARs as part of the drug discovery process.

Comments:

The passage you provided highlights the application of Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology in identifying potential therapies for lysosomal storage disorders (LSDs), specifically related to β-galactosidase (β-Gal) deficiency caused by mutations in the GLB1 gene. The study utilized structurally targeted allosteric regulators (STARs) identified through this technology to enhance the transport and delivery of both wildtype and mutated β-Gal enzymes.

Here's a breakdown:

1. **Technology Overview:** SEE-Tx® is described as a versatile tool applicable to proteins with known three-dimensional structures, aiding in drug discovery across various diseases.

2. **Target and Research Focus:** The study focused on lysosomal hydrolase β-Gal and its deficiency due to GLB1 mutations, causing LSDs.

3. **Methodology:** The HaloTag cleavage assay was used to monitor the delivery of both normal and mutated forms of β-Gal in the presence and absence of two STAR compounds. These compounds were compared to an existing competitive pharmacological chaperone, NN-DGJ, commonly used in this field.

4. **Results:** The identified STAR compounds showed promising results:
- Enhanced stabilization and improved lysosomal transport of wildtype β-Gal and disease-causing variants compared to NN-DGJ.
- Reduction in intracellular accumulation of GM1 ganglioside, which wasn’t observed with NN-DGJ.

5. **Implications:** This study serves as a proof of concept for the SEE-Tx® platform's effectiveness in identifying STARs as potential treatments for LSDs. Additionally, the developed HaloTag assay was valuable in assessing the impact of STARs on enzyme transport and lysosomal delivery. The proposal for automating and scaling up this assay could significantly benefit the screening of STARs in the drug discovery process.

Overall, this research demonstrates the potential of the SEE-Tx® platform and the efficacy of STAR compounds in addressing lysosomal storage disorders by enhancing the transport and activity of β-Gal enzymes, thereby offering hope for therapeutic interventions in these conditions.