A new resorufin-based alpha-glucosidase assay for high-throughput screening

Area of Science:

• Biochemistry
• Enzymology
• Lysosomal Storage Disorders

Background:

• Pompe disease, a lysosomal storage disorder, results from alpha-glucosidase mutations causing glycogen accumulation.
• Small molecule chaperones represent a therapeutic strategy for lysosomal storage disorders by correcting protein misfolding.
• Alpha-glucosidase is also a target for type II diabetes, with inhibitors used clinically.

Purpose of the Study:

• To develop a new fluorogenic substrate for alpha-glucosidase enzyme assays.
• To improve upon existing substrates by reducing interference and enabling continuous kinetic assays.
• To facilitate compound screening for novel Pompe disease therapies.

Main Methods:

• Development of a novel fluorogenic substrate, resorufin alpha-d-glucopyranoside.
• Characterization of the substrate's product emission at 590 nm.
• Enzyme kinetic assays utilizing the new substrate, assessing continuous measurement capabilities due to lower pK(a).

Main Results:

• The new substrate, resorufin alpha-d-glucopyranoside, exhibits product emission at 590 nm, minimizing interference compared to existing substrates.
• The substrate allows for continuous enzyme kinetic assays without requiring a stop solution due to its product's lower pK(a).
• The developed substrate is a valuable tool for alpha-glucosidase enzyme assays.

Conclusions:

• The novel fluorogenic substrate offers enhanced performance for alpha-glucosidase assays.
• This tool is expected to accelerate the screening of compounds for developing new Pompe disease treatments.
• Improved enzyme assay methods are crucial for advancing therapies for lysosomal storage disorders.