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

Methods Development

Abstract

Mutations in alpha-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, alpha-glucosidase is a therapeutic target for type II diabetes, and alpha-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the alpha-glucosidase enzyme assay, resorufin alpha-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-alpha-D-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK(a) of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the alpha-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.

Authors

Motabar, Omid; Shi, Zhen-Dan; Goldin, Ehud; Liu, Ke; Southall, Noel; Sidransky, Ellen; Austin, Christopher; Griffiths, Gary L; Zheng, Wei;

Keywords

  • Benzoxazines/ analysis
  • Benzoxazines/ chemical synthesis
  • Benzoxazines/ chemistry
  • Fluorescent Dyes/ analysis
  • Fluorescent Dyes/ chemistry
  • Glucosides/ analysis
  • Glucosides/ chemical synthesis
  • Glucosides/ chemistry
  • Glycogen Storage Disease Type II/ diagnosis
  • Humans
  • Kinetics
  • Mutation
  • Oxazines/ analysis
  • Oxazines/ chemical synthesis
  • Oxazines/ chemistry
  • Spectrometry, Fluorescence
  • alpha-Glucosidases/ metabolism

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