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

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Abstract

Mutations in α-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, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-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-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pKa of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.

Section snippets

Reagents and chemicals

4MU-α-glc, NB–DNJ, a known inhibitor of GAA, and the buffer components were purchased from Sigma–Aldrich (St. Louis, MO, USA). GAA was obtained from residual solution after clinical infusions of Myozyme (Genzyme). The enzyme solution was mixed with 30% glycerol, and small aliquots were stored at −80 °C for up to 2 years.

The assay buffer was composed of 50 mM citric acid, 115 mM K2PO4, 110 mM KCl, 10 mM NaCl, 1 mM MgCl2, and 0.01% Tween 20 at pH 5.0. It was stored at 4 °C for up to 6 months. A solution

Results and discussion

On hydrolysis by GAA, fluorogenic res-α-glc forms two products: glucose and resorufin. Resorufin has a pKa of approximately 6.0 and emits red fluorescence at a peak of 590 nm (Fig. 1). This substrate has advantages over the existing substrate, 4MU-α-glc, which emits blue fluorescence at a peak of 440 nm, as explained below. The fluorescence enzyme assay is usually more sensitive than the chromogenic enzyme assay and is a better choice for assay miniaturization for HTS. However, a number of

Acknowledgments

This research was supported by the Molecular Libraries Initiative of the National Institutes of Health (NIH) Roadmap for Medical Research and the Intramural Research Programs of the National Human Genome Research Institute. The authors thank Paul Shinn for assistance with compound management.

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