Cell-Based Screening of Active-Site Specific Chaperone for the Treatment of Fabry Disease

Publisher Summary This chapter describes a screening method for active-site specific chaperones (ASSCs) as exemplified for misfolded α-galactosidase A (α-Gal A) using a transient expression system with COS-7 cells. ASSCs have been shown to be effective in the specific rescuing of misfolded mutant α-Gal Ain human fibroblasts derived from patients of Fabry disease, which is an inherited disorder resulting from deficient lysosomal α-Gal A activity. This method can be applied for the screening of ASSCs effective for other genetic disorders caused by the misfolding of proteins. Screening of ASSCs is achieved and discussed by assaying for intracellular enzyme activity, Western-blot analysis of the enzyme in cell homogenates, and immunocytochemistry of cultivated COS-7 cells transiently expresses mutant α-Gal A in the presence of potential ASSC compounds. Because an ASSC may inhibit the enzyme activity at the enzyme assay stage, the ASSC concentration is examined.