Furthermore, AT2220 improved the catalytic activity of the precursor forms of multiple GAA mutants prior to proteolytic processing into its mature lysosomal forms. While increases in glycogen Nicaraven hydrolysis have been attributed to increased levels of mature GAA, our studies indicate that the synthesis of precursor forms in the presence of AT2220 can result in catalytic improvement against 4MUG. The AT2220-mediated improvements in mutant GAA activity may be due to changes in de novo folding that result in more efficient substrate turnover. A similar effect was noted with isofagomine on mutant glucocerebrosidase in Gaucher patient-derived fibroblasts. While isofagomine increased the specific activity of N370S GCase by approximately 30%, the effect of AT2220 on the specific activity of mutant GAA was much more pronounced, with increases ranging from 50% to 600% depending on the mutant form. AT2220 also promotes the trafficking of multiple mutant forms of GAA, permitting exit from the ER, passage through the secretory pathway, and delivery to lysosomes where processing to the mature 76 and 70 kDa forms occurs. Together these results suggest that AT2220 improves the folding of mutant GAA, resulting in increased catalytic activity, passage through the ER quality control, and enhanced stability in lysosomes. In fact, a recent survey of Pompe disease-related mutant forms of GAA revealed a strong correlation between residual enzyme activity and trafficking to lysosomes as evident through proteolytic processing. Over 80% of the GAA mutants tested that had less than 2% of wild-type activity showed no indication of lysosomal trafficking, while over 70% of mutants tested that had greater that 2% of wildtype activity did show lysosomal trafficking. These findings suggest that the Tetracycline hydrochloride AT2220-induced conformational changes that promote mutant GAA trafficking may also enhance its catalytic activity. Our studies also indicate that AT2220 increases the stability of mutant GAA precursors that are secreted into the culture media, as well as the mature GAA isoforms in lysosomes. For the GAA precursors that were secreted from transfected COS-7 cells, AT2220 increased the specific activity nearly ten-fold.