Figure 5.

In vitro and in situ inhibition of GAA and GANAB. (a) 5 inhibits labeling of GAA and GANAB by fluorescent ABP 13 in fibroblast lysates in a concentration and time dependent manner. (b) 5 inhibits labeling of several α-glucosidases in mouse intestine lysate by 13. Sucrase-isomaltase (Sis), maltase-glucoamylase (MGAM), GAA, and GANAB are labeled by 13, as well as some off-target β-glucosidase labeling (LPH and GBA). α-Glucosidase labeling can be abrogated by preincubation with 5, while β-glucosidase labeling persists, demonstrating the superior selectivity of 5 compared to 13. (c) In situ inhibition of GAA and GANAB in fibroblasts at pH 4.0 and 7.0 respectively by 5 at incubation times of 2 and 24 h, followed by labeling of GAA and GANAB by cyclophellitol aziridine Cy5 probe 13. (d) Apparent IC₅₀s for in situ inhibition of GAA and GANAB enzyme activity by 5. Reported IC₅₀s are mean ± standard deviation from two biological replicates, each with three technical replicates.