Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria
J. Clin. Invest. Angel L. Pey, et al. 118:2858 doi:10.1172/JCI34355 [Go to this article.]

Figure 5
Effect of stabilizing hit compounds on the activity, protein level, and kinetics of degradation/synthesis of WT-PAH and PKU mutants. (A and B) Effect of compounds III (black bars) and IV (white bars) on activity (A) and protein measured by Western blot (B) of WT-PAH and PKU mutants transiently expressed (24 hours) in A293 cells. Values are given as mean ± SD of 3–4 independent expression experiments and are compared with controls for the corresponding PAH protein, with specific activities (nmol l-Tyr/min × mg): 7.1 ± 2.3 (WT-PAH), 1.7 ± 0.6 (I65T-PAH), 3.3 ± 0.4 (R68S-PAH), and 4.9 ± 1.9 (R261Q-PAH). The activity of R252W-PAH was <0.01 nmol l-Tyr/min × mg protein (the detection limit) under all conditions tested and thus not considered reliable. ANOVA tests, *P < 0.05; **P < 0.01. (C) Western blot analysis of the degradation of WT-PAH and I65T-PAH after transient transfection of A293 cells (18 hours) followed by inhibition of protein translation by puromycin (10 μg/ml). The half-lives (t_1/2) for I65T-PAH (in hours) were 5.0 ± 1.0 (control), 4.8 ± 1.1 (with compound III), and 6.8 ± 0.8 (with compound IV). (D) Kinetics of synthesis of WT-PAH in cell-free RTS without and with compounds III and IV. Data (given as mean ± SD of 3–4 independent experiments) refer to labeled PAH synthesized after 15.0 (black), 22.5 (light gray), and 30.0 (dark gray) minutes, measured by densitometry and normalized with respect to the corresponding control sample after 30-minute synthesis in the absence of compounds (100%). In all experiments, compounds are present at 40 μM in 1% DMSO, which is also added in control samples.