Fig. 4.

Correlation between stabilization and inhibitor potency. Compounds are identified in Table 1. (A) The stabilizing effect of PTC124 analogs, as demonstrated by ΔT_m, on 3.6 μM Fluc in the presence of 2 mM ATP correlates with their IC₅₀s for FLuc inhibition in vitro. The average maximum ΔT_m is plotted and error bars represent the SD from two determinations. Compounds showing decreasing ΔT_m also show lower inhibitor potency against the enzyme. (B) The ΔT_m values for PTC124 (1), PTC 124 + 2 mM ATP, and the synthetically prepared PTC124-AMP adduct (6) are shown in either PBS (i) or Tris-acetate buffer (ii). The higher ΔT_m value obtained for the synthetic adduct is likely due to the fact that this adduct is measured in the absence of ATP (2 mM). Adding an equivalent concentration of ATP to 6 yielded the same ΔT_m value (12.6 °C) as was observed with PTC124 and ATP. (C) A correlation plot of the EC₅₀ values in the cell-based FLuc nonsense codon suppression assay plotted against their IC₅₀ values in the purified FLuc enzymatic assay for PTC124, a regioisomer, and related analogs (values from Table 1). Inhibition of purified FLuc in an enzymatic assay follows the same trend as apparent activation in a cell-based FLuc reporter gene assay. (D) Fold-shift in IC₅₀ values for PTC124 analogs relative to PTC124 (1) in the FLuc enzyme assay (i) and the nonsense codon suppression cell-based assay (ii) indicate that the potency trends remain largely similar in both types of assay.