Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

Abstract

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 A cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

Fig. 1.

Chemical and cocrystal structures. (A) Comparison of the structure of the mixed anhydrides of AMP (Left) with PTC124, D- luciferin (LH₂), dehydroluciferin (L), and DLSA. (B) Fo-Fc omit map of the PTC124-AMP mixed anhydride contoured at 3σ. (C) Hydrogen-bonded interactions between PTC124-AMP MAI (6, ball and stick) and luciferase residue (sticks) are indicated as dashed lines. (D) Cocrystal X-ray structure of FLuc and PTC124-AMP MAI.

Fig. 2.

Substrate binding pocket of FLuc with PTC124-AMP bound. (A, B) Two pocket orientations. (C) Structure of FLuc (Gray Ribbon) liganded with PTC124-AMP (Turquoise) overlaid on the structure of DLSA (Gold) bound to LcrLuc (PDB: 2D1S) showing the close similarity of ligand binding modes. Leu286 in FLuc corresponds to Ile288 in LcrLuc. These residues are located at the end of the respective ligand binding pockets, but whereas Ile288 moves upon ligand binding, Leu286 does not. (D) Overlay of the structures of apo-FLuc (Magenta) and FLuc liganded with the PTC124-AMP MAI adduct (6, Turquoise) showing movement of a loop between residues Ser314 and Leu319 upon binding of ligand (ball and stick).

Fig. 3.

NAC analysis of carboxylate PTC124 regioisomers. (A) Schematic of bond formation as the m-carboxylate of PTC124 displaces the pyrophosphate from ATP. R = 5-(2-fluorophenyl)-1,2,4-oxadiazole. (B) NAC modeling of PTC124/analogs within the FLuc active site. The m-carboxylate of PTC124 (1) provides an optimal NAC as the 3,5-diaryl oxadiazole is optimally positioned within the luciferin binding pocket. However, the p-carboxylate analog (7), must rotate by ∼20° within the constraints of a productive NAC model to be able to form a NAC and react with ATP. For the o-carboxylate analog (10) to maintain the NAC, a classic biaryl steric clash between the o-carboxylate and the adjacent oxadiazole occurs necessitating out of plane rotation which effectively pushes the analog out of the luciferin binding pocket, preventing MAI formation. (C, D) Graphs show formation of adenylate adducts by FLuc as analyzed by LC-MS. Total ion current (TIC) data for regioisomers of PTC124 mass (C), or the corresponding AMP adduct mass (D), following incubation of 20 μM PTC124 in the presence of 2 mM ATP for the regioisomers (indicated in D). Percentages represent the percent remaining of the parent ion peak, calculated from the ratio of the peak areas for the compound incubated with ATP in the presence and absence of FLuc.

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.