The effect of LZP25 (A) and LZP6 (B) on PTP1B-catalyzed pNPP hydrolysis. The Lineweaver-Burk plot displayed the characteristic intersecting line pattern, consistent with noncompetitive inhibition. The experiment was performed at 25 °C and pH 7.0. (A) LZP25 concentrations were 0 (●), 2.5 (○), 5 (▼), and 7.5 μM (▽), respectively. (B) LZP6 concentrations were 0 (●), 1.25 (○), 2.5 (▼), and 3.75 μM (▽), respectively.

Crystal structure of PTP1B in complex with LZP6. (A). Electron density map of LZP6 was contoured at 1.2 δ level in blue. Atoms were colored according to atom type (carbon in yellow, oxygen in red, and nitrogen in blue). The P-loop and WPD loop are shown in red and blue, respectively. (B). Binding modes of LZP6 in the active site of PTP1B: Atomic colors are as follows: oxygen - red, carbon — cyan, sulfur - orange, nitrogen - blue. Ligand’s carbon atoms are colored yellow. Key interactions are highlighted with dash lines as follows: electrostatic interactions with the ligand are shown in red, and hydrophobic interactions with ligands are shown in black. (C) Superposition of PTP1B•LZP6 and PTP1B•pTyr, the superposition was calculated with active site residues without the ligands. Active site of PTP1B in the open conformation is shown by transparent surface representation, and key residues are shown in stick model. Phenyl rings P_B1 and P_B2 of LZP6 are not shown. Atomic colors are as follows: oxygen - red, carbon — white, sulfur - orange, nitrogen - blue. Carbon atoms of LZP6 are colored yellow, and pTyr’s carbon atoms are colored green. (D) A symmetry related interaction between LZP6 monomer B and PTP1B. LZP6 is shown as a stick model, PTP1B is shown by transparent surface representation. Color scheme for surface: carbon, white; oxygen, red; nitrigen, blue.

Cellular activity of aryl diketoacid derivatives. (A) Effect of LZP25 and LZP6 on insulin-mediated IRβ phosphorylation in HepG2 cells. (B) Effect of LZP70 and LZP71 on insulin-mediated IRβ phosphorylation in HepG2 cells. (C) Concentration dependence of LZP6 on insulin-mediated IRβ phosphorylation in HepG2 cells.. Subconfluent HepG2 cells were preincubated with either LZP25 or LZP6 20 min, followed by a 5 min treatment with or without 100 nM insulin. Cell lysates were subjected to SDS-PAGE and the resolved proteins transferred to nitrocellulose membranes, which were probed with anti-phospho-IRβ antibodies. β-actin was used as a loading control.

Structures of the aryl diketoacid derivatives.

Crystal structure of PTP1B in complex with LZP25. (A) Electron density map at the active site contoured at 1.0 σ level. The P-loop and WPD loop are shown in red and blue, respectively. (B) Binding mode of LZP25 in the active site of PTP1B. Atomic colors are as follows: oxygen - red, carbon — cyan, sulfur - orange, nitrogen - blue. Ligand’s carbon atoms are colored yellow. Key interactions are highlighted with dash lines as follows: electrostatic interactions with the ligand are shown in red, and hydrophobic interactions with ligands are shown in black. P-loop of PTP1B is shown with transparent green cartoon. (C) Superposition of PTP1B•LZP25 and PTP1B•pTyr, the superposition was calculated with active site residues without the ligands. Residues marked with * are in the WPD loop closed conformation. Atomic colors are as follows: oxygen - red, carbon — white, sulfur - orange, nitrogen - blue. LZP25’s carbon atoms are colored yellow, while carbon atoms from PTP1B•pY (PDB entry code 1EEN) are colored green.

Modeling of pNPP binding to PTP1B in the presence of LZP25 (A) or LZP6 (B). The structures of PTP1B•LZP25 and PTP1B•LZP6 were utilized as the starting points molecular modeling. pNPP was manually docked into active site of PTP1B in the presence of LZP25 or LZP6, and energy minimized using program CNS.23

Surface representations of PTP1B in the WPD loop closed (A) and open (B) conformation. The figure was prepared using GRASP.42