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. 2011 Jan 27;54(2):562-71.
doi: 10.1021/jm101004d. Epub 2010 Dec 29.

Inhibition of Lymphoid Tyrosine Phosphatase by Benzofuran Salicylic Acids

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Free PMC article

Inhibition of Lymphoid Tyrosine Phosphatase by Benzofuran Salicylic Acids

Torkel Vang et al. J Med Chem. .
Free PMC article

Abstract

The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 μM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds.

Figures

Figure 1
Figure 1
Co-crystal structure of Lyp with inhibitor 478 (PDB code 2QCT). Left panel: Ribbon diagram of Lyp catalytic domain with 478 in stick representation (magenta); the catalytic cysteine (C227) as part of the P-loop, as well as amino acid residues that define the second binding site, are highlighted in stick representation. Right panel: Same view as left, but Lyp shown in surface representation, colored by electrostatic potential (red, most negative; blue, most positive). The protein structure was corrected by adding the missing side chains for 10 residues as predicted with Schrödinger Prime in the presence of ligand. The structure of the ligand (478) was corrected by adding the missing phenyl ring in 2-position of the benzofuran ring, correcting the valency of the triazole ring, and deprotonating the salicylic acid group. The protein ligand complex was then minimized with Schrödinger Macromodel (force field: OPLS-2005; solvent: water; dielectric constant = 1; constraints: naphthalene ring and benzofurane ring kept fixed during minimization). The graphics were prepared in PyMol (http://www.pymol.org/).
Figure 2
Figure 2
In silico docking studies with the crystal structure of Lyp (PDB code 2QCT). Docking poses of compounds 584 (cyan), 522 (yellow), and 478 (magenta). Substituents in para-position (R2) of the phenyl ring in 584 (methoxy) and 522 (methyl) favorably interact with side chains of Asp-195 and Glu-277. Protein structures were prepared with Prime, and docking was performed with Glide (Schrödinger Suite 2009; Schrödinger, LLC). The graphics were prepared in PyMol (http://www.pymol.org/).
Figure 3
Figure 3
Dual luciferase reporter assays in Jurkat TAg T cells to measure the effects of Lyp inhibitors on TCR-induced activation of the proximal IL-2 promoter (containing NFAT and AP-1 sites). A. Screening of generated benzofuran salicylic acids at 5 μM concentration. Compounds giving over 2-fold (200%) NFAT/AP-1 activation in the TCR-stimulated sample, compared to a DMSO control, were considered as hits. Compounds yielding higher levels of transcriptional activity in the unstimulated vs. TCR-stimulated sample were discarded due to unspecific effects. B. Dose-response studies of compounds 584, 525, 522, 630, and 486 on TCR-induced NFAT/AP-1 activation. All samples were run in triplicate (average ± standard deviation), and values are relative to a TCR-stimulated control sample pre-treated with DMSO (vehicle control).
Figure 4
Figure 4
Effects of compounds on Lyp's direct substrates Lck-pY394 and the phosphorylated ITAMs of the ζ-chains associated with the TCR, as well as on Lck's negative regulatory site pY505, which is controlled by the CD45 phosphatase. Jurkat TAg T cells were treated with inhibitor or vehicle (DMSO) for 45 min and then TCR-stimulated with OKT3 for 0-1-5-15 min. Subsequently, cell extracts were subjected to immunoblotting with the indicated antibodies.
Figure 5
Figure 5
In silico docking studies with compound 486 and the crystal structures of Lyp (PDB code 2QCT) and PTP1B (PDB code 2CM2). Upper panel: Stereo ribbon diagram close-up of active sites of Lyp (green) and PTP1B (white) with docking poses for 486 in stick representation (with Lyp, yellow; with PTP1B, blue). Lower panel: Lyp and PTP1B shown in surface representation, colored by electrostatic potential (red, most negative; blue, most positive). Docking poses for 486 in stick representation as above. Protein structures were prepared with Prime, and docking was performed with Glide (Schrödinger Suite 2009; Schrödinger, LLC). The graphics were prepared in PyMol (http://www.pymol.org/).
Scheme 1
Scheme 1
Click chemistry synthesis scheme of benzofuran salicylic acids.
Scheme 2
Scheme 2
Chemical synthesis of the alkynes.
Scheme 3
Scheme 3
Chemical synthesis of the azides.
Scheme 4
Scheme 4
Example of Cu-catalyzed click chemistry assembly of final compounds.

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