Discovery of a novel series of inhibitors of lymphoid tyrosine phosphatase with activity in human T cells

Abstract

The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, is a critical regulator of signaling in T cells and recently emerged as a candidate target for therapy of autoimmune diseases. Here, by library screening, we identified a series of noncompetitive inhibitors of LYP that showed activity in primary T cells. Kinetic analysis confirmed that binding of the compounds to the phosphatase is nonmutually exclusive with respect to a known bidentate competitive inhibitor. The mechanism of action of the lead inhibitor compound 4e was studied by a combination of hydrogen/deuterium-exchange mass spectrometry and molecular modeling. The results suggest that the inhibitor interacts critically with a hydrophobic patch located outside the active site of the phosphatase. Targeting of secondary allosteric sites is viewed as a promising yet unexplored approach to develop pharmacological inhibitors of protein tyrosine phosphatases. Our novel scaffold could be a starting point to attempt development of "nonactive site" anti-LYP pharmacological agents.

Figure 1

Intracellular inhibition of LYP by compound 4. (A, B) Compound 4 increases phosphorylation of Lck Y394 in T cells. (A) JTAg cells were treated with 50 μM of the top four compounds in Table 1 (lanes 3 and 4 of each panel) or DMSO (lanes 1 and 2 of each panel) and either left unstimulated (lanes 1 and 3 of each panel) or stimulated (lanes 2 and 4 of each panel) with C305 for 2 min. Top panels show anti-pSrc(Y416) immunoblots. Bottom panels show anti-Lck blots of same samples. (B) Thymocytes from Ptpn22−/− (left panels) or Ptpn22+/+ (right panels) mice were treated with 50 μM compound 4 (lanes 3 and 4 in each panel) or DMSO (lanes 1 and 2 in each panel) and either left unstimulated (lanes 1 and 3 in each panel) or stimulated (lanes 2 and 4 in each panel) with biotinylated anti-CD3 and anti-CD4, followed by cross-linking with streptavidin for 1.5min. Top panels show anti-pSrc(Y416) immunoblots. Bottom panels show anti-Lck blots of same samples. Arrows indicate the position of Lck in each panel. The ratio of pSrc(Y416)/Lck band intensity as determined by densitometric scanning of the blots is indicated above each lane. (C–E) Compound 4 affects TCR signaling downstream Lck. (C) Jurkat TAg cells were treated with 50 μM compound 4 (lanes 3 and 4 in each panel) or left untreated (lanes 1 and 2 in each panel) and were either left unstimulated (lanes 1 and 3 in each panel) or stimulated (lanes 2 and 4 in each panel) with C305 for 2 min. Panels show blots of total lysates with the following antibodies: top panel, anti-pSrc(Y416); second from top, control anti-Lck; third from top, antipZAP70(Y319); third from bottom, control anti-ZAP70; second from bottom, anti-pERK; bottom panel, control anti-ERK. (D) Single-cell analysis of TCR-induced phosphorylation of SLP-76 by phospho-flow cytometry. Jurkat TAg cells were treated with 50 μM compound 4 (continuous line and black graphs) or DMSO (dashed-line and gray graphs) and either left unstimulated (white graphs) or stimulated with C305 for 2 min (shaded graphs). Cells were fixed and stained with a PE-conjugated anti-pSLP76(Y128) antibody. Graphs show relative pSLP76 levels as detected by flow cytometry after gating on high forward and high side scatter (=cells with high SLP76 phosphorylation) cells. (E). Primary human T cells were treated with 50 μM compound 4 (black histograms) or DMSO (gray histograms) and were either left unstimulated or stimulated with anti-CD3 and anti-CD28 Abs and cross-linked for 1 min. Graph shows relative levels of pLAT(Y191) as assessed by ELISA. All data in this figure are representative of at least two independent experiments.

Figure 2

Compound 4 and analogues are nonactive site LYP inhibitors. (A) Plots of the IC₅₀ of compound 4 (black circles), compound 4e (black diamonds), compound 4g (black triangles), and compound 13 (black squares) on 25 nM LYP-catalyzed hydrolysis of the pY peptide versus substrate concentration/K_M. Plots show the IC₅₀±95% confidence intervals. Lines are fitting of the data to a linear regression. (B) Activity of 25 nM LYP on the pY peptide in the presence of increasing concentrations of compound 4 (DMSO, white squares; 5 μM, light gray squares; 7.5 μM, dark gray squares; 12.5 μM, black squares). Points are the average±SD of the activity of LYP plotted vs substrate concentration. Lines are fitting of data to the Michaelis–Menten equation. V_max and K_M values for each concentration of inhibitor are shown below the graph. (C) Activity of 25 nM LYP on the pY peptide in the presence of increasing concentrations of compound 13 (DMSO, white squares; 22 μM, light gray squares; 27 μM, dark gray squares; 32 μM, black squares; structure of compound 13 is also shown). Points are the average±SD of the activity of LYP plotted vs substrate concentration. Lines are fitting of data to the Michaelis–Menten equation. V_max and K_M values for each concentration of inhibitor are shown below the graph. (D–F) Inhibition of LYP by compounds 4e and 4g is nonmutually exclusive with compound 13. (D) Dual inhibition plot of compound 4e versus compound 13. The reciprocal rate of 25 nM LYP-catalyzed hydrolysis of the pY peptide was plotted at a series of compound 13 concentrations (DMSO, white circles; 65 μM, gray circles; 75 μM, black circles) in the presence of increasing concentration of compound 4e. Lines are fitting of the data to a linear regression. (E) Dual inhibition plot of compound 4g vs compound 13. The reciprocal rate of 25 nM LYP-catalyzed hydrolysis of the pY peptide was plotted at a series of compound 13 concentrations (DMSO, white circles; 65 μM, gray circles; 75 μM, black circles) in the presence of increasing concentration of compound 4g. Lines are fitting of the data to a linear regression. (F) Dual inhibition plot of compound 4e versus 4g. The reciprocal rate of 25 nM LYP-catalyzed hydrolysis of the pY peptide was plotted at a series of compound 4g concentrations (DMSO, white circles; 10 μM, gray circles; 20 μM, black circles) in the presence of increasing concentration of compound 4e. Lines are fitting of the data to a linear regression. All data in this figure are representative of at least two independent experiments.

Figure 3

Effect of compound 4e on deuteration exchange of LYP. (A) The percent in deuterium exchange between LYP with DMSO or compound 4e is shown. Each histogram represents a peptide in which deuterium exchange was quantified. Deuterium exchange for four different time points was collected (ranging from 30 to 1000 s), and the time point for each peptide showing the greatest change is shown. For different ionic forms of peptides the average variation was calculated. Any change greater than 10% was considered significant, and such peptides are shown in yellow. Peptides that showed a difference less than 10% are shown in turquoise. For different peptides that covered the same segment, only the longest peptide is shown. Data are representative of two independent experiments, and the variation between the same peptide between the two experiments was less than 5%. (B) Deuteration change of LYP upon binding of compound 4e. Plot shows percent deuteration of two peptides shown in (A) versus time for LYP incubated with compound 4e (yellow squares) or DMSO (blue squares). (C) Primary structure of the catalytic domain of LYP, with secondary structure superimposed. Yellow and turquoise coding is the same as in (A). The WPD loop is shown in purple, and the P loop is shown in blue. (D) Three-dimensional representation of the backbone of LYP according to PDB code 3H2X. Coding is the same as in (C). Selected helices are shown.

Figure 4

Three-dimensional surface representation of the potential site of interaction between LYP and compound 4. (A) Three-dimensional surface representation of the catalytic domain of LYP according to PDB code 3H2X. Candidate hydrophobic pockets are indicated as site 1, site 2, and site 3. Exposed hydrophobic residues within each site are shown in red. Other residues that contribute to form the pocket are shown in pink. Deuteration of the protein is coded as in Figure 3. (B) Magnification and 180° rotation of the backbone from (A) (green inset) which showed a significant docking score. Key residues forming the pocket are shown. Residues that coordinate with the compound are named. (C) Compounds that inhibited LYP show significantly higher GOLD docking score compared to inactive compounds. Correlation between inhibitory activity and GOLD score and statistical significance is shown. Compounds that inhibited LYP are shown as red symbols; inactive compounds are shown as black symbols. (D–E) Docking of compound 4e (D) and compound 4 (E) into site 1 of LYP as shown in (A). Key residues shown in (C) are named. See text for further explanation. (F, G) Effect of LYP L29A mutation on the inhibitory activity of compound 4e and compound 4. Graph shows dose-response inhibition of LYP WT (black squares and continuous line) or LYP L29A (red squares and dashed line) by compound 4e (F) and compound 4 (G). Plot shows the average ± SD percent activity of 25 nM LYP-catalyzed hydrolysis of the pY peptide versus inhibitor concentration. Lines are fitting of the data to a one-phase exponential decay equation in order to calculate the IC₅₀. Data from (F) and (G) are each representative of two independent experiments.