Discovery and Characterization of Two Classes of Selective Inhibitors of the Suppressor of the TCR Signaling Family of Proteins

Abstract

The suppressor of T-cell receptor signaling (Sts) proteins, Sts-1, has recently emerged as a potential immunostimulatory target for drug development. Genetic inactivation of the Sts proteins dramatically increases host survival of systemic infection and leads to improved pathogen clearance. The protein tyrosine phosphatase (PTP) activity of these proteins arises from a C-terminal 2-histidine phosphatase (HP) domain. To identify new inhibitors of the HP activity of Sts-1, we miniaturized a phosphatase assay to a 1536-well format and conducted a 20 580 compound screen. Among the hits were two classes of structurally related compounds, tetracycline variants and sulfonated azo dyes. These hits had low micromolar to nanomolar IC₅₀ values. Orthogonal screening confirmed the validity of these inhibitors and demonstrated that both act competitively on Sts-1 phosphatase activity. When tested on other PTPs, PTP1B and SHP1, it was found that the tetracycline PTP1B, SHP1, the tetracycline variant (doxycycline), and the sulfonated azo dye (Congo red) are selective inhibitors of Sts-1_HP, with selectivity indices ranging from 19 to as high as 200. The planar polyaromatic moieties present in both classes of compounds suggested a common binding mode. The mutation of either tryptophan 494 or tyrosine 596, located near the active site of the protein, reduced the K_i of the inhibitors from 3- to 18-fold, indicating that these residues may help to promote the binding of substrates with aromatic groups. This work provides new insights into substrate selectivity mechanisms and describes two classes of compounds that can serve as probes of function or as a basis for future drug discovery.

Keywords: PTP; Sts; TULA; UBASH3; tetracycline.

Figure 1.

Pilot screen. The % response (A) for the 1,280 LOPAC library compounds, conducted in triplicate, is shown. The high control is plotted in red, the low control is plotted in green, the PHPS1 standard is plotted in magenta, and the samples are plotted in black. Compounds that had a response greater than three times the standard deviation (31.8% response in this case) were selected as active. The potent Sts-1 inhibitor, PHPS1, which has an IC₅₀ of 17.6 μM (B), was used as a positive control. A correlation plot (C) of the compounds screened in duplicate plates is shown (R² = 0.933). The hit cutoffs are designated by red lines.

Figure 2.

20,580 compound high-throughput screen. The 20K screen, summarized in (A), yielded 115 initial hits using a standard cutoff (23.12% response, in this case) (B). After retesting and counterscreens, 51 active compounds (with IC₅₀ < 10 μM) were identified and advanced for further testing. All of the assays performed well, yielding reasonable Z´ and signal-to-background values (C).

Figure 3.

Tetracycline derivatives identified in the screen. Shown are the structures and names of the top tetracycline active compounds (IC₅₀ < 10 μM) selected in the screening campaign.

Figure 4.

Sulfonated azo dyes. Shown are the structures and names of the top sulfonated azo dye variants that were identified as active compounds (IC₅₀ < 10 μM) in the screening campaign

Figure 5.

Mechanism of action. Double reciprocal plots for inhibition of Sts-1_HP by the tetracycline derivative doxycycline (A) and the azo dye Congo red (B). For each, three concentrations of the inhibitor were used (shown as inset on each graph) and a linear fit was used. The lines fit to the data intersect the X-axis at a value close to zero, in both cases, indicating a competitive mode of inhibition.

Figure 6.

Active site of the histidine phosphatase domain of Sts-1. In addition to the catalytic histidine residues (H379 and H565; shown in ball-and-stick representation with green carbon atoms and blue nitrogen atoms) and conserved arginine residues (R379, R383 and R462; shown in ball-and-stick representation with green carbon atoms and blue nitrogen atoms), two aromatic residues (W494 and Y596; shown in ball-and-stick representation with black carbon atoms, blue nitrogen atoms and red oxygen atoms) occupy the phosphatase active site of Sts-1 (A). A sulfate molecule (shown in ball-and-stick representation with the sulfate atom shown in orange and oxygen atoms shown in red) in the structure acts as a phosphate surrogate and marks the site where catalysis occurs. An overall view of the Sts-1_HP dimer (B); the left protomer shows a surface representation where the conserved His and Arg residues are colored blue, while W494 and Y596 are colored black) illustrates how Trp494 and Tyr596 are at the periphery of the active site and could serve a role in positioning planar aromatic inhibitors to block access to the active site.