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

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.