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

Abstract

T cells play critical roles in the recognition and elimination of foreign pathogens in the host immune system. The T cell receptor (TCR) is responsible for activating T cells and discriminating between foreign antigens and inappropriate expression of endogenous proteins. Control of T cell signaling occurs through both positive and negative regulation. Two members of the suppressor of TCR signaling (Sts) family of proteins, Sts‐1 and Sts‐2, have been shown to be functionally redundant negative regulator of signaling pathways downstream of the TCR. Sts‐ 1 contains a C‐terminal histidine phosphatase (HP) catalytic domain. This domain of mouse Sts‐ 1 has been shown to have an intrinsic phosphatase activity to dephosphorylate Zap‐70, which contributes to the negative regulation of signaling pathways downstream of the TCR. Moreover, Sts‐knockout mice displayed significantly enhanced survival after infection with the fungal pathogen C. Albicans. The goal of my research is to establish human Sts‐1 as a viable drug target, and identify novel small molecule inhibitors of Sts‐1 as lead compounds for future development into adjuvant therapeutics for systemic fungal infections. To better understand the molecular determinants of function and structure of Sts‐1 in humans, the structure and steady‐state kinetics of the histidine phosphatase domains of human Sts‐1 (Sts‐ 1HP) have been characterized. We determined the X‐ray crystal structures of Sts‐1HP, unliganded and in complex with sulfate to 2.5 Å and 1.9 Å, respectively. The steady‐state kinetic analysis revealed that human and mouse Sts‐1 have similar kinetic properties. In addition, comparison of phosphatase activity of the full‐length Sts‐1 protein to that of Sts‐1HP reveals that Sts‐1HP is a functional surrogate for the native protein. Additionally, we demonstrated that human Sts‐1HP has robust phosphatase activity against Zap‐70 in a cell‐based assay. To identify and validate novel small molecule inhibitors of Sts‐1, we tested a set of known phosphatase inhibitors and identified that the SHP‐1 inhibitor, PHPS1, is a potent inhibitor of Sts‐1. We then conducted a 20,580‐compound high throughput screening using an optimized 1536‐well format phosphatase assay with human Sts‐1HP. The screen yielded 51 active compounds (IC50 < 10 μM) that were inactive in an enzyme‐minus counter‐screen. There were two main groups of compounds with similar structural scaffolds among these 51 hits. Using kinetic assays, we determined that these two classes of compounds are competitive inhibitors of Sts‐1HP. Comparison of Ki values for Sts‐1HP to canonical protein tyrosine phosphatases, including PTP1B and SHP1, indicates that these two groups of compounds can inhibit Sts‐1HP selectively. In addition, several mutations at the active site were made to explore which active site features of the enzyme contribute to inhibitor binding. These mutations decrease the inhibitory activity by 3 to 18 fold while not dramatically altering the enzyme activity. Taken together, these data suggest that the human...

High-Throughput Screening for the Discovery of Enzyme Inhibitors

“…Intuitively, a higher Z ′ factor (a larger difference between the signals obtained for positive and negative controls and/or smaller standard deviations in their signals) is expected to result in a higher hit rate, although this is also dependent on the statistical cutoff used to determine a hit . In order to confirm whether this expected correlation existed, the confirmed % hit rate for campaigns identified in Table , ,,,,,− Table , ,,,,,− ,,,,,,,− ,,, and several additional papers − was plotted as a function of Z ′ factor (Figure ).…”

“…In order to investigate this, data were assembled from studies reported in Table , ,,,,,− Table , ,,,,,− ,,,,,,,− ,,,, and several additional papers − as noted above. Confirmed % hit rates were plotted as a function of library size (Figure ).…”

TULA Proteins in Men, Mice, Hens, and Lice: Welcome to the Family

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Rebamipide and Derivatives are Potent, Selective Inhibitors of Histidine Phosphatase Activity of the Suppressor of T Cell Receptor Signaling Proteins

“…Despite this opportunity, little progress has been made toward the development of Sts inactivators. Prior studies have yielded nonspecific PTP inhibitors, low activity compounds, and nondrug-like hits. , While these demonstrate the druggability of the protein, they do not provide a clear direction for further development nor a foundation upon which to build a lead series. To identify novel inhibitors of the HP activity of Sts-1 (Sts-1 HP ), we recently conducted a 590,000 compound high-throughput screen at The Molecular Screening Center at UF Scripps .…”