Inhibitor Screen Identifies Covalent Inhibitors of the Protein Histidine Phosphatase PHPT1.

The protein histidine phosphatase PHPT1 is implicated in a variety of cellular signaling pathways. However, little is known about the precise biological roles of this enzyme and a dearth of chemical tools for studying histidine phosphorylation and dephosphorylation has hampered progress in the field. With the goal of identifying the first inhibitors of PHPT1 activity, we carried out an inhibitor screen using a facile fluorogenic assay for PHPT1 activity recently developed in our laboratory. From a panel of approximately 4000 compounds obtained from the Microsource Spectrum Collection and the NCI Diversity Set IV, we identified several potential hits with significant selectivity for inhibiting PHPT1 activity over other phosphatases. Of these, norstictic acid was the most potent inhibitor of PHPT1 activity with an IC50 value of 7.9 ± 0.8 µM under our assay conditions. Norstictic acid is a time-dependent, covalent inhibitor of PHPT1 activity with K I = 90 ± 20 µM and k inact = 1.7 ± 0.1 min-1.

Fluorogenic probes for imaging cellular phosphatase activity.

The ability to visualize enzyme activity in a cell, tissue, or living organism can greatly enhance our understanding of the biological roles of that enzyme. While many aspects of cellular signaling are controlled by reversible protein phosphorylation, our understanding of the biological roles of the protein phosphatases involved is limited. Here, we provide an overview of progress toward the development of fluorescent probes that can be used to visualize the activity of protein phosphatases. Significant advances include the development of probes with visible and near-infrared (near-IR) excitation and emission profiles, which provides greater tissue and whole-animal imaging capabilities. In addition, the development of peptide-based probes has provided some selectivity for a phosphatase of interest. Key challenges involve the difficulty of achieving sufficient selectivity for an individual member of a phosphatase enzyme family and the necessity of fully validating the best probes before they can be adopted widely.

In Vitro Assays for Measuring Protein Histidine Phosphatase Activity.

In order to obtain a detailed kinetic characterization, identify inhibitors, and elucidate the biological roles of an enzyme, it is advantageous to have a facile, sensitive enzyme assay protocol. Here we present a brief overview of the techniques available to monitor histidine phosphatase activity and provide protocols for measuring the activity and inhibition of PHPT1 in vitro using the fluorescent probe 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). This assay uses small quantities of commercially available materials, making its use feasible for most laboratories.

Synthesis and PTP Inhibitory Activity of Illudalic Acid and Its Methyl Ether, with Insights into Selectivity for LAR PTP over Other Tyrosine Phosphatases under Physiologically Relevant Conditions.

The protein tyrosine phosphatase (PTP) family of enzymes includes many attractive therapeutic targets, such as those in the leukocyte common antigen-related (LAR) subfamily of receptor PTPs. Synthesis and PTP inhibitory activity of illudalic acid and its methyl ether are described, with a focus on selective inhibition of LAR PTP relative to a small collection of other representative PTPs. The synthesis comprises 16 steps and provides illudalic acid in up to 12% overall yield from neopentylene-fused benzoate 1 (20 steps from commercial materials). Illudalic acid dose-dependently (measured IC50 = 2.1 ± 0.2 µM) and time-dependently inhibits LAR consistent with previous reports of covalent binding. The kinetics of LAR inhibition by illudalic acid are consistent with a two-step mechanism in which the inhibitor and enzyme first interact noncovalently (KI = 130 ± 50 µM), followed by covalent ligation at a rate kinact = 1.3 ± 0.4 min-1. The kinact/KI ratio of 104 corresponds to a t∞1/2 of 0.5 min, as discussed herein. The phenol methyl ether of illudalic acid was found to be less potent in our dose-response assays (measured IC50 = 55 ± 6 µM) but more selective for LAR, with a weaker initial noncovalent interaction and faster covalent ligation of LAR as compared to illudalic acid itself. A truncated analogue of illudalic acid that lacks the neopentylene ring fusion was found to be devoid of significant activity under our assay conditions, in contrast to previous reports. These observations collectively help inform further development of illudalic acid analogues as potent and selective inhibitors of the LAR subfamily of tyrosine phosphatases.

Facile, Fluorogenic Assay for Protein Histidine Phosphatase Activity.

Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved as compared with other PHPT1 assays in the literature, with a kcat of 0.39 ± 0.02 s-1, a Km of 220 ± 30 µM, and a kcat/ Km of 1800 ± 200 M-1 s-1. In addition, the assay is significantly more sensitive as a result of using a fluorescent probe, requiring only 109 nM enzyme as compared with 2.4 µM as required by previously published assays. In the process of assay optimization, we discovered that PHPT1 is sensitive to a reducing environment and inhibited by transition-metal ions, with one apparent Cu(II) binding site with IC50 value of 500 ± 20 µM and two apparent Zn(II) binding sites with IC50 values of 25 ± 1 and 490 ± 20 µM.

Dual colorimetric and fluorogenic probes for visualizing tyrosine phosphatase activity.

Two resorufin-based substrates for protein tyrosine phosphatase (PTP) activity have been synthesized. These substrates provide sensitive fluorogenic readouts of PTP activity in vitro and in living cells at both acidic and neutral pH. In addition, the presence of the pathogenic bacteria Staphylococcus aureus was detected visually using a colorimetric readout.