Amy M. Barrios

The human protein histidine phosphatase PHPT1 is involved in several important cellular pathways and has been implicated in various cancers. However, the biological roles of this enzyme are not well understood due to a lack of chemical tools that enable its study. Herein we have identified phenylarsonic acids as general scaffolds which inhibit PHPT1 activity. Notably, phenylarsonic acids can be embedded into peptide sequences, providing the first known peptide-based inhibitors of PHPT1. In a counterscreen against a small panel of phosphatases, we demonstrate that these compounds exhibit some selectivity for PHPT1. Moreover, we show that these compounds exhibit mixed inhibition. We provide evidence that reduction of the phenylarsonic acids in situ by reducing agents like dithiothreitol (DTT) to provide phenylarsine species gives rise to the observed PHPT1 inhibition. These As(III) species are known to be thiophilic and can interact with solvent-exposed cysteine residues of proteins. Finally, we demonstrate that mutating the three cysteine residues of PHPT1 to alanine results in a significant decrease in enzyme inhibition by the phenylarsonic acids, suggesting that these compounds likely interact at least in part with Cys residues in PHPT1.

Fluorogenic substrates are essential tools for studying the activity of many enzymes including the protein tyrosine phosphatases (PTPs). Here, we have taken the first step toward the development of genetically encodable sensors for PTP activity using fluorescent and fluorogen‐activating proteins. The Fluorescence‐Activating and absorption Shifting Tag (FAST) is a small protein that becomes fluorescent upon binding to a small molecule dye. We demonstrate that FAST protein can be used as a sensor for PTP‐mediated dephosphorylation of phosphorylated dye molecules. Phosphorylated 4‐hydroxybenzylidene rhodanine (pHBR) is not able to bind to the FAST protein and induce fluorescence, but provides a sensitive assay for PTP activity, readily detecting 100 pM concentrations of PTP1B in the presence of FAST with a k_cat value of 19±1 s⁻¹ and a K_M value of 93±3 μM. In addition, while phosphorylation of the C‐terminal peptide of split GFP does not result in appreciable change in fluorescence of the reconstituted protein, phosphorylation of the C‐terminal peptide of the split FAST protein abrogates fluorescence. Upon PTP‐mediated dephosphorylation of the C‐terminal peptide, the ability of the N‐ and C‐terminal components to form a fluorescent complex with the small molecule dye is restored, leading to fluorescence.

PHPT1 is a protein histidine phosphatase that has been implicated in several disease pathways, but the chemical tools necessary to study the biological roles of this enzyme and investigate its utility as a therapeutic target have yet to be developed. To this end, the discovery of PHPT1 inhibitors is an area of significant interest. Here, we report an investigation of illudalic acid and illudalic acid analog‐based inhibition of PHPT1 activity. Four of the seven analogs investigated had IC₅₀ values below 5 μM, with the most potent compound (IA1‐8H2) exhibiting an IC₅₀ value of 3.4±0.7 μM. Interestingly, these compounds appear to be non‐covalent, non‐competitive inhibitors of PHPT1 activity, in contrast to other recently reported PHPT1 inhibitors. Mutating the three cysteine residues to alanine has no effect on inhibition, indicating that cysteine is not critical for interactions between inhibitor and enzyme.

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.

Chondroitin sulfate proteoglycans (CSPGs) prevent sympathetic nerve regeneration in the heart after myocardial infarction and prevent central nerve regrowth after traumatic brain injury and spinal cord injury. Currently, there are no small-molecule therapeutics to promote nerve regeneration through CSPG-containing scars. CSPGs bind to monomers of receptor protein tyrosine phosphatase sigma (PTPσ) on the surface of neurons, enhancing the ability of PTPσ to bind and dephosphorylate tropomyosin receptor kinases (Trks), inhibiting their activity and preventing axon outgrowth. Targeting PTPσ–Trk interactions is thus a potential therapeutic target. Here, we describe the development and synthesis of small molecules (HJ-01 and HJ-02) that disrupt PTPσ interactions with Trks, enhance Trk signaling, and promote sympathetic nerve regeneration over CSPGs.

A convergent 5-step synthesis (LLS) of illudalic acid allows for concise preparation of analogues for pharmacological evaluation.

By using a proteolysis-targeting chimera (PROTAC) strategy that couples an allosteric, reversible PTP inhibitor with an E3 ligase targeting ligand through a well-designed linker, the first molecule to successfully inhibit PTP activity through degradation has been developed.