In Vitro Assays for Measuring Protein Histidine Phosphatase Activity

McCullough, Brandon S.; Barrios, Amy

doi:10.1007/978-1-4939-9884-5_8

Cited by 2 publications

(3 citation statements)

References 30 publications

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“…We began by investigating the PHPT1 inhibitory activity of several previously reported illudalic acid analogs [35,36] (Figure 1) using an assay previously developed and validated in our lab [27,37] . Illudalic acid (IA1) and the didesmethyl illudalic acid analog (IA1‐8H2) are the most potent inhibitors of PHPT1 activity identified to date, with IC 50 values of 3.5±0.6 and 3.4±0.7 μM (Table 1 and Figure S1).…”

Section: Resultsmentioning

confidence: 99%

Derivatives of the Fungal Natural Product Illudalic Acid Inhibit the Activity of Protein Histidine Phosphatase PHPT1

et al. 2023

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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.

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Section: Resultsmentioning

confidence: 99%

Derivatives of the Fungal Natural Product Illudalic Acid Inhibit the Activity of Protein Histidine Phosphatase PHPT1

et al. 2023

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“…Our laboratory recently developed a facile, fluorogenic assay for monitoring PHPT1 activity. 18,19 Building upon this work, we validated the assay for use in high throughput screening (HTS). A robust HTS assay is defined as having a Z′ and signal window greater than 0.4 and 2, respectively.…”

mentioning

confidence: 99%

“…Our laboratory recently developed a facile, fluorogenic assay for monitoring PHPT1 activity. , Building upon this work, we validated the assay for use in high throughput screening (HTS). A robust HTS assay is defined as having a Z′ and signal window greater than 0.4 and 2, respectively. , Using ZnCl 2 as the inhibitor, our assay was determined to have an average Z′ of 0.82 and a signal window of 14, assuming that only a single replicate will be used during screening (see Figure S1 for representative data).…”

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confidence: 99%

Inhibitor Screen Identifies Covalent Inhibitors of the Protein Histidine Phosphatase PHPT1

McCullough

Barrios

2022

ACS Med. Chem. Lett.

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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.

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In Vitro Assays for Measuring Protein Histidine Phosphatase Activity

Cited by 2 publications

References 30 publications

Derivatives of the Fungal Natural Product Illudalic Acid Inhibit the Activity of Protein Histidine Phosphatase PHPT1

Derivatives of the Fungal Natural Product Illudalic Acid Inhibit the Activity of Protein Histidine Phosphatase PHPT1

Inhibitor Screen Identifies Covalent Inhibitors of the Protein Histidine Phosphatase PHPT1

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