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. 2024 Jun 10;13:RP93094. doi: 10.7554/eLife.93094

Figure 2. Characterization of the 7,8-dihydroxyflavone (7,8-DHF)/pyridoxal 5’-phosphate phosphatase (PDXP) interaction.

(a) Determination of half-maximal inhibitory constants (IC50) of 7,8-DHF (2D structure shown on top) for purified murine or human PDXP, using pyridoxal 5’-phosphate (PLP) as a substrate. Phosphatase activities in the presence of 7,8-DHF were normalized to the respective enzyme activities measured in the presence of the DMSO solvent control. Data are mean values ± SD of n=3 (human PDXP) and n=4 (murine PDXP) biologically independent experiments. (b) IC50 values of different flavones for purified murine PDXP with PLP as a substrate. Phosphatase activities in the presence of flavones were normalized to the respective enzyme activities in the presence of the DMSO solvent control. All data are mean values ± SD. The inhibition of PDXP by 3,7,8-trihydroxyflavone-4’-hydroxyphenyl (2D structure shown on top) was assessed in n=6 biologically independent experiments. All other data are from n=3 biologically independent experiments. Apparently missing error bars are hidden by the symbols. (c) Biolayer interferometry (BLI) measurements of the interaction of 7,8-DHF with purified murine PDXP. Left panel, example sensorgram overlayed with the global 1:1 binding model (red) and the negative control (gray). The dashed line indicates the start of the dissociation phase. Right panel, steady-state dose-response analysis for 7,8-DHF based on n=4 technically independent measurements. (d) Sensitivity of the indicated phosphatases to 7,8-DHF. Phosphatase activities in the presence of 7,8-DHF were normalized to the respective enzyme activities measured in the presence of the DMSO solvent control. Data are mean values ± SD of n=4 (PGP) or n=3 biologically independent experiments (all other phosphatases). Phosphatase substrates and haloacid dehalogenase (HAD) phosphatase cap types are indicated in parentheses. PDXP, pyridoxal 5’-phosphate phosphatase (pyridoxal 5’-phosphate, C2); PGP, phosphoglycolate phosphatase (2-phosphoglycolate; C2); LHPP, phospholysine phosphohistidine inorganic pyrophosphate phosphatase (imidodiphosphate; C2); NT5C1A, soluble cytosolic 5'-nucleotidase 1A (AMP; C1); NANP, N-acetylneuraminate 9-phosphate phosphatase (6-phosphogluconate; C1); PHOP2, phosphatase orphan 2 (pyridoxal 5’-phosphate; C1); PSPH, phosphoserine phosphatase (O-phospho-L-serine; C1); PNKP, polynucleotide kinase phosphatase (3-phospho-oligonucleotide; C0); MDP1, magnesium-dependent phosphatase-1 (D-ribose-5-phosphate; C0); PTP1B (protein tyrosine phosphatase 1B; EGFR phospho-peptide); PP2B, protein phosphatase 2B/calcineurin (PKA regulatory subunit type II phospho-peptide); CIP, calf intestinal phosphatase (pNPP). Source data are available for this figure.

Figure 2—source data 1. Phosphatase activity assays (to Figure 2a).
Effect of 7,8-dihydroxyflavone (7,8-DHF) on murine and human pyridoxal 5’-phosphate phosphatase (PDXP) activity. Data are OD values and normalized data of malachite green assays, using pyridoxal 5’-phosphate (PLP) as a PDXP substrate.
Figure 2—source data 2. Phosphatase activity assays (to Figure 2b).
Effects of the indicated flavones on the phosphatase activity of murine pyridoxal 5’-phosphate phosphatase (PDXP). Data are OD values and normalized data of malachite green assays, using pyridoxal 5’-phosphate (PLP) as a PDXP substrate.
Figure 2—source data 3. Biolayer interferometry (BLI) measurements with 7,8-dihydroxyflavone (7,8-DHF) and murine pyridoxal 5’-phosphate phosphatase (PDXP) (to Figure 2c).
Data of the association and dissociation curves for fitting.
elife-93094-fig2-data3.xlsx (350.1KB, xlsx)
Figure 2—source data 4. Effect of 7,8-dihydroxyflavone (7,8-DHF) on the phosphatase activity of different phosphatases (to Figure 2d).
Data are OD values and normalized data of malachite green assays. For calf intestinal phosphatase (CIP), kinetic data are OD values from pNPP-dephosphorylation assays.
Figure 2—source data 5. Pyridoxal 5’-phosphate phosphatase (PDXP) inhibitor hits.
Determination of half-maximal inhibitory constants (IC50) of 11 PDXP inhibitory compounds (see InChI Key for chemical substance identification) using purified murine PDXP and pyridoxal 5’-phosphate (PLP) as a substrate. Data marked with an asterisk (*) are results of n=3 biologically independent experiments. Because of the limited quantity of most compounds available for these assays, all other data are results of n=1 determinations. 7,8-DHF, 7,8-dihydroxyflavone.

Figure 2.

Figure 2—figure supplement 1. Identification of pyridoxal 5’-phosphate phosphatase (PDXP) inhibitors.

Figure 2—figure supplement 1.

A primary screen was conducted using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as an artificial substrate. Out of 41,182 screened compounds, 256 compounds were discarded that showed very high autofluorescence (as recognized by elevated fluorescence at the start of the kinetic curve); 26 compounds showed statistically significant PDXP activation, and 255 compounds showed PDXP inhibition (as recognized by an elevated or decreased slope of the kinetic curve, respectively). The average Z’-factor of the screen was 0.75±0.112. These 281 compounds were selected for DiFMUP-based concentration-dependent validation, and the 46 most potent compounds were selected. A counter-screening was conducted in parallel, also in a concentration-dependent fashion, against the PDXP paralog and closest relative phosphoglycolate phosphatase (PGP). The 11 compounds that were inactive against PGP were validated in a secondary assay, using the PDXP substrate pyridoxal 5’-phosphate (PLP). One PDXP inhibitor hit blocked PLP dephosphorylation by ≥50%. Source data are available for this figure.
Figure 2—figure supplement 1—source data 1. Screening campaign for pyridoxal 5’-phosphate phosphatase (PDXP) inhibitors: IC50 data of the PDXP_PDXP primary screen.
Figure 2—figure supplement 1—source data 2. Screening campaign for pyridoxal 5’-phosphate phosphatase (PDXP) inhibitors: IC50 data of the PGP_PDXP counter-screen.
Figure 2—figure supplement 2. Biolayer interferometry (BLI) measurements of the interaction of 7,8-dihydroxyflavone (7,8-DHF) with purified murine pyridoxal 5’-phosphate phosphatase (PDXP).

Figure 2—figure supplement 2.

Sensorgrams of three additional experiments overlayed with the global 1:1 binding model (red) and the negative control (gray). The dashed line indicates the start of the dissociation phase. Source data are available for this figure.