Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration

Abstract

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.

Keywords: Anticancer Drug; Enzyme Inhibitor; Migration; Phosphatase; Transcription Coactivator.

FIGURE 1.

Eya2 ED enzyme kinetic experiments indicate that the N-arylidenebenzohydrazide-containing compounds are non-competitive inhibitors at high Mg²⁺ concentrations (5 mm). A, chemical structures of representative compounds identified directly in HTS (MLS000544460 and MLS000585814), a commercially available compound that has slightly higher potency (NCGC00249327), and a structurally related but inactive compound (NCGC00241224). B, saturation curve for Eya2 ED showing initial rate with FDP concentration at each concentration of inhibitor NCGC00249327 (top). A Lineweaver-Burk plot shows non-competitive inhibition by NCGC00249327 given that the plots have the same x intercept as uninhibited enzyme and differing slopes and y intercepts. RFU, relative fluorescence units.

FIGURE 2.

The N-arylidenebenzohydrazide-containing compounds selectively inhibits Eya2. A, MLS000544460 inhibits Eya2 but not Eya3 in an OMFP-based phosphatase assay. B, MLS000544460 binds to Eya2 ED (K_D, 2.0 ± 0.3 μm) but not Eya3 ED in an ITC experiment. RFU, relative fluorescence units.

FIGURE 3.

MLS000544460 inhibits Eya2-dependent cell migration. Compound MLS000544460 (10 μm) inhibits Eya2 phosphatase-dependent migration of MCF10A cells, in a gap closure migration assay for 6 h, whereas the structurally similar but inactive compound NCGC00241224 has no effect. *, p < 0.05; ***, p < 0.001, two-way analysis of variance.

FIGURE 4.

The interaction between Eya2 ED and the N-arylidenebenzohydrazide-containing compound does not require Mg²⁺. A, structures of two active compounds with heteroatoms that can potentially coordinate Mg²⁺ shown in red. B, UV absorption spectrum of compound MLS000544460 in the presence of Mg²⁺. λ_max shifted from 322 nm to 334 nm with increasing concentrations of Mg²⁺. C, the λ_max shift observed in B is quantified as a function of Mg²⁺ concentration. D, the UV absorption spectrum of MLS000544460 in the presence of Na⁺. λ_max remained at 323 nm when titrated with Na⁺. E, UV absorption spectrum of the low activity analog MLS000585814 in the presence of Mg²⁺. λ_max shifted from 316 to 327 nm with increasing Mg²⁺ concentrations. F, the λ_max shift observed in E is quantified as a function of Mg²⁺ concentration. G, the T_m increase caused by the binding of compounds in the presence of 5 mm EDTA correlates with their IC₅₀ values, suggesting that these compounds stabilize Eya2 ED when Mg²⁺ is removed. H, MLS000544460 binds with lower affinity in the presence of 5 mm Mg²⁺ (K_D = 6.0 ± 1.2 μm). I, MLS000544460 has higher affinity toward Eya2 ED when Mg²⁺ is removed with 10 mm EDTA (K_D, 0.80 ± 0.04 μm). J, the structurally similar but inactive compound NCGC00241224 does not bind Eya2 ED. Abs, absorbance; a.u., absorbance units.

FIGURE 5.

MLS000544460 is a reversible inhibitor. 1 nm Eya2 ED incubated with various concentrations of MLS000544460 demonstrates that the IC₅₀ of MLS000544460 is ∼4 μm under this condition (first three columns in each panel). The enzymatic activity of Eya2 ED at 1 or 3 h following a 100-fold dilution of 100 nm Eya2 ED incubation with or without MLS000544460 indicate that MLS000544460 is a reversible inhibitor with a slow off rate. Cpd, compound.

FIGURE 6.

HSQC of ¹⁵N-labeled Eya2 ED in the presence of vehicle (DMSO), inactive compound (NCGC00241224), and active compound (MLS000544460). Select regions are expanded to provide spectrum details. MLS000544460 but not NCGC00241224 generated significant changes in the Eya2 ED HSQC spectrum.

FIGURE 7.

Eya2 inhibitors likely do not bind in the active site. A, a representative docking model of MLS000544460 in the active site. B, phosphatase activity of representative active site mutants of Eya2 ED in the presence of DMSO (vehicle control) or EDTA (fully inhibited). C, representative dose response curves of active site mutations demonstrate no effect on the ability of MLS000544460 to inhibit the mutant Eya2 ED. The phosphatase activity of Eya2 ED in the presence of DMSO is normalized to 1, and the activity in the presence of EDTA is normalized to 0 in these plots. RFU, relative fluorescence units.

FIGURE 8.

Sequence alignment of Eya2 ED and Eya3 ED. Residues colored gray are not in the ED (which begins at the bar) but were in the Eya construct used for phosphatase, ITC, and NMR experiments. Symbols beneath sequences represent identical residues (*), strongly similar properties (colon), weakly similar properties (semicolon), or no similarity (no symbol). Residues highlighted in red are surface amino acids that were mutated in Fig. 9.

FIGURE 9.

Mutagenesis based on differences between Eya2 and Eya3 ED identifies a potential compound binding site. A, a surface representation of the Eya2 ED structure with surface residues that differ between Eya2 and Eya3 highlighted in different colors. B, Eya2 ED mutants generated to change corresponding Eya2 residues to those of Eya3. C, phosphatase activity of representative mutants of Eya2 ED in the presence of DMSO (vehicle control) or EDTA (fully inhibited). D, dose response curves of Eya2 mutants. One mutant, L423S/T426G/H427T, demonstrated significantly reduced inhibition by MLS000544460, indicating a potential binding site. The phosphatase activity of Eya2 ED in the presence of DMSO is normalized to 1, and the activity in the presence of EDTA is normalized to 0 in these plots. E, ITC experiments demonstrate dramatically reduced affinity between the L423S/T426G/H427T and MLS000544460. RFU, relative fluorescence units.

FIGURE 10.

MLS000544460 likely binds to an allosteric site. A, the proposed allosteric compound binding site is on the opposite face of the active site. Compound is shown as a green ball-and-stick model, and the Mg²⁺ ion in the active site is represented by a green sphere. B, a docking model demonstrating residues in the allosteric binding pocket surrounding the compound. C, phosphatase activity of representative allosteric site mutants of Eya2 ED in the presence of DMSO (vehicle control) or EDTA (fully inhibited). Various mutants were characterized at different times so they are presented separately with their corresponding WT controls in the same experiment. D, effect of mutations in the allosteric binding pocket on the ability of MLS000544460 to inhibit the mutant Eya2 ED. The phosphatase activity of Eya2 ED in the presence of DMSO is normalized to 1, and the activity in the presence of EDTA is normalized to 0 in these plots. E, ITC experiments show that MLS000544460 does not bind Eya2 L425N and L417W.

FIGURE 11.

Possible mechanisms of allosteric inhibition by MLS000544460. A, Eya2 is a HAD family member consisting of a catalytic core (orange) and a HBM cap (cyan). The allosteric compound binding site is located at the interface between these two motifs (α7-α8 loop) (compound is shown in a green ball-and-stick model). B, surface representation of the catalytic core (orange) and HBM (cyan) showing MLS000544460 at the interface. C, a ribbon diagram demonstrating that the compound (green) binds directly behind the catalytic residues centered around the Mg²⁺ ion (green sphere). The compound binding at the allosteric site can potentially induce rigidity or conformational changes of the active site pocket.