Identification of a selective small-molecule inhibitor series targeting the eyes absent 2 (Eya2) phosphatase activity

Abstract

Eya proteins are essential coactivators of the Six family of homeobox transcription factors and also contain a unique protein tyrosine phosphatase activity, belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for a subset of Six1-mediated transcription, making this a unique type of transcriptional control. It is also responsible for directing cells to the repair instead of apoptosis pathway upon DNA damage. Furthermore, the phosphatase activity of Eya is critical for transformation, migration, invasion, and metastasis of breast cancer cells. Thus, inhibitors of the Eya phosphatase activity may be antitumorigenic and antimetastatic, as well as sensitize cancer cells to DNA damage-inducing therapies. In this article, we identified a previously unknown chemical series using high-throughput screening that inhibits the Eya2 phosphatase activity with IC(50)s ranging from 1.8 to 79 µM. Compound activity was confirmed using an alternative malachite green assay and H2AX, a known Eya substrate. Importantly, these Eya2 phosphatase inhibitors show specificity and do not significantly inhibit several other cellular phosphatases. Our studies identify the first selective Eya2 phosphatase inhibitors that can potentially be developed into chemical probes for functional studies of Eya phosphatase or into anticancer drugs in the future.

FIGURE 1

Purification and characterization of the phosphatase activity of Eya2 ED. (A) Superdex-200 elution profile of Eya2 ED and SDS PAGE of purified Eya2 ED. (B) EGTA effectively inhibits Eya2 phosphatase activity with an IC₅₀ of 65 μM and can be used as a positive control for inhibition. (C) DMSO (up to 10%) has no effect on the phosphatase activity of Eya2 ED. (D) Common phosphatase inhibitors do not significantly inhibit Eya2 ED. Compounds showing inhibition display high IC₅₀ values: 11.3 mM for Na₂MnO₄, 8.2 mM for β-glycerophosphate, 6.6 mM for NaF, and 1.8 mM for Na₃VO₄. Okadaic acid, L-phenylalanine, cyclosporine A, 1,10-phenanthroline, and phenylarsine oxide do not show inhibition at concentrations tested.

FIGURE 2

HTS assay optimization for the Eya2 phosphatase assay using OMFP as a substrate. (A) Scattered plot of a DMSO plate test of the Eya2 phosphatase assay in a 1536-well plate. The wells in column 2 contain 1 mM EGTA that was used as a positive control. All other wells contain DMSO as the negative (no inhibition) control. (B) Z’ and signal/background (S/B) values of a pilot screen using the LOPAC library. (C) The pilot screen revealed three compounds that inhibit Eya2 phosphatase activity. (D) Chemical structures of the three compounds in C. (E) Two pilot screen compounds inhibit Eya2 phosphate activity using the phospho-peptide substrate, pH2AX.

FIGURE 3

A new series of Eya2 ED phosphatase inhibitors were identified in the HTS that inhibit Eya2's phosphatase activity. (A) Structures of compounds that demonstrate inhibitory activity in the large scale HTS. (B) Activity of this series was confirmed in 12-point dose response curves. IC₅₀ values of each compound are listed. (C) Structures of four additional commercially available compounds that have the same N-arylidenebenzohydrazide core. (D) The four compounds tested display varying levels of inhibition towards Eya2 ED.

FIGURE 4

A secondary, malachite green based phosphatase assay using a phospho-H2AX peptide as the substrate confirmed the inhibition of Eya2's phosphatase activity by the best compounds of this class.

FIGURE 5

Identified lead compounds specifically inhibit the Eya2 Tyr phosphatase activity, and do not significantly inhibit other cellular phosphatases, including the Mg²⁺-dependent phosphatase PPM1A (A), protein tyrosine phosphatase 1B (PTP1B) (B), and HAD family member Scp1 (C) in an OMFP-based phosphatase assay.

FIGURE 6

Enzymatic kinetic experiments indicate that the compounds are mixed-mode inhibitors.