Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

<div><p>STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC₅₀ of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and <i>in vivo</i> in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.</p></div

Compound 3 fractionation and initial characterization.

<p>(A) Commercially purchased Compound <b>3</b> was dissolved in methanol at 10 mg/mL, and 300 µL portions were injected onto a Zorbax (Agilent) 5 µm 300SB-C18 column (0.94×25 cm, 3 mL/min 75% methanol/25% pH 4.0 0.1 M ammonium acetate). Thirty-five fractions (3 mL each) were collected, evaporated, and reconstituted in 100 µL of DMSO. Fractions were tested with pNPP assays to determine inhibition of STEP activity by using 0.1 µL of each fraction and 100 nM of STEP protein in 96-well plates. DMSO alone was used as a control. Shown in the insert is a representative chromatogram (UV absorbance detection, 350 nm). Peaks A, B, and C indicate early unretained material, Compound 3, and the unknown compound. (B) Structure of S₈, the benzopentathiepin core, and 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153). (C and D) Dose–response curves for S₈ and TC-2153. (C) The IC₅₀ for S₈ was determined to be 17.2±0.4 nM (mean ± s.e.m., <i>n</i> = 4). (D) The IC₅₀ for TC-2153 was determined to be 24.6±0.8 nM (mean ± s.e.m., <i>n</i> = 4).</p

TC-2153 targets the active site cysteine of STEP.

<p>(A) STEP activity was measured with pNPP and IC₅₀s were 24.6±0.8 nM and 8.79±0.43 µM in the absence and presence of 1 mM GSH (mean ± s.e.m., <i>n</i> = 2). (B) STEP (200 nM) and TC-2153 (1 µM) (or DMSO control) were incubated for 60 min to inhibit enzymatic activity prior to dialysis. Aliquots were tested against pNPP (mean ± s.e.m., <i>n</i> = 4). (C) The progress curve method was used to determine the second-order rate constant: <i>k</i>_inact/<i>K</i>_i = 153,000±15,000 M⁻¹s⁻¹ (mean ± s.e.m., <i>n</i> = 4). (D) STEP (200 nM) and TC-2153 (5 µM) were incubated for 10 min and then incubated with GSH or DTT (1 mM each) or water (no reductant) for 0, 15, 30, or 60 min, and the enzymatic activity of STEP was measured using the pNPP assay (mean ± s.e.m., <i>n</i> = 4). (E) Detection of trisulfide bridge formation between C⁴⁶⁵ and C⁴⁷². The peptide sequence in (1) illustrates the trisulfide bridge along with the b and y-ion assignments detected in the MS/MS fragmentations spectrum (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001923#pbio.1001923.s006" target="_blank">Figure S6</a>). (2) compares the 3D elution profile of the trisulfide peptide (mass  = 2,746.242 Da). The trisulfide bridge (modified) peptide is only detected in the WT STEP in the presence of TC-2153. The corresponding disulfide (non-modified) peptide (mass  = 2,714.254 Da) was detected in WT STEP (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001923#pbio.1001923.s006" target="_blank">Figure S6</a>).</p

S₈ and TC-2153 increases the Tyr phosphorylation of STEP substrates in neuronal cultures and <i>in vivo</i>.

<p>Cortical neuronal cultures were treated with (A) S₈ and vehicle (Veh) or (B) TC-2153 and vehicle (0.05, 0.1, 1, and 10 µM) for 1 h. Phosphorylation of GluN2B (Y¹⁴⁷²), Pyk2 (Y⁴⁰²), and ERK1/2 (Y^204/187) were significantly higher after treatment of cultures with S₈ (A) or TC-2153 (B) (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, one-way ANOVA with post hoc Bonferroni test). Data represent the phospho-signal normalized to total protein and then to GAPDH (mean ± s.e.m., <i>n</i> = 4). C57BL/6 mice (3–6 mo) were injected with (C) S₈ (0.5, 1, 3 mg/kg, i.p.) or (D) TC-2153 (i.p., 1, 3, 6, 10 mg/kg, i.p.) and were sacrificed 3 h later. Cortices were microdissected and lysates spun down to P2 fraction and prepared for Western blotting. Tyrosine phosphorylation status was probed with phospho-specific antibodies to pGluN2B: Tyr¹⁴⁷², pPyk2: Tyr⁴⁰², and pERK1/2: Tyr^204/187 (*<i>p</i><0.05; **<i>p</i><0.01; one-way ANOVA with post hoc Bonferroni test). Data represent the phospho-signal normalized to the total protein signal and then to GAPDH (mean ± s.e.m., <i>n</i> = 3).</p

Selectivity of TC-2153 <i>in Vitro</i>.

a<p>from NCBI database; ^bcalculated using GraphPad Prism 5. mean ± s.e.m. (<i>n</i> = 3).</p

TC-2153 does not induce neuronal cell death.

<p>Cortical cells were incubated with TC-2153 (1, 10, and 100 µM) for 1 h along with positive controls: glutamate (100 µM), SDS (0.02%), and Triton-X-100 (0.15%) (A), and at multiple time points (1 h, 3 h, 24 h, and 48 h) (B). Bovine LDH was used as a LDH-positive control. The media was collected and analyzed for LDH. The assay quantitatively measures LDH, a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured with a 30-min coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells.</p

TC-2153 improves cognitive deficits in 3xTg-AD mice.

<p>WT and 3xTg-AD mice (male, 6 mo old) were treated with vehicle or TC-2153 (10 mg/kg, i.p.) and tested in the Y-maze, NOR, and MWM tasks. (A and B) Y-maze, number of arm entries and percentage spontaneous alternations were calculated (*<i>p</i><0.05, paired <i>t</i> test, AD-TC versus AD-Veh) (WT, <i>n</i> = 20/group; AD, <i>n</i> = 11/group). (C) NOR, the DI of each group was calculated (***<i>p</i><0.001, AD-TC versus AD-Veh) (WT, <i>n</i> = 9/group; AD, <i>n</i> = 16/group). (D) MWM, the 3xTg-AD mice injected with vehicle (<i>n</i> = 6) showed longer escape latency before finding the hidden platform (3 trials/day; 60 s; 30 m intertrial interval) when compared to AD mice treated with TC-2153 (<i>n</i> = 7) or WT mice injected with vehicle (<i>n</i> = 12) or TC-2153 (<i>n</i> = 13) (three-way ANOVA). * and ⁺ represents a statistical significant variation between AD-Veh mice and AD-TC or WT-Veh, respectively. (E) Swim speed at each training day was not significantly different between groups (three-way ANOVA). (F) Number of entries in a circular zone positioned around the previous platform location and in the opposite quadrants. * represents a statistical significant variation between AD-TC mice and other groups for the target quadrant. ⁺ indicates a difference for the target and opposite quadrant within each group. Data are mean ± s.e.m. *,⁺<i>p</i><0.05; **,⁺⁺<i>p</i><0.01; ***,⁺⁺⁺<i>p</i><0.01.</p