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

Figure 5

TC-2153 targets the active site cysteine of STEP.

(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., n = 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., n = 4). (C) The progress curve method was used to determine the second-order rate constant: kinact/Ki = 153,000±15,000 M−1s−1 (mean ± s.e.m., n = 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., n = 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 Figure S6). (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 Figure S6).

Figure 5