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Substrate-based fragment identification for the development of selective, nonpeptidic inhibitors of striatal-enriched protein tyrosine phosphatase.

ABSTRACT: High levels of striatal-enriched protein tyrosine phosphatase (STEP) activity are observed in a number of neuropsychiatric disorders such as Alzheimer's disease. Overexpression of STEP results in the dephosphorylation and inactivation of many key neuronal signaling molecules, including ionotropic glutamate receptors. Moreover, genetically reducing STEP levels in AD mouse models significantly reversed cognitive deficits and decreased glutamate receptor internalization. These results support STEP as a potential target for drug discovery for the treatment of Alzheimer's disease. Herein, a substrate-based approach for the discovery and optimization of fragments called substrate activity screening (SAS) has been applied to the development of low molecular weight (<450 Da) and nonpeptidic, single-digit micromolar mechanism-based STEP inhibitors with greater than 20-fold selectivity across multiple tyrosine and dual specificity phosphatases. Significant levels of STEP inhibition in rat cortical neurons are also observed.

SUBMITTER: Baguley TD

PROVIDER: S-EPMC3875168 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Substrate-based fragment identification for the development of selective, nonpeptidic inhibitors of striatal-enriched protein tyrosine phosphatase.

Baguley Tyler D TD Xu Hai-Chao HC Chatterjee Manavi M Nairn Angus C AC Lombroso Paul J PJ Ellman Jonathan A JA

Journal of medicinal chemistry 20131001 19

High levels of striatal-enriched protein tyrosine phosphatase (STEP) activity are observed in a number of neuropsychiatric disorders such as Alzheimer's disease. Overexpression of STEP results in the dephosphorylation and inactivation of many key neuronal signaling molecules, including ionotropic glutamate receptors. Moreover, genetically reducing STEP levels in AD mouse models significantly reversed cognitive deficits and decreased glutamate receptor internalization. These results support STEP ...[more]

PMID: 24083656

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Project description:The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTP? dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTP? and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTP? by directly dephosphorylating PTP? at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTP? to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTP? at Tyr(789), as well as increased translocation of PTP? to synaptic membranes. Activation of PTP? and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by EtOH leads to the activation of PTP?, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTP? and suggest that STEP and PTP? coordinate the regulation of Fyn. STEP61 , PTP?, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTP? is a novel substrate for STEP61. Upon ethanol exposure, STEP61 is phosphorylated and inactivated by protein kinase A (PKA) signaling in the DMS. As a result of STEP61 inhibition, there is an increase in the phosphorylation of PTP?, which translocates to lipid rafts and activates Fyn and subsequent NMDAR signaling. The results demonstrate a synergistic regulation of Fyn-NMDAR signaling by STEP61 and PTP?, which may contribute to the regulation of ethanol-related behaviors. NMDA, N-methyl-D-aspartate; PTP?, receptor-type protein tyrosine phosphatase alpha; STEP, STriatal-Enriched protein tyrosine Phosphatase.

Project description:Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward para-nitrophenyl phosphate, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase-specific sequence of STEP were required for ERK interaction. In addition to the N-terminal kinase-specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. Regulation of phospho-ERK by STEP underlies important neuronal activities. A detailed enzymologic characterisation and cellular studies of STEP revealed that specific residues in KIM and active site mediated ERK recognition. Structural differences between the KIM-ERK interfaces and the active site among different ERK phosphatases could be targeted to develop specific STEP inhibitor, which has therapeutic potential for neurological disorders. PKA, protein kinase A & NGF, nerve growth factor.

Dataset Information

Substrate-based fragment identification for the development of selective, nonpeptidic inhibitors of striatal-enriched protein tyrosine phosphatase.

Publications

Substrate-based fragment identification for the development of selective, nonpeptidic inhibitors of striatal-enriched protein tyrosine phosphatase.

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