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Inhibition of striatal-enriched protein tyrosine phosphatase by targeting computationally revealed cryptic pockets.

ABSTRACT: Cryptic pockets, which are not apparent in crystallographic structures, provide promising alternatives to traditional binding sites for drug development. However, identifying cryptic pockets is extremely challenging and the therapeutic potential of cryptic pockets remains unclear. Here, we reported the discovery of novel inhibitors for striatal-enriched protein tyrosine phosphatase (STEP), a potential drug target for multiple neuropsychiatric disorders, based on cryptic pocket detection. By combining the use of molecular dynamics simulations and fragment-centric topographical mapping, we identified transiently open cryptic pockets and identified 12 new STEP inhibition scaffolds through structure-based virtual screening. Site-directed mutagenesis verified the binding of ST3 with the predicted cryptic pockets. Moreover, the most potent and selective inhibitors could modulate the phosphorylation of both ERK1/2 and Pyk2 in PC12 cells.

SUBMITTER: Hou X

PROVIDER: S-EPMC7163917 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Inhibition of striatal-enriched protein tyrosine phosphatase by targeting computationally revealed cryptic pockets.

Hou Xuben X Sun Jin-Peng JP Ge Lin L Liang Xiao X Li Kangshuai K Zhang Yingkai Y Fang Hao H

European journal of medicinal chemistry 20200211

Cryptic pockets, which are not apparent in crystallographic structures, provide promising alternatives to traditional binding sites for drug development. However, identifying cryptic pockets is extremely challenging and the therapeutic potential of cryptic pockets remains unclear. Here, we reported the discovery of novel inhibitors for striatal-enriched protein tyrosine phosphatase (STEP), a potential drug target for multiple neuropsychiatric disorders, based on cryptic pocket detection. By comb ...[more]

PMID: 32078861

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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. PTPα dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTPα 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 PTPα by directly dephosphorylating PTPα at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTPα at Tyr(789), as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα 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 PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. STEP61 , PTPα, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTPα 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 PTPα, 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 PTPα, which may contribute to the regulation of ethanol-related behaviors. NMDA, N-methyl-D-aspartate; PTPα, 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.

Project description:ObjectiveSTriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific tyrosine phosphatase. Membrane-bound STEP61 is the only isoform expressed in hippocampus and cortex. Genetic deletion of STEP enhances excitatory synaptic currents and long-term potentiation in the hippocampus. However, whether STEP61 affects seizure susceptibility is unclear. Here we investigated the effects of STEP inhibitor TC-2153 on seizure propensity in a murine model displaying kainic acid (KA)-induced status epilepticus and its effect on hippocampal excitability.MethodsAdult male and female C57BL/6J mice received intraperitoneal injection of either vehicle (2.8% dimethylsulfoxide [DMSO] in saline) or TC-2153 (10 mg/kg) and then either saline or KA (30 mg/kg) 3 h later before being monitored for behavioral seizures. A subset of female mice was ovariectomized (OVX). Acute hippocampal slices from Thy1-GCaMP6s mice were treated with either DMSO or TC-2153 (10 μM) for 1 h, and then incubated in artificial cerebrospinal fluid (ACSF) and potassium chloride (15 mM) for 2 min prior to live calcium imaging. Pyramidal neurons in dissociated rat hippocampal culture (DIV 8-10) were pre-treated with DMSO or TC-2153 (10 µM) for 1 h before whole-cell patch-clamp recording.ResultsTC-2153 treatment significantly reduced KA-induced seizure severity, with greater trend seen in female mice. OVX abolished this TC-2153-induced decrease in seizure severity in female mice. TC-2153 application significantly decreased overall excitability of acute hippocampal slices from both sexes. Surprisingly, TC-2153 treatment hyperpolarized resting membrane potential and decreased firing rate, sag voltage, and hyperpolarization-induced current (Ih ) of cultured hippocampal pyramidal neurons.SignificanceThis study is the first to demonstrate that pharmacological inhibition of STEP with TC-2153 decreases seizure severity and hippocampal activity in both sexes, and dampens hippocampal neuronal excitability and Ih . We propose that the antiseizure effects of TC-2153 are mediated by its unexpected action on suppressing neuronal intrinsic excitability.

Dataset Information

Inhibition of striatal-enriched protein tyrosine phosphatase by targeting computationally revealed cryptic pockets.

Publications

Inhibition of striatal-enriched protein tyrosine phosphatase by targeting computationally revealed cryptic pockets.

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