Structural and kinetic characterization of DUSP5 with a Di-phosphorylated tripeptide substrate from the ERK activation loop.

Introduction: Dual specific phosphatases (DUSPs) are mitogen-activated protein kinase (MAPK) regulators, which also serve as drug targets for treating various vascular diseases. Previously, we have presented mechanistic characterizations of DUSP5 and its interaction with pERK, proposing a dual active site.

Methods: Herein, we characterize the interactions between the DUSP5 phosphatase domain and the pT-E-pY activation loop of ERK2, with specific active site assignments.

The novel estrogen receptor beta agonist EGX358 and genotype influence memory, vasomotor, and anxiety outcomes in an Alzheimer's mouse model.

Introduction: Alzheimer's disease (AD) prevalence and severity are associated with increased age, female sex, and apolipoprotein E4 (APOE4) genotype. Although estrogen therapy (ET) effectively reduces symptoms of menopause including hot flashes and anxiety, and can reduce dementia risk, it is associated with increased risks of breast and uterine cancer due to estrogen receptor alpha (ERα)-mediated increases in cancer cell proliferation. Because ERβ activation reduces this cell proliferation, selective targeting of ERβ may provide a safer method of improving memory and reducing hot flashes in menopausal women, including those with AD.

Real-time thiol detection in iPSC-derived neuron cultures using SemKur-IM, a novel fluorescent dithio probe.

Neurological disorders associated with inflammation and oxidative stress show reduced glutathione (GSH) levels in the human brain. Drug discovery efforts and pharmacological studies would benefit from tools (e.g.

Solution phase refinement of active site structure using 2D NMR and judiciously C-labeled cytochrome P450.

The Cytochrome P450 (CYP450) superfamily has been the subject of intense research for over six decades. Here the HU227 strain of E. coli, lacking the δ-aminolevulinic acid (δ-ALA) synthase gene, was employed, along with [5-C] δ-ALA, in the heterologous expression of P450cam harboring a prosthetic group labeled with C at the four methine carbons (C) and pyrrole C positions.

Discovery of two novel (4-hydroxyphenyl) substituted polycyclic carbocycles as potent and selective estrogen receptor beta agonists.

Two (4-hydroxyphenyl) substituted polycyclic carbocycles were prepared and assayed for estrogen receptor activity. 4-(4-Hydroxyphenyl)tricyclo[3.3.

Long-term oral administration of a novel estrogen receptor beta agonist enhances memory and alleviates drug-induced vasodilation in young ovariectomized mice.

Development of estrogen therapies targeting the β (ERβ) but not α (ERα) estrogen receptor is critically needed for the treatment of negative menopausal symptoms, as ERα activation increases health risks like cancer. Here, we determined the effects of long-term oral treatment with EGX358, a novel highly selective ERβ agonist, on memory, vasodilation, and affect in young ovariectomized mice. Mice were orally gavaged daily for 9 weeks with vehicle, 17β-estradiol (E), the ERβ agonist diarylpropionitrile (DPN), or EGX358 at doses that enhance memory when delivered acutely.

Synthesis and evaluation of 17α-triazolyl and 9α-cyano derivatives of estradiol.

A variety of 17α-triazolyl and 9α-cyano derivatives of estradiol were prepared and evaluated for binding to human ERβ in both a TR-FRET assay, as well as ERβ and ERα agonism in cell-based functional assays. 9α-Cyanoestradiol (5) was nearly equipotent as estradiol as an agonist for both ERβ and ERα. The potency of the 17α-triazolylestradiol analogs is considerably more variable and depends on the nature of the 4-substituent of the triazole ring.

Discovery and Characterization of Halogenated Xanthene Inhibitors of DUSP5 as Potential Photodynamic Therapeutics.

Dual specific phosphatases (DUSPs) are an important class of mitogen-activated protein kinase (MAPK) regulators, and are drug targets for treating vascular diseases. Previously we had shown that DUSP5 plays a role in embryonic vertebrate vascular patterning. Herein, we screened a library of FDA-approved drugs and related compounds, using a -nitrophenylphosphate substrate (NPP)-based assay.

Role of Conserved Histidine and Serine in the HCXXXXXRS Motif of Human Dual-Specificity Phosphatase 5.

Background: The mitogen-activated protein kinase (MAPK) pathway is functionally generic and critical in maintaining physiological homeostasis and normal tissue development. This pathway is under tight regulation, which is in part mediated by dual-specific phosphatases (DUSPs), which dephosphorylate serine, threonine, and tyrosine residues of the ERK family of proteins. DUSP5 is of high clinical interest because of mutations we identified in this protein in patients with vascular anomalies.

Synthesis and evaluation of 4-cycloheptylphenols as selective Estrogen receptor-β agonists (SERBAs).

A short and efficient route to 4-(4-hydroxyphenyl)cycloheptanemethanol was developed, which resulted in the preparation of a mixture of 4 stereoisomers. The stereoisomers were separated by preparative HPLC, and two of the stereoisomers identified by X-ray crystallography. The stereoisomers, as well as a small family of 4-cycloheptylphenol derivatives, were evaluated as estrogen receptor-beta agonists.

A-C Estrogens as Potent and Selective Estrogen Receptor-Beta Agonists (SERBAs) to Enhance Memory Consolidation under Low-Estrogen Conditions.

Estrogen receptor-beta (ERβ) is a drug target for memory consolidation in postmenopausal women. Herein is reported a series of potent and selective ERβ agonists (SERBAs) with in vivo efficacy that are A-C estrogens, lacking the B and D estrogen rings. The most potent and selective A-C estrogen is selective for activating ER relative to seven other nuclear hormone receptors, with a surprising 750-fold selectivity for the β over α isoform and with ECs of 20-30 nM in cell-based and direct binding assays.

Dual Specificity Phosphatase 5-Substrate Interaction: A Mechanistic Perspective.

The mammalian genome contains approximately 200 phosphatases that are responsible for catalytically removing phosphate groups from proteins. In this review, we discuss dual specificity phosphatase 5 (DUSP5). DUSP5 belongs to the dual specificity phosphatase (DUSP) family, so named after the family members' abilities to remove phosphate groups from serine/threonine and tyrosine residues.

Endocannabinoid Transport Proteins: Discovery of Tools to Study Sterol Carrier Protein-2.

The endocannabinoid (eCB) neurotransmitter system regulates diverse neurological functions including stress and anxiety, pain, mood, and reward. Understanding the mechanisms underlying eCB regulation is critical for developing targeted pharmacotherapies to treat these and other neurologic disorders. Cellular studies suggest that the arachidonate eCBs, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are substrates for intracellular binding and transport proteins, and several candidate proteins have been identified.

Serendipitous discovery of light-induced (In Situ) formation of an Azo-bridged dimeric sulfonated naphthol as a potent PTP1B inhibitor.

Background: Protein tyrosine phosphatases (PTPs) like dual specificity phosphatase 5 (DUSP5) and protein tyrosine phosphatase 1B (PTP1B) are drug targets for diseases that include cancer, diabetes, and vascular disorders such as hemangiomas. The PTPs are also known to be notoriously difficult targets for designing inihibitors that become viable drug leads. Therefore, the pipeline for approved drugs in this class is minimal.

Docking into Mycobacterium tuberculosis Thioredoxin Reductase Protein Yields Pyrazolone Lead Molecules for Methicillin-Resistant Staphylococcus aureus.

The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against . Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the thioredoxin reductase.

Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs.

DUSP5 is an inducible nuclear dual-specificity phosphatase that specifically interacts with and deactivates extracellular signal-regulated kinases ERK1 and ERK2, which are responsible for cell proliferation, differentiation, and survival. The phosphatase domain (PD) of DUSP5 has unique structural features absent from other nuclear DUSPs, such as the presence of a secondary anion-binding site in the proximity of the reaction center and a glutamic acid E264 positioned next to the catalytic cysteine C263, as well as a remote intramolecular disulfide linkage. The overall 400 ns molecular dynamics simulations indicate that the secondary binding site of DUSP5 PD acts as an allosteric regulator of the phosphatase activity of DUSP5.

A Novel Scoring Based Distributed Protein Docking Application to Improve Enrichment.

Molecular docking is a computational technique which predicts the binding energy and the preferred binding mode of a ligand to a protein target. Virtual screening is a tool which uses docking to investigate large chemical libraries to identify ligands that bind favorably to a protein target. We have developed a novel scoring based distributed protein docking application to improve enrichment in virtual screening.

Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets.

Background: Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. We present a p-nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5.

Methods: pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.

Generation of Molecular Complexity from Cyclooctatetraene: Preparation of Aminobicyclo[5.1.0]octitols.

A series of eight stereoisomeric N-(tetrahydroxy bicyclo-[5.1.0]oct-2S*-yl)phthalimides were prepared in one to four steps from N-(bicyclo[5.

Novel Uses of In Vitro Data to Develop Quantitative Biological Activity Relationship Models for in Vivo Carcinogenicity Prediction.

The availability of large in vitro datasets enables better insight into the mode of action of chemicals and better identification of potential mechanism(s) of toxicity. Several studies have shown that not all in vitro assays can contribute as equal predictors of in vivo carcinogenicity for development of hybrid Quantitative Structure Activity Relationship (QSAR) models. We propose two novel approaches for the use of mechanistically relevant in vitro assay data in the identification of relevant biological descriptors and development of Quantitative Biological Activity Relationship (QBAR) models for carcinogenicity prediction.

Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins.

Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function.

Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria.

Sterol carrier protein-2: binding protein for endocannabinoids.

The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs "on-demand," thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water.

Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: a combined synthesis, binding and docking study.

Various estrogen analogs were synthesized and tested for binding to human ERα using a fluorescence polarization displacement assay. Binding affinity and orientation were also predicted using docking calculations. Docking was able to accurately predict relative binding affinity and orientation for estradiol, but only if a tightly bound water molecule bridging Arg394/Glu353 is present.

Protein structure in context: the molecular landscape of angiogenesis.

A team of students, educators, and researchers has developed new materials to teach cell signaling within its cellular context. Two nontraditional modalities are employed: physical models, to explore the atomic details of several of the proteins in the angiogenesis signaling cascade, and illustrations of the proteins in their cellular environment, to give an intuitive understanding of the cellular context of the pathway. The experiences of the team underscore the use of these types of materials as an effective mode for fostering students' understanding of the molecular world and the scientific method used to define it.