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. chemical probes) that detect changes to oxidative stress, from the perspective of physiologically-relevant reporters like cellular thiols, including GSH. To this end, we have developed a fluorescence visualization assay using iPSC-derived cortical glutamatergic neurons that were loaded with 25 µM of a novel thiol-detection fluorescent probe, SemKur-IM. This probe enables visualization of cellular thiol level changes in the neuronal somas and neurites, in response exposure to N-acetyl-cysteine (NAC). Cellular thiol redox state was observed to change, based on an increase in green fluorescence (485 nm excitation maximum; 525 nm emission maximum) due to changes in thiol levels, from 0 to 40 mM. Interestingly, prior to treatment with NAC, cells did not appear to have significant levels of reduced thiols. Our studies demonstrate the utility of SemKur-IM in the detection of thiol levels in live cells in response to chemical exposures, such as from drugs that return the cell to a healthier reduced state. An initial application to screening the effects of an Alzheimer's disease drug candidate, Posiphen, using fluorescence cell sorting is presented. Other potential applications include high throughput screening of central nervous system (CNS) drugs thought to work by affecting cellular redox state in neurons.

Solution phase refinement of active site structure using 2D NMR and judiciously 13C-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 [5-13C] δ-ALA, in the heterologous expression of P450cam harboring a prosthetic group labeled with 13C at the four methine carbons (Cm) and pyrrole Cα positions. The product was utilized as a proof of principle strategy for defining and refining solution phase active site structure in cytochrome P450cam, providing proton-to-proton distances from 13CmH to protons on bound substrate or nearby amino acid residues, using short mixing time 2D or 3D NOESY-HMQC methods. The results reveal the interesting finding that 2D 13C-filtered NOESY-HMQC can be used to obtain distances between protons on labeled 13C to positions of protons nearby in the active site, confirming the utility of this NMR-based approach to probing active site structure under physiological conditions. Such 13C-heme-filtered NOE data complement X-ray crystallographic and T1-based NMR measurements; and, may also be of potentially significant utility in furnishing experimental distance constraints in validations of docking routines commonly employed for determining the relative affinities and binding orientations of drug candidates with CYP450s.

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[3.3.1.13,7]decane-1-methanol (5a/b) and 7-(4-hydroxyphenyl)spiro[3.5]nonan-2-ol ((±)-11) were found to be potent ERß agonists (1.9 ± 0.4 nM and 6.2 ± 1.4 nM respectively) in a cell-based functional assay. Furthermore, both 5a/b and 11 were highly selective for ERß over ERα (377 and 1,100-fold selective respectively). While neither compound inhibited CYP2D6 or CYP3A4 at concentrations up to 62.5 µM, 5a/b did have weak binding to CYP2C9 with an IC50 of 10 ± 0.5 µM. Computational assessment of 5a/b and 11 predicted the most probable site of metabolism would be ortho to the phenolic hydroxyl group.

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 ß (ERß) 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 ERß agonist, on memory, vasodilation, and affect in young ovariectomized mice. Mice were orally gavaged daily for 9 weeks with vehicle, 17ß-estradiol (E2), the ERß agonist diarylpropionitrile (DPN), or EGX358 at doses that enhance memory when delivered acutely. Tail skin temperature was recorded as a proxy for vasodilation following injection of vehicle or senktide, a tachykinin receptor 3 agonist used to model hot flashes. Anxiety-like behavior was assessed in the open field (OF) and elevated plus maze (EPM), and depression-like behavior was measured in the tail suspension (TST) and forced swim tests (FST). Finally, memory was assessed in object recognition (OR) and object placement (OP) tasks. E2, DPN, and EGX358 reduced senktide-mediated increases in tail skin temperature compared to vehicle. All three treatments also enhanced memory in the OR and OP tasks, whereas vehicle did not. Although E2 increased time spent in the center of the OF, no other treatment effects were observed in the OF, EPM, TST, or FST. These data suggest that long-term ERß activation can reduce hot flash-like symptoms and enhance spatial and object recognition memories in ovariectomized mice. Thus, the highly selective ERß agonist EGX358 may be a promising avenue for reducing menopause-related hot flashes and memory dysfunction.

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 ERß in both a TR-FRET assay, as well as ERß and ERα agonism in cell-based functional assays. 9α-Cyanoestradiol (5) was nearly equipotent as estradiol as an agonist for both ERß 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. While rigid protein docking simulations exhibited significant steric clashing, induced fit docking providing more protein flexibility revealed that the triazole linker of analogs 2d and 2e extends outside of the traditional ligand binding domain with the benzene ring located in the loop connecting helix 11 to helix 12.

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 para-nitrophenylphosphate substrate (pNPP)-based assay. This assay identified merbromin (also known as mercurochrome) as targeting DUSP5; and, we subsequently identified xanthene-ring based merbromin analogs eosin Y, erythrosin B, and rose bengal, all of which inhibit DUSP5 in vitro. Inhibition was time-dependent for merbromin, eosin Y, 2',7'-dibromofluorescein, and 2',7'-dichlorofluorescein, with enzyme inhibition increasing over time. Reaction progress curve data fit best to a slow-binding model of irreversible enzyme inactivation. Potency of the time-dependent compounds, except for 2',7'-dichlorofluorescein, was diminished when dithiothreitol (DTT) was present, suggesting thiol reactivity. Two additional merbromin analogs, erythrosin B and rose bengal also inhibit DUSP5, but have the therapeutic advantage of being less sensitive to DTT and exhibiting little time dependence for inhibition. Inhibition potency is correlated with the xanthene dye's LUMO energy, which affects ability to form light-activated radical anions, a likely active inhibitor form. Consistent with this hypothesis, rose bengal inhibition is light-dependent and demonstrates the expected red shifted spectrum upon binding to DUSP5, with a Kd of 690 nM. These studies provide a mechanistic foundation for further development of xanthene dyes for treating vascular diseases that respond to DUSP5 inhibition, with the following relative potencies: rose bengal > merbromin > erythrosin B > eosin Y.

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. Unlike other DUSPs, DUSP5 has unique specificity toward substrate pERK1/2. Using molecular docking and simulation strategies, we previously showed that DUSP5 has two pockets, which are utilized in a specific fashion to facilitate specificity toward catalysis of its substrate pERK1/2. Remarkably, most DUSPs share high similarity in their catalytic sites. Studying the catalytic domain of DUSP5 and identifying amino acid residues that are important for dephosphorylating pERK1/2 could be critical in developing small molecules for therapies targeting DUSP5. RESULTS: In this study, we utilized computational modeling to identify and predict the importance of two conserved amino acid residues, H262 and S270, in the DUSP5 catalytic site. Modeling studies predicted that catalytic activity of DUSP5 would be altered if these critical conserved residues were mutated. We next generated independent Glutathione-S-Transferase (GST)-tagged full-length DUSP5 mutant proteins carrying specific mutations H262F and S270A in the phosphatase domain. Biochemical analysis was performed on these purified proteins, and consistent with our computational prediction, we observed altered enzyme activity kinetic profiles for both mutants with a synthetic small molecule substrate (pNPP) and the physiological relevant substrate (pERK) when compared to wild type GST-DUSP5 protein. CONCLUSION: Our molecular modeling and biochemical studies combined demonstrate that enzymatic activity of phosphatases can be manipulated by mutating specific conserved amino acid residues in the catalytic site (phosphatase domain). This strategy could facilitate generation of small molecules that will serve as agonists/antagonists of DUSP5 activity.

Synthesis and evaluation of 4-cycloheptylphenols as selective Estrogen receptor-ß 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. The lead compound, 4-(4-hydroxyphenyl)cycloheptanemethanol was selective for activating ER relative to seven other nuclear hormone receptors, with 300-fold selectivity for the ß over α isoform and with EC50 of 30-50 nM in cell-based and direct binding assays.

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

Estrogen receptor-beta (ERß) is a drug target for memory consolidation in postmenopausal women. Herein is reported a series of potent and selective ERß 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 ß over α isoform and with EC50s of 20-30 nM in cell-based and direct binding assays. Comparison of potency in different assays suggests that the ER isoform selectivity is related to the compound's ability to drive the productive conformational change needed to activate transcription. The compound also shows in vivo efficacy after microinfusion into the dorsal hippocampus and after intraperitoneal injection (0.5 mg/kg) or oral gavage (0.5 mg/kg). This simple yet novel A-C estrogen is selective, brain penetrant, and facilitates memory consolidation.

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. We provide a comparison of DUSP5's structure to other DUSPs and, using molecular modeling studies, provide an explanation for DUSP5's mechanistic interaction and specificity toward phospho-extracellular regulated kinase, its only known substrate. We also discuss new insights from molecular modeling studies that will influence our current thinking of mitogen-activated protein kinase signaling. Finally, we discuss the lessons learned from identifying small molecules that target DUSP5, which might benefit targeting efforts for other phosphatases. © 2017 American Physiological Society. Compr Physiol 7:1449-1461, 2017.

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. Initial evidence from our laboratory indicates that the lipid transport protein, sterol carrier protein 2 (SCP-2), binds to the eCBs and can regulate their cellular concentrations. Here, we present methods for evaluating SCP-2 binding of eCBs and their application to the discovery of the first inhibitor lead molecules. Using a fluorescent probe displacement assay, we found SCP-2 binds the eCBs, AEA (Ki=0.68±0.05µM) and 2-AG (Ki=0.37±0.02µM), with moderate affinity. A series of structurally diverse arachidonate analogues also bind SCP-2 with Ki values between 0.82 and 2.95µM, suggesting a high degree of tolerance for arachidonic acid head group modifications in this region of the protein. We also report initial structure-activity relationships surrounding previously reported inhibitors of Aedis aegypti SCP-2, and the results of an in silico high-throughput screen that identified structurally novel SCP-2 inhibitor leads. The methods and results reported here provide the basis for a robust probe discovery effort to fully elucidate the role of facilitated transport mediated by SCP-2 in eCB regulation and function.

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. Furthermore, drug screening for targets like PTPs often produce false positive and false negative results. RESULTS: Studies presented herein provide important insights into: (a) how to detect such artifacts, (b) the importance of compound re-synthesis and verification, and (c) how in situ chemical reactivity of compounds, when diagnosed and characterized, can actually lead to serendipitous discovery of valuable new lead molecules. Initial docking of compounds from the National Cancer Institute (NCI), followed by experimental testing in enzyme inhibition assays, identified an inhibitor of DUSP5. Subsequent control experiments revealed that this compound demonstrated time-dependent inhibition, and also a time-dependent change in color of the inhibitor that correlated with potency of inhibition. In addition, the compound activity varied depending on vendor source. We hypothesized, and then confirmed by synthesis of the compound, that the actual inhibitor of DUSP5 was a dimeric form of the original inhibitor compound, formed upon exposure to light and oxygen. This compound has an IC50 of 36 µM for DUSP5, and is a competitive inhibitor. Testing against PTP1B, for selectivity, demonstrated the dimeric compound was actually a more potent inhibitor of PTP1B, with an IC50 of 2.1 µM. The compound, an azo-bridged dimer of sulfonated naphthol rings, resembles previously reported PTP inhibitors, but with 18-fold selectivity for PTP1B versus DUSP5. CONCLUSION: We report the identification of a potent PTP1B inhibitor that was initially identified in a screen for DUSP5, implying common mechanism of inhibitory action for these scaffolds.

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 Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics.

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. Our studies have identified E264 as a critical constituent of the dual binding pocket, which regulates the catalytic activity of DUSP5 by forming a salt bridge with arginine R269. Molecular dynamics studies showed that initial occupation of the secondary binding pocket leads to the breakage of the salt bridge, which then allows the occupation of the active site. Indeed, biochemical analysis using the pERK assay on mutant E264Q demonstrated that mutation of glutamic acid E264 leads to an increase in the DUSP5 catalytic activity. The role of the secondary binding site in assembling the DUSP5-pERK pre-reactive complex was further demonstrated by molecular dynamics simulations that showed that the remote C197-C219 disulfide linkage controls the structure of the secondary binding pocket based on its redox state (i.e., disulfide/dithiol) and, in turn, the enzymatic 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. The application addresses the issue of time and cost of screening in contrast to conventional systematic parallel virtual screening methods in two ways. Firstly, it automates the process of creating and launching multiple independent dockings on a high performance computing cluster. Secondly, it uses a Nȧi̇ve Bayes scoring function to calculate binding energy of un-docked ligands to identify and preferentially dock (Autodock predicted) better binders. The application was tested on four proteins using a library of 10,573 ligands. In all the experiments, (i). 200 of the 1,000 best binders are identified after docking only ~14 percent of the chemical library, (ii). 9 or 10 best-binders are identified after docking only ~19 percent of the chemical library, and (iii). no significant enrichment is observed after docking ~70 percent of the chemical library. The results show significant increase in enrichment of potential drug leads in early rounds of 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 7.6 ± 0.4 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual-phosphorylated ERK2 substrate tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate. RESULTS: The most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 µM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD (3) _2320) with IC50 of 33 µM that is similar to NTS and does not aggregate. CONCLUSIONS: The new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs.

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[5.1.0]octa-3,5-dien-2-yl)phthalimide (±)-7, which is readily available from cyclooctatetraene (62 % yield). The structural assignments of the stereoisomers were established by (1) H NMR spectral data as well as X-ray crystal structures for certain members. The outcomes of several epoxydiol hydrolyses, particularly ring contraction and enlargement, are of note. The isomeric phthalimides as well as the free amines did not exhibit ß-glucosidase inhibitory activity at a concentration of less than 100 µM.

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. We demonstrate that in vitro assay data can be used to develop QBAR models for in vivo carcinogenicity prediction via two case studies corroborated with firm scientific rationale. The case studies demonstrate the similarities between QBAR and QSAR modeling in: (i) the selection of relevant descriptors to be used in the machine learning algorithm, and (ii) the development of a computational model that maps chemical or biological descriptors to a toxic endpoint. The results of both the case studies show: (i) improved accuracy and sensitivity which is especially desirable under regulatory requirements, and (ii) overall adherence with the OECD/REACH guidelines. Such mechanism based models can be used along with QSAR models for prediction of mechanistically complex toxic endpoints.

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. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. CONCLUSION: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.