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July 2013 - present
July 2011 - July 2013
July 2002 - July 2011
Publications
Publications (122)
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 can...
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 intera...
Background
The prevalence and severity of Alzheimer’s disease (AD) are increased by female sex, apolipoprotein E (APOE) genotype, and age, such that postmenopausal female carriers of the Apoe4 allele are at greater risk of developing AD than age‐matched males. Although estrogen therapy shows promise in preventing and reducing menopause‐ and AD‐rela...
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...
Estrogen receptors (ER) are nuclear hormone receptors which are responsible for sex hormone signaling in women. A series of (1,4-disubstituted)-1,2,3-triazoles 5–21 were prepared by reaction of azidophenols with terminal alkynes under Fokin reaction conditions. The products were purified by column chromatography or recrystallization and characteriz...
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 ERβ agonists (1.9 ± 0.4 nM and 6.2 ± 1.4 nM respectively) in a cell-based functional assay. Fu...
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 chronic oral treatment with EGX358, a novel highly selective ERβ agonist, on memory, vasodilation,...
Background
Women are at greater risk of Alzheimer’s Disease (AD) than men, and symptoms associated with the menopausal loss of circulating estrogens, including hot flashes, exacerbate cognitive decline and AD risk. Although estrogen‐based therapies reduce hot flashes and mitigate AD risk early in the menopausal transition, these treatments can incr...
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...
Estrogen receptor-beta (ER-beta) is a drug target for memory consolidation in postmenopausal women, while estrogen receptor-alpha (ER-alpha) is linked with the proliferation of certain breast cancer cell lines. While the ligand-binding domains of ER-beta and ER-alpha share less than 60% sequence homology, the ligand-binding pockets of the two subty...
Estrogens in the brain are critical in the protection of neural pathways, and in the enhancement of hippocampal memory consolidation. The estrogen receptor exists in two forms: alpha (ERα) and beta (ERβ), the latter of which is predominant in the hippocampus and a drug target for several cognitive disorders. Thus, we have developed a selective ERβ...
Background:
The mitogen-activated protein kinases (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), wherein they dephosphorylate serine, threonine and tyrosine residues o...
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-nit...
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 derivativ...
Estrogen receptor-beta (ERβ) is a drug target for memory consolidation in post-menopausal 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...
Leaves of Sesamum angustifolium (Oliv.) Engl. have traditionally been used to treat infections of the skin, throat, eyes and inflamed mouth membranes. The aim of this study was to isolate and identify active metabolites from solvent extracts of S. angustifolium (Oliv.) Engl. initially by checking their anti-infective bioactivities, after purity con...
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...
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...
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 eC...
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...
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 foc...
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 in other nuclear DUSPs, such as pres...
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 prote...
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.
pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km...
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...
Dual specific 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. pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds with a K m of 7.6 + 0.5 mM. Docking follo...
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 Relat...
Disclosed are dithio compounds that include a quenched fluorophore and a non-fluorophore peptide linked via a dithio bond to the fluorophore. The dithio compounds may be used in methods for detecting thiol-containing compounds or dithio-containing compounds. The dithio compounds also may be used as cellular probes where the peptide portion of the c...
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 hypothesiz...
The prevalence of type 2 diabetes mellitus (T2DM) is increasing. As insulin resistance is a major feature of T2DM, this will necessitate the increased use of drugs that restore insulin sensitivity. Thiazolidinediones (TDZs) comprise one such class of drugs and are thought to act through peroxisome proliferator‐activated receptor gamma (PPARγ); howe...
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 acti...
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 molecul...
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,...
Here we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C-terminal r...
The aim of this work was to identify gaps in undergraduate students’ biochemistry knowledge and to create novel educational tools to address these gaps. We present a case study in which new teaching methods, including 3‐D protein models, were employed in the teaching of protein structure and function in an upper‐division biochemistry course. Final...
The cytochromes P450 (CYPs) play a central role in a variety of important biological oxidations, such as steroid synthesis and the metabolism of xenobiotic compounds, including most drugs. Because CYPs are frequently assayed as drug targets or as anti-targets, tools that provide confirmation of active-site binding and information on binding orienta...
Inherent complexity of the proteome often demands that it be studied as manageable subsets, termed subproteomes. A subproteome can be defined in a number of ways, although a pragmatic approach is to define it based on common features in an active site that lead to binding of a common small molecule ligand (e.g., a cofactor or a cross-reactive drug...
The high cost of drug discovery and development requires more efficient approaches to the identification and inhibition of tractable protein targets. One strategy is to pursue families of proteins that already possess affinity for a drug lead scaffold, where that scaffold plays the dual role of serving (a) when tethered to a resin, as a ligand to p...
Drugs exert desired and undesired effects based on their binding interactions with protein target(s) and off-target(s), providing evidence for drug efficacy and toxicity. Pioglitazone and rosiglitazone possess a common functional core, glitazone, which is considered a privileged scaffold upon which to build a drug selective for a given target-in th...
Mycobacterium tuberculosis ( M. tb ) resists oxidative killing in part by using the thioredoxin (Trx) system. Trx's catalyzes thioldisulfide exchange reactions using redox active cysteine thiols to reduce disulfides of other essential proteins, including metabolically essential enzymes. Oxidized Trx is reduced by thioredoxin reductase (TrxR) in an...
The insulin‐sensitizing drugs rosiglitazone and pioglitazone are currently the only two thiazolidinedione (TZD)‐based drugs on the market to treat type II diabetes in the U.S. Both drugs are thought to be selective for the peroxisome proliferator‐activated receptor gamma. Despite their chemical and mechanistic similarities, there are large cohort s...
The purpose of this in vitro study was to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite (NaOCl) and chlorhexidine (CHX) and the relative molecular weight of the components.
Using commercially available CHX gluconate, a 2% solution was formed and mixed in a 1:1 ratio with commercially available NaOCl prod...
Most diseases produce some perturbation of the levels and redox state of cellular thiols. Herein, we report an improved version of our previously reported fluorescent‐dithio probes for detecting cellular thiols, referred to as PMR‐CYS‐FITC, where PMR is p‐methyl red (aka dabcyl), a fluorescein quencher. Unlike most thiol‐reactive probes, PMR‐CYS‐FI...
One‐third of the world's population is infected by Mycobacterium tuberculosis (M.tb) . Two million people die each year from tuberculosis (TB), the disease caused by this bacterium. TB primarily affects the lungs and is easily transmitted. One way to kill M. tb might be to inhibit the enzyme dihydrofolate reductase (DHFR). DHFR catalyzes the produc...
Phosphomevalonate kinase (PMK) catalyzes phosphoryl transfer from adenosine triphosphate (ATP) to mevalonate 5-phosphate (M5P) on the pathway for synthesizing cholesterol and other isoprenoids. To permit this reaction, its substrates must be brought proximal, which would result in a significant and repulsive buildup of negative charge. To facilitat...
The purpose of this in vitro study was to determine whether para-chloroaniline (PCA) is formed through the reaction of mixing sodium hypochlorite (NaOCl) and chlorhexidine (CHX).
Initially, commercially available samples of chlorhexidine acetate (CHXa) and PCA were analyzed with 1H nuclear magnetic resonance (NMR) spectroscopy. Two solutions, NaOCl...
Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the gamma-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especiall...
Thio containing compounds like glutathione and protein cysteines are essential for the proper function of a cell. The levels of thiols and their oxidation state determine the cellular redox‐potential, which is associated with oxidative stress, and disease state of a cell. We recently reported synthesis and preliminary characterization of a coumarin...
Heart disease is one of the most prominent causes of death in the United States. Hypercholesterolemia is an underlying factor leading to heart disease. By inhibiting the cholesterol synthesis pathway, scientists can develop ways to reduce the number of heart disease‐related deaths. One way could be by inhibiting the actions of phosphomevalonate kin...
The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism
of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that
provide: (a) confirmation of active site binding and (b) structural data, would be of great utility...
Dihydrodipicolinate reductase (DHPR) is a homotetramer that catalyzes reduction of dihydrodipicolinate (DHP). We recently reported a biligand inhibitor ( K i = 100 nM) of DHPR, comprised of fragments that bind in the NADH (CRAA = catechol rhodanine acetic acid) and DHP (PDC = pyridine dicarboxylate) binding sites. Herein, we characterize binding sy...
Drugs typically exert their desired and undesired biological effects by virtue of binding interactions with protein target(s) and antitarget(s), respectively. Strategies are therefore needed to efficiently manipulate and monitor cross-target binding profiles (e.g., imatinib and isoniazid) as an integrated part of the drug design process. Herein we...
The current development of NMR methods facilitates the study of the mechanism by which enzymes catalyze reactions. The focus of this study is the dynamics and geometry of cofactor NAD(H) upon its binding to a dehydrogenase. To facilitate NMR analysis, the NAD(H) carboxamide was labeled with ¹⁵ N ( ¹⁵ N‐CA). CPMG relaxation dispersion measurements f...
Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, involving the transfer of a phosphoryl group from ATP to mevalonate‐5‐phosphate (M5P), producing mevalonate‐5‐diphosphate. This is the sole pathway for biosynthesis of steroids and isoprenoids in mammals. Despite the importance of this pathway in human cardiovascu...
The total concentration of cellular thiols is about 30 mM, and use of current fluorescent chemical probes to quantitate these thiols inside cells is impractical. We recently reported fluorescent dithio probes (donor‐S‐S‐acceptor, DSSA) with unusually low reduction potential (− 0.6 V) that can detect changes in intracellular thiols and cross membran...
F1Fo-ATP synthase is a large multiprotein complex, including at least 10 subunits in the membrane-bound Fo-sector. One of these Fo proteins is subunit e (Su e), involved in the stable dimerization of F1Fo-ATP synthase, and required for the establishment of normal cristae membrane architecture. As a step toward enabling structure-function studies of...
Histidine protein kinases (HPKs) are a class of receptor proteins found in bacterial two-component signal transduction systems, which allow bacteria to respond to changes in their external environment. To date, there are few potent inhibitors of histidine kinases, despite their potential ability to weaken bacteria against antibiotic treatment. EnvZ...
A catechol rhodanine (CR)-based privileged scaffold, tailored to dehydrogenase enzymes, has recently been reported. This scaffold was used as a template in a focused combinatorial library, designed using the NMR SOLVE methodology, to prepare potent (50-200nM) biligand inhibitors for multiple dehydrogenases. It is reported here that this CR scaffold...
P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active...
Organic amines are prevalent in nature and in drugs, especially the psychotherapeutic agents, and a major defense against potentially toxic amines is metabolism by CYP2D6. In order to understand better the constraints on the broad specificity of CYP2D6, 4207 amines were docked into the binding site of this enzyme. Docking poses were found predomina...
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium...
Cytochrome P450 2D6 (CYP2D6) is used to develop an approach for predicting affinity and relevant binding conformation(s) for highly flexible binding sites. The approach combines the use of docking scores and compound properties as attributes in building a neural network (NN) model. It begins by identifying segments of CYP2D6 that are important for...
Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPbetaS (sulfur on the beta-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the autho...
Historical Developments Leading to NMR-based Fragment AssemblyTheoretical Foundation for the Linking EffectNMR-based Identification of Fragments that Bind Proteins Fragment Library Design ConsiderationsThe “SHAPES” NMR Fragment LibraryThe “SAR by NMR” Fragment LibraryFragment-based Classification of protein TargetsNMR-based Screening for Fragment B...
Thiols play a central role in maintaining biological homeostasis. Their levels can change dramatically in response to oxidative stress associated with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i)...
Homology models were constructed for the ligand-binding domains of zebrafish estrogen receptors (zfERs) alpha, beta(1), and beta(2). Estradiol-binding sites are nearly identical in zfERs and their human homologs, suggesting that zebrafish will serve as a good model system for studying human ER-binding drugs. Conversely, studies of endocrine disrupt...
Proteomics efforts have created a need for better strategies to functionally categorize newly discovered proteins. To this end, we have employed saturation transfer difference NMR with pools of closely related cofactors, to determine cofactor preferences. This approach works well for dehydrogenases and has also been applied to cyclic nucleotide-bin...
Chemical proteomic strategies strive to probe and understand protein-ligand interactions across gene families. One gene family of particular interest in drug and xenobiotic metabolism are the cytochromes P450 (CYPs), the topic of this article. Although numerous tools exist to probe affinity of CYP-ligand interactions, fewer exist for the rapid expe...
Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the lev...
Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidore...
Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidore...
IntroductionNMR with Very Large Proteins Protein Perdeuteration and SEA-TROSY (Solvent Exposed Amides with Transverse Relaxation Optimized Spectroscopy)Protein Perdeuteration and Selective Amino Acid LabelingNMR-Based Drug Design Techniques NMR-DOC (Nuclear Magnetic Resonance Docking of Compounds)NMR-SOLVE (Nuclear Magnetic Resonance Structurally O...
A novel method to organize protein structural information based solely on sequence is presented. The method clusters proteins into families that correlate with the three-dimensional protein structure and the conformation of the bound ligands. This procedure was applied to nicotinamide adenine dinucleotide [NAD(P)]-utilizing enzymes to identify a to...
EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectrosco...
NMR spectroscopy has evolved into an important technique in support of structure-based drug design. Here, we survey the principles that enable NMR to provide information on the nature of molecular interactions and, on this basis, we discuss current NMR-based strategies that can identify weak-binding compounds and aid their development into potent,...
Genomic research on target identification and validation has created a great need for methods that rapidly provide detailed structural information on protein-ligand interactions. We developed a suite of NMR experiments as rapid and efficient tools to provide descriptive structural information on protein-ligand complexes. The methods work with large...
Triad Therapeutics’ proprietary technologies enable dramatic increases in both the speed of lead compound generation and the quality (binding affinity and specificity) of the compounds produced. Triad's approach, called Integrated Object-oriented PharmacoEngineering (IOPE™), focuses on designing inhibitors for entire protein families, such as oxido...
Genome sequencing projects have dramatically increased our understanding of the pathways and proteins involved in biological systems. The NAD(P)-utilizing family of enzymes is one of the most important gene families representing 15% of all known enzyme functions. We have performed a complete characterization of this gene family on a sequence, struc...
