Chemistry Faculty Publications

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    Glycan based detection and drug susceptibility of influenza virus
    (2014-01-01) Dinh, Hieu; Zhang, Xiaohu; Sweeney, Joyce; Yang, Yang; He, Yun; Dhawane, Abasaheb; Iyer, Suri S.; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    ABSTRACT: We have developed a panel of synthetic glycans as receptor mimics for the specific capture of influenza viruses. The glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. The microarray was evaluated for its ability to capture three different strains of influenza A virus, two H1N1, A/Brisbane/59/2007 and A/Solomon Islands/3/2006 and one H3N2, A/Aichi/2/1968. We observed an excellent detection ability with some compounds exhibiting clinically relevant (101 plaque forming units) limit of detection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using this microarray and could determine antiviral resistance for these strains.

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    Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides
    (2014-01-01) Huang, Zhen; Abdur, Rob; Gerlits, Oksana; Gan, Jianhua; Salon, Jozef; Kovalevsky, Andrey Y.; Chumanevich, Alexander A.; Weber, Irene; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University
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    Limbic Hyperactivation During Processing of Neutral Facial Expressions in Children with Bipolar Disorder
    (2006-01-01) Rich, Brendan A; Vinton, Deborah T; Roberson-Nay, Roxann; Hommer, Rebecca E; Berghorst, Lisa H; McClure, Erin B; Fromm, Stephen J; Pine, Daniel S; Leibenluft, Ellen; NIH; NIH; Virginia Commonwealth University; NIH; NIH; Georgia State University; NIH; NIH; NIH

    A major paradigm shift in mental health has led to the ascendance of the view that chronic psychopathology results from perturbed neural development. While most work in this area examines schizophrenia, the current report extends the paradigm to bipolar disorder (BD) in youth, thus demonstrating traction (not sure I understand what you mean here) in the developmental-psychobiology perspective. To study the role of amygdala dysfunction, we examined the neural mechanisms mediating face processing in 22 youth (mean age 14.21 + 3.11 years) with BD and 21 controls of comparable age, gender, and IQ. Event-related fMRI compared neural activation when attention was directed to emotional aspects of faces (hostility, subjects’ own fearfulness) vs. non-emotional aspects (nose width). Compared to controls, patients perceived greater hostility in neutral faces, and reported more fear when viewing them. Also, compared to controls, patients had greater activation in the left amygdala, accumbens, putamen, and ventral prefrontal cortex when rating face hostility, and greater activation in the left amygdala and bilateral accumbens when rating their own fear of the face. There were no between-group behavioral or neural differences in the non-emotional conditions. Results implicate deficient emotion-attention interactions in the pathophysiology of BD in youth, and suggest that developmental psychobiology approaches to chronic mental illness have broad applicability.

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    Understanding mixed sequence DNA recognition by novel designed compounds: the kinetic and thermodynamic behavior of azabenzimidazole diamidines
    (2015-01-01) Paul, Ananya; Wilson, W. David; Boykin, David; Chai, Yun; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    Sequence-specific recognition of DNA by small organic molecules offers a potentially effective approach for the external regulation of gene expression and is an important goal in cell biochemistry. Rational design of compounds from established modules can potentially yield compounds that bind strongly and selectively with specific DNA sequences. An initial approach is to start with common A·T bp recognition molecules and build in G·C recognition units. Here we report on the DNA interaction of a synthetic compound that specifically binds to a G·C bp in the minor groove of DNA by using an azabenzimidazole moiety. The detailed interactions were evaluated with biosensor-surface plasmon resonance (SPR), isothermal calorimetric (ITC), and mass spectrometry (ESI-MS) methods. The compound, DB2277, binds with single G·C bp containing sequences with subnanomolar potency and displays slow dissociation kinetics and high selectivity. A detailed thermodynamic and kinetic study at different experimental salt concentrations and temperatures shows that the binding free energy is salt concentration dependent but essentially temperature independent under our experimental conditions, and binding enthalpy is temperature dependent but salt concentration independent. The results show that in the proper compound structural context novel heterocyclic cations can be designed to strongly recognize complex DNA sequences.

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    Ventrolateral Prefrontal Cortex Activation and Attentional Bias in Response to Angry Faces in Adolescents with Generalized Anxiety Disorder
    (2006-01-01) Monk, Christopher; Nelson, Eric E; McClure, Erin B; Mogg, Karin; Bradley, Brendan P; Leibenluft, Ellen; Blair, James R; Chen, Geng; Charney, Dennis S; Ernst, Monique; Pine, Daniel S; NIMH, NIH/DHHS; NIMH, NIH/DHHS; Georgia State University; NIMH, NIH/DHHS; NIMH, NIH/DHHS; NIMH, NIH/DHHS; NIMH, NIH/DHHS; Georgia State University; NIMH, NIH/DHHS; NIMH, NIH/DHHS; NIMH, NIH/DHHS

    Objective: While adolescent anxiety disorders represent prevalent, debilitating conditions, few studies explore their brain physiology. Using event-related functional MRI (fMRI) and a behavioral measure of attention to angry faces, we evaluated differences in response between healthy adolescents and adolescents with generalized anxiety disorder (GAD). Method: In the primary trials of interest, 18 adolescents with GAD and 15 comparisons of equivalent age/gender/IQ viewed angry/neutral face pairs during fMRI acquisition. Following the presentation of each face pair, subjects pressed a button to a probe that was either on the same (congruent) or opposite (incongruent) side as the angry face. Reaction time differences between congruent and incongruent face-trials provided a measure of attention bias to angry faces. Results: Relative to controls, patients with GAD manifested greater right ventrolateral prefrontal cortex (VLPFC) activation to trials containing angry faces. Compared with controls, patients with GAD also showed greater attentional bias away from angry faces. VLPFC activation differences were independent of differences in attentional bias. Conclusions: Adolescents with GAD show greater right VLPFC activation and attentional bias away from angry faces than controls. Enhanced VLPFC engagement may directly relate to anxiety, or may regulate abnormal functioning in another region.

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    DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents
    (2014-02-01) Koeller, Kevin J.; Harris, G Davis; Aston, Karl; He, Gaofei; Castaneda, Carlos H.; Thornton, Melissa A.; Edwards, Terri G.; Wang, Shuo; Nanjunda, Rupesh; Wilson, W. David; Fisher, Chris; Bashkin, James K.; University of Missouri-St. Louis; University of Missouri-St. Louis; University of Missouri-St. Louis; University of Missouri-St. Louis; University of Missouri-St. Louis; University of Missouri-St. Louis; Georgia State University; Georgia State University; Georgia State University; University of Missouri-St. Louis

    There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of β-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.

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    Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes
    (2014-03-01) Maganti, Nagini; Moody, Tomika; Truax, Agnieszka D; Thakkar, Meghna; Spring, Alexander M.; Germann, Markus W; Greer, Susanna F.; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    Accumulating evidence shows the 26S proteasome is involved in the regulation of gene expression. We and others have demonstrated that proteasome components bind to sites of gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear, although prior observations suggest both proteolytic and non-proteolytic activities. Here, we define novel, non-proteolytic, roles for each of the three 19S heterodimers, represented by the 19S ATPases Sug1, S7, and S6a, in mammalian gene expression using the inflammatory gene CIITApIV. These 19S ATPases are recruited to induced CIITApIV promoters and also associate with CIITA coding regions. Additionally, these ATPases interact with elongation factor PTEFb complex members CDK9 and Hexim-1 and with Ser5 phosphorylated RNA Pol II. Both the generation of transcripts from CIITApIV and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by siRNA mediated knockdown of these 19S ATPases. Together, these results define novel roles for 19S ATPases in mammalian gene expression and indicate roles for these ATPases in promoting transcription processes.

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    Crystallographic and Spectroscopic Snapshots Reveal a Dehydrogenase in Action
    (2015-01-01) Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin; Georgia State University; University of Pennsylvania; Brookhaven National Laboratory; Georgia State University; Kansai University; Kansai University; Brookhaven National Laboratory; Georgia State University

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NADþ-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.

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    Effect of Ca2+ on the Steady-State and Time-Resolved Emission Properties of the Genetically Encoded Fluorescent Sensor CatchER
    (2014-05-01) Zhou, You; Solntsev, Kyril M.; Reddish, Florence; Tang, Shen; Yang, Jenny J.; Georgia State University; Georgia Institute of Technology; Georgia State University; Georgia State University

    We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ.

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    Iron(II)-Catalyzed Intermolecular Amino-Oxygenation of Olefins through the N−O Bond Cleavage of Functionalized Hydroxylamines
    (2014-08-01) Lu, Deng-Fu; Zhu, Cheng-Liang; Jia, Zhen-Xin; Xu, Hao; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    An iron-catalyzed diastereoselective intermolecular olefin amino-oxygenation reaction is reported, which proceeds via an iron-nitrenoid generated by the N− O bond cleavage of a functionalized hydroxylamine. In this reaction, a bench-stable hydroxylamine derivative is used as the amination reagent and oxidant. This method tolerates a range of synthetically valuable substrates that have been all incompatible with existing amino-oxygenation methods. It can also provide amino alcohol derivatives with regio- and stereochemical arrays complementary to known amino-oxygenation methods.

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    Hydrogen Sulfide Prodrugs—A review
    (2015-09-01) Zheng, Yueqin; Ji, Xingyue; Ji, Kaili; Wang, Binghe; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    Abstract Hydrogen sulfide (H2S) is recognized as one of three gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO). As a signaling molecule, H2S plays an important role in physiology and shows great potential in pharmaceutical applications. Along this line, there is a need for the development of H2S prodrugs for various reasons. In this review, we summarize different H2S

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    Spectrophotometric and Spectrofluorimetric Studies on Azilsartan Medoxomil and Chlorthalidone to Be Utilized in Their Determination in Pharmaceuticals
    (2014-01-01) Ebeid, Walid M.; Elkady, Ehab F.; El-Zaher, Asmaa A.; El-Bagary, Ramzia I.; Patonay, Gabor; Georgia State University; Cairo University; Cairo University; Cairo University; Georgia State University

    The recently approved angiotensin II receptor blocker, azilsartan medoxomil (AZL), was determined spectrophotometrically and spectrofluorimetrically in its combination with chlorthalidone (CLT) in their combined dosage form. The UV-spectrophotometric technique depends on simultaneous measurement of the first derivative spectra for AZL and CLT at 286 and 257 nm, respectively, in methanol. The spectrofluorimetric technique depends on measurement of the fourth derivative of the synchronous spectra intensities of AZL in presence of CLT at 298 nm in methanol. The effects of different solvents on spectrophotometric and spectrofluorimetric responses were studied. For, the spectrofluorimetric study, the effect of pH and micelle-assisted fluorescence enhancement were also studied. Linearity, accuracy, and precision were found to be satisfactory over the concentration ranges of 8–50 μg mL-1 and 2–20 μg mL-1 for AZL and CLT, respectively, in the spectrophotometric method as well as 0.01–0.08 μg mL-1 for AZL in the spectrofluorimetric method. The methods were successfully applied for the determination of the studied drugs in their co-formulated tablets. The developed methods are inexpensive and simple for the quality control and routine analysis of the cited drugs in bulk and in pharmaceuticals.

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    A Novel Approach Using Electrospray Ionization Mass Spectrometry to Study Competitive Binding of Small Molecules with Mixed DNA Sequences
    (2014-10-01) Laughlin, Sarah R; Wang, Siming; Kumar, Arvind; Boykin, David; Wilson, W. David; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    Minor groove binding compounds have been shown to induce changes in global DNA conformation, allosterically inhibiting DNA-protein interactions necessary for transcriptional processes. Many minor groove binders are specific for AT-base pairs but have little preference over alternating AT or A-tract sequences. Few compounds, other than polyamides, show selectivity for mixed sequences with AT and GC base pairs. Electrospray ionization mass spectrometry (ESI-MS) can provide insight on the stoichiometry and relative affinities in minor groove recognition of different DNA sequences with a library of minor groove binders. A goal in our current research is to develop new compounds that recognize mixed sequences of DNA. In an effort to optimize screening for compounds that target mixed AT and GC base pair sequences of DNA, ESI-MS was used to study the competitive binding of compounds with a mixed set of DNA sequences. The method identified preferred binding sites, relative affinities, and concentration-dependent binding stoichiometry for the minor groove binding compounds netropsin and DB75 with AT-rich sequences, and DB293 with ATGA and AT-sites.

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    EcoScale, a Semi-quantitative Tool to Select an Organic Preparation Based on Economical and Ecological Parameters
    (2006-01-01) Van Aken, Koen; Strekowski, Lucjan; Patiny, Luc; Georgia State University

    A novel post-synthesis analysis tool is presented which evaluates quality of the organic preparation based on yield, cost, safety, conditions and ease of workup/purification. The proposed approach is based on assigning a range of penalty points to these parameters. This semi-quantitative analysis can easily be modified by other synthetic chemists who may feel that some parameters should be assigned different relative penalty points. It is a powerful tool to compare several preparations of the same product based on safety, economical and ecological features.

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    The Unusual Monomer Recognition of Guanine-Containing Mixed Sequence DNA by a Dithiophene Heterocyclic Diamidine
    (2014-02-01) Munde, Manoj; Kumar, Arvind; Peixoto, Paul; Depauw, Sabine; Ismail, Mohamed A.; Farahat, Abdelbasset A.; Paul, Ananya; Say, Martial V.; David-Cordonnier, Marie-Hélène; Boykin, David; Wilson, W. David; Georgia State University; Georgia State University; Jean-Pierre Aubert Research Center; Jean-Pierre Aubert Research Center; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    DB1255 is a symmetrical diamidinophenyl-dithiophene that exhibits cellular activity by binding to DNA and inhibiting binding of ERG, an ETS family transcription factor that is commonly overexpressed or translocated in leukemia and prostate cancer [Nhili, R., Peixoto, P., Depauw, S., Flajollet, S., Dezitter, X., Munde, M. M., Ismail, M. A., Kumar, A., Farahat, A. A., Stephens, C. E., Duterque-Coquillaud, M., Wilson, W. D., Boykin, D. W., and David-Cordonnier, M. H. (2013) Nucleic Acids Res. 41, 125−138]. Because transcription factor inhibition is complex but is an attractive area for anticancer and antiparasitic drug development, we have evaluated the DNA interactions of additional derivatives of DB1255 to gain an improved understanding of the biophysical chemistry of complex function and inhibition. DNase I footprinting, biosensor surface plasmon resonance, and circular dichroism experiments show that DB1255 has an unusual and strong monomer binding mode in minor groove sites that contain a single GC base pair flanked by AT base pairs, for example, 5′-ATGAT-3′. Closely related derivatives, such as compounds with the thiophene replaced with furan or selenophane, bind very weakly to GC-containing sequences and do not have biological activity. DB1255 is selective for the ATGAT site; however, a similar sequence, 5′-ATGAC-3′, binds DB1255 more weakly and does not produce a footprint. Molecular docking studies show that the two thiophene sulfur atoms form strong, bifurcated hydrogen bond-type interactions with the G-N-H sequence that extends into the minor groove while the amidines form hydrogen bonds to the flanking AT base pairs. The central dithiophene unit of DB1255 thus forms an excellent, but unexpected, single-GC base pair recognition module in a monomer minor groove complex.

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    The Unusual Monomer Recognition of Guanine-Containing Mixed Sequence DNA by a Dithiophene Heterocyclic Diamidine
    (2014-02-01) Munde, Manoj; Kumar, Arvind; Peixoto, Paul; Depauw, Sabine; Ismail, Mohamed A.; Farahat, Abdelbasset A.; Paul, Ananya; Say, Martial V.; David-Cordonnier, Marie-Hélène; Boykin, David; Wilson, W. David; Georgia State University; Georgia State University; Jean-Pierre Aubert Research Center; Jean-Pierre Aubert Research Center; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University; Georgia State University

    DB1255 is a symmetrical diamidinophenyl-dithiophene that exhibits cellular activity by binding to DNA and inhibiting binding of ERG, an ETS family transcription factor that is commonly overexpressed or translocated in leukemia and prostate cancer [Nhili, R., Peixoto, P., Depauw, S., Flajollet, S., Dezitter, X., Munde, M. M., Ismail, M. A., Kumar, A., Farahat, A. A., Stephens, C. E., Duterque-Coquillaud, M., Wilson, W. D., Boykin, D. W., and David-Cordonnier, M. H. (2013) Nucleic Acids Res. 41, 125−138]. Because transcription factor inhibition is complex but is an attractive area for anticancer and antiparasitic drug development, we have evaluated the DNA interactions of additional derivatives of DB1255 to gain an improved understanding of the biophysical chemistry of complex function and inhibition. DNase I footprinting, biosensor surface plasmon resonance, and circular dichroism experiments show that DB1255 has an unusual and strong monomer binding mode in minor groove sites that contain a single GC base pair flanked by AT base pairs, for example, 5′-ATGAT-3′. Closely related derivatives, such as compounds with the thiophene replaced with furan or selenophane, bind very weakly to GC-containing sequences and do not have biological activity. DB1255 is selective for the ATGAT site; however, a similar sequence, 5′-ATGAC-3′, binds DB1255 more weakly and does not produce a footprint. Molecular docking studies show that the two thiophene sulfur atoms form strong, bifurcated hydrogen bond-type interactions with the G-N-H sequence that extends into the minor groove while the amidines form hydrogen bonds to the flanking AT base pairs. The central dithiophene unit of DB1255 thus forms an excellent, but unexpected, single-GC base pair recognition module in a monomer minor groove complex.

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    O-GlcNAcylation of G6PD Promotes the Pentose Phosphate Pathway and Tumor Growth
    (2015-09-01) Rao, Xiongjian; Duan, Xiaotao; Mao, Weimin; Li, Xuexia; Li, Zhonghua; Li, Qian; Zheng, Zhiguo; Xu, Haimiao; Chen, Min; Wang, Peng George; Wang, Yingjie; Shen, Binghui; Yi, Wen; Zhejiang University; Beijing Institute of Pharmacology and Toxicology; Zhejiang Cancer Research Institute; Zhejiang University; Zhejiang University; Zhejiang University; Zhejiang Cancer Research Institute; Zhejiang Cancer Research Institute; School of Life Science and the State Key Laboratory of Microbial Technology,; Georgia State University; The First Affiliated Hospital of Zhejiang University; City of Hope National Medical Center; Zhejiang University

    The pentose phosphate pathway (PPP) plays a critical role in macromolecule biosynthesis and maintaining cellular redox homoeostasis in rapidly proliferating cells. Upregulation of the PPP has been shown in several types of cancer. However, how the PPP is regulated to confer a selective growth advantage on cancer cells is not well understood. Here we show that glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, is dynamically modified with an O-linked b-N-acetylglucosamine sugar in response to hypoxia. Glycosylation activates G6PD activity and increases glucose flux through the PPP, thereby providing precursors for nucleotide and lipid biosynthesis, and reducing equivalents for antioxidant defense. Blocking glycosylation of G6PD reduces cancer cell proliferation in vitro and impairs tumor growth in vivo. Importantly, G6PD glycosylation is increased in human lung cancers. Our findings reveal a mechanistic understanding of how O-glycosylation directly regulates the PPP to confer a selective growth advantage to tumours.

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    Simultaneous Determination of Sitagliptin and Metformin in Pharmaceutical Preparations by Capillary Zone Electrophoresis and its Application to Human Plasma Analysis
    (2012-07-01) Salim, Mohamed; El-Enany, Nahed; Belal, Fathalah; Walash, Mohomad; Patonay, Gabor; Georgia State University; University of Mansoura; University of Mansoura; University of Mansoura; Georgia State University

    A novel, quick, reliable and simple capillary zone electrophoresis CZE method was developed and validated for the simultaneous determination of sitagliptin (SG) and metformin (MF) in pharmaceutical preparations. Separation was carried out in fused silica capillary (50.0 cm total length and 43.0 cm effective length, 49 μm i.d.) by applying a potential of 15 KV (positive polarity) and a running buffer containing 60 mM phosphate buffer at pH 4.0 with UV detection at 203 nm. The samples were injected hydrodynamically for 3 s at 0.5 psi and the temperature of the capillary cartridge was kept at 25 °C. Phenformin was used as internal standard (IS). The method was suitably validated with respect to specificity, linearity, limit of detection and quantitation, accuracy, precision, and robustness. The method showed good linearity in the ranges of 10–100 μg/mL and 50–500 μg/mL with limits of detection of 0.49, 2.11 μg/mL and limits of quantification of 1.48, 6.39 μg/mL for SG and MF, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their synthetic mixtures and co-formulated tablets without interfering peaks due to the excipients present in the pharmaceutical tablets. The method was further extended to the in-vitro determination of the two drugs in spiked human plasma. The estimated amounts of SG/MF were almost identical with the certified values, and their percentage relative standard deviation values (% R.S.D.) were found to be #1.50% (n = 3). The results were compared to a reference method reported in the literature and no significant difference was found statistically.

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    Structures of Darunavir-Resistant HIV‑1 Protease Mutant Reveal Atypical Binding of Darunavir to Wide Open Flaps
    (2014-04-01) Zhang, Ying; Chang, Yu-Chung E.; Louis, John M.; Wang, Yuan Fang; Harrison, Robert W.; Weber, Irene; Georgia State University; Georgia State University; National Institutes of Health; Georgia State University; Georgia State University; Georgia State University

    The molecular basis for high resistance to clinical inhibitors of HIV-1 protease (PR) was examined for the variant designated PRP51 that was selected for resistance to darunavir (DRV). High resolution crystal structures of PRP51 with the active site D25N mutation revealed a ligand-free form and an inhibitor-bound form showing a unique binding site and orientation for DRV. This inactivating mutation is known to increase the dimer dissociation constant and decrease DRV affinity of PR. The PRP51‑D25N dimers were in the open conformation with widely separated flaps, as reported for other highly resistant variants. PRP51‑D25N dimer bound two DRV molecules and showed larger separation of 8.7 Å between the closest atoms of the two flaps compared with 4.4 Å for the ligand-free structure of this mutant. The ligand-free structure, however, lacked van der Waals contacts between Ile50 and Pro81′ from the other subunit in the dimer, unlike the majority of PR structures. DRV is bound inside the active site cavity; however, the inhibitor is oriented almost perpendicular to its typical position and exhibits only 2 direct hydrogen bond and two water-mediated interactions with atoms of PRP51‑D25N compared with 11 hydrogen bond interactions seen for DRV bound in the typical position in wild-type enzyme. The atypical location of DRV may provide opportunities for design of novel inhibitors targeting the open conformation of PR drug-resistant mutants.

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    Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease- Associated ‘‘Toggle’’ Calcium-Sensing Receptor Mutations
    (2014-11-01) Zhang, Chen; Mulpuri, Nagaraju; Hannan, Fadil M.; Nesbit, M. Andrew; Thakker, Rajesh V.; Hamelberg, Donald; Brown, Edward M.; Yang, Jenny J.; Georgia State University; Georgia State University; University of Oxford; University of Oxford; University of Oxford; Georgia State University; Brigham and Women's Hospital; Georgia State University

    The Ca2+-sensing receptor (CaSR) regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+ ([Ca2+]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-offunction CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.