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Publication Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors(MDPI, 2019-02-25) Chang, Kyeong-Ok; Kim, Yunjeong; Lovell, Scott; Rathnayake, Athri D.; Groutas, William C.Proteases are a major enzyme group playing important roles in a wide variety of biological processes in life forms ranging from viruses to mammalians. The aberrant activity of proteases can lead to various diseases; consequently, host proteases have been the focus of intense investigation as potential therapeutic targets. A wide range of viruses encode proteases which play an essential role in viral replication and, therefore, constitute attractive targets for the development of antiviral therapeutics. There are numerous examples of successful drug development targeting cellular and viral proteases, including antivirals against human immunodeficiency virus and hepatitis C virus. Most FDA-approved antiviral agents are peptidomimetics and macrocyclic compounds that interact with the active site of a targeted protease. Norovirus proteases are cysteine proteases that contain a chymotrypsin-like fold in their 3D structures. This review focuses on our group’s efforts related to the development of norovirus protease inhibitors as potential anti-norovirus therapeutics. These protease inhibitors are rationally designed transition-state inhibitors encompassing dipeptidyl, tripeptidyl and macrocyclic compounds. Highly effective inhibitors validated in X-ray co-crystallization, enzyme and cell-based assays, as well as an animal model, were generated by launching an optimization campaign utilizing the initial hit compounds. A prodrug approach was also explored to improve the pharmacokinetics (PK) of the identified inhibitors.Publication Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress(Wiley, 2014-04) Vu, Hieu Sy; Roth, Mary R.; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelzc, Eric A.; Williams, Todd D.; Welti, RuthFormation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection, and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.Publication Discrimination of soluble and aggregation-prone proteins based on sequence information(Royal Society of Chemistry, 2013-04-05) Fang, Yaping; Fang, JianwenUnderstanding the factors governing protein solubility is a key to grasp the mechanisms of protein solubility and may provide insight into protein aggregation and misfolding related diseases such as Alzheimer’s disease. In this work, we attempt to identify factors important to protein solubility using feature selection. Firstly, we calculate 1438 features including physicochemical properties and statistics for each protein. Random Forest algorithm is used to select the most informative and the minimal subset of features based on their predictive performance. A predictive model is built based on 17 selected features. Compared with previous models, our model achieves better performance with a sensitivity of 0.82, specificity 0.85, ACC 0.84, AUC 0.91 and MCC 0.67. Furthermore, a model using redundancy-reduced dataset (sequence identity <= 30%) achieves the same performance as the model without redundancy reduction. Our results provide not only a reliable model for predicting protein solubility but also a list of features important to protein solubility. The predictive model is implemented as a freely available web application at http://shark.abl.ku.edu/ProS/.Publication Production, purification, and characterization of recombinant hFSH glycoforms for functional studies(Elsevier, 2015-04-15) Butnev, Viktor Y.; Butnev, Vladimir Y.; May, Jeffrey V.; Shuai, Bin; Tran, Patrick; White, William K.; Brown, Alan; Smalter Hall, Aaron; Harvey, David J.; Bousfield, George R.Previously, our laboratory demonstrated the existence of a β-subunit glycosylation-deficient human FSH glycoform, hFSH21. A third variant, hFSH18, has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH21 abundance in individual female pituitaries progressively decreased with increasing age. Hypo-glycosylated glycoform preparations are significantly more active than fully-glycosylated hFSH preparations. The purpose of this study was to produce, purify and chemically characterize both glycoform variants expressed by a mammalian cell line. Recombinant hFSH was expressed in a stable GH3 cell line and isolated from serum-free cell culture medium by sequential, hydrophobic and immunoaffinity chromatography. FSH glycoform fractions were separated by Superdex 75 gel-filtration. Western blot analysis revealed the presence of both hFSH18 and hFSH21 glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH21/18 -derived β-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHβ subunits, as both Asn7 and Asn24 were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH21/18 exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH24. Thus, the age-related reduction in hypo-glycosylated hFSH significantly reduces circulating levels of FSH biological activity that may further compromise reproductive function. Taken together, the ability to express and isolate recombinant hFSH glycoforms opens the way to study functional differences between them both in vivo and in vitro.Publication Constrained TRPV1 agonists synthesized via silver-mediated intramolecular azo-methine ylide cycloaddition of α-iminoamides(Elsevier, 2014-02-01) Painter, Thomas O.; Kaszas, Krisztian; Gross, Jacklyn; Douglas, Justin T.; Day, Victor W.; Iadarola, Michael J.; Santini, ConradAs part of an effort to identify agonists of TRPV1, a peripheral sensory nerve ion channel, high throughput screening of the NIH Small Molecule Repository (SMR) collection identified MLS002174161, a pentacyclic benzodiazepine. A synthesis effort was initiated that ultimately afforded racemic seco analogs 12 of the SMR compound via a silver mediated intramolecular [3+2] cycloaddition of an azo-methine ylide generated from α-iminoamides 11. The cycloaddition set four contiguous stereocenters and, in some cases, also spontaneously afforded imides 13 from 12. The synthesis of compounds 12, the features that facilitated the conversion of 12–13, and their partial agonist activity against TRPV1 are discussed.Publication Synthesis of a Family of Spirocyclic Scaffolds; Building Blocks for the Exploration of Chemical Space(American Chemical Society, 2013-07-05) Kumar, Sarvesh; Thornton, Paul; Painter, Thomas O.; Jain, Prashi; Downard, Jared; Douglas, Justin T.; Santini, ConradThis report describes the preparation of a series of 17 novel racemic spirocyclic scaffolds that are intended for the creation of compound libraries by parallel synthesis for biological screening. Each scaffold features two points of orthogonal diversification. The scaffolds are related to each other in four ways: Through stepwise changes in the size of the nitrogen bearing ring. Through the oxidation state of the carbon centered point of diversification. Through the relative stereochemical orientation of the two diversification sites in those members that are stereogenic. Through the provision of both saturated and unsaturated versions of the furan ring in the scaffold series derived from 3-piperidone. The scaffolds provide incremental changes in the relative orientation of the diversity components that would be introduced onto them. The scaffolds feature high sp3 carbon content which is essential for the three dimensional exploration of chemical space. This characteristic is particularly evident in those members of this family that bear two stereocenters, i.e. the two series derived from 3-piperidone and 3-pyrrolidinone. In the series derived from 3-piperidone we were able to “split the difference” between the two diastereomers by preparation of their corresponding unsaturated version.Publication Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?(Elsevier, 2008-12-27) Sharov, Victor S.; Galeva, Nadezhda A.; Dremina, Elena S.; Williams, Todd D.; Schoneich, ChristianThere is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function (Sharov et al., Exp. Gerontol. 41, 407–416; 2006); in another report, the nitration of one specific residue, Tyr613, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation (Dairou et al., J. Mol. Biol. 372, 1009–1021; 2007). In the present study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite.Publication LC-MS/MS quantification of salvinorin A from biological fluids(Royal Society of Chemistry, 2014-12-21) Caspers, Michael J.; Williams, Todd D.; Lovell, Kimberly M.; Lozama, Anthony; Butelman, Eduardo R.; Kreek, Mary Jeanne; Johnson, Matthew W.; Griffiths, Roland R.; MacLean, Katherine A.; Prisinzano, Thomas E.A facile method for quantifying the concentration of the powerful and widely available hallucinogen salvinorin A (a selective kappa opioid agonist) from non-human primate cerebrospinal fluid (CSF) and human plasma has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive electrospray ionization (ESI) mode. With CSF solid phase extraction can be avoided completely by simply diluting each sample to 10 % (v/v) acetonitrile, 1 % (v/v) formic acid and injecting under high aqueous conditions for analyte focusing. Extensive plasma sample preparation was investigated including protein precipitation, SPE column selection, and plasma particulate removal. Human plasma samples were centrifuged at 21,000 × gravity for 4 minutes to obtain clear particulate-free plasma, from which 300 μl was spiked with internal standard and loaded onto a C18 SPE column with a 100 mg mL−1 loading capacity. Guard columns (C18, hand packed 1 mm × 20 mm) were exchanged after backpressure increased above 4600psi, about 250 injections. A shallow acetonitrile/water gradient was used, 29 to 33% CH3CN over 8 minutes to elute salvinorin A. Reduction of chemical noise was achieved using tandem mass spectrometry with multiple reaction monitoring while sensitivity increases were observed using a 50 μL injection volume onto a small bore analytical column (C18, 1 mm ID × 50 mm) thus increasing peak concentration. Limits of quantification were found to be 0.0125 ng mL−1 (CSF) and 0.05 ng mL−1 (plasma) with interday precision and accuracy below 1.7 % and 9.42 % (CSF) and 3.47 % and 12.37 % (plasma) respectively. This method was used to determine the concentration of salvinorin A from an in vivo Rhesus monkey study and a trial of healthy human research participants, using behaviorally active doses.Publication A Methodology for Simultaneous Fluorogenic Derivatization and Boronate Affinity Enrichment of 3-Nitrotyrosine Containing Peptides(Elsevier Masson, 2012-11-15) Dremina, Elena S.; Li, Xiaobao; Galeva, Nadezhda A.; Sharov, Victor S.; Stobaugh, John F.; Schoneich, ChristianWe synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO2)LER, was employed as a model for method validation. Further, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of non-derivatized peptides, and to enrich them for fluorescent detection and MS identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.Publication A label-free mass spectrometry method for the quantification of protein isotypes(Elsevier Masson, 2013-07-15) Winefield, Robert D.; Williams, Todd D.; Himes, Richard H.Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIIItubulin represented 3.2% of the total HeLa β-tubulin.Publication Opioid Receptor Probes Derived from Cycloaddition of the Hallucinogen Natural Product Salvinorin A(Journal of Natural Products, 2011-02-21) Lozama, Anthony; Cunningham, Christopher W.; Caspers, Michael J.; Douglas, Justin T.; Dersch, Christina M.; Rothman, Richard B.; Prisinzano, Thomas E.As part of our continuing efforts toward more fully understanding the structure−activity relationships of the neoclerodane diterpene salvinorin A, we report the synthesis and biological characterization of unique cycloadducts through [4+2] Diels−Alder cycloaddition. Microwave-assisted methods were developed and successfully employed, aiding in functionalizing the chemically sensitive salvinorin A scaffold. This demonstrates the first reported results for both cycloaddition of the furan ring and functionalization via microwave-assisted methodology of the salvinorin A skeleton. The cycloadducts yielded herein introduce electron-withdrawing substituents and bulky aromatic groups into the C-12 position. Kappa opioid (KOP) receptor space was explored through aromatization of the bent oxanorbornadiene system possessed by the cycloadducts to a planar phenyl ring system. Although dimethyl- and diethylcarboxylate analogues 5 and 6 retain some affinity and selectivity for KOP receptors and are full agonists, their aromatized counterparts 13 and 14 have reduced affinity for KOP receptors. The methods developed herein signify a novel approach toward rapidly probing the structure−activity relationships of furan-containing natural products.Publication Antiproliferative Withanolides from Datura wrightii(Journal of Natural Products, 2012-12-19) Zhang, Huaping; Bazzill, Joseph; Gallagher, Robert J.; Subramanian, Chitra; Grogan, Patrick T.; Day, Victor W.; Kindscher, Kelly; Cohen, Mark S.; Timmermann, Barbara N.A new withanolide, named withawrightolide (1), and four known withanolides (2−5) were isolated from the aerial parts of Datura wrightii. The structure of compound 1 was elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withametelin L (2) was confirmed by X-ray crystallographic analysis. Using MTS viability assays, withanolides 1−5 showed antiproliferative activities against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cells with IC50 values in the range between 0.56 and 5.6 μM.