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1.  Multiplex Cytological Profiling Assay to Measure Diverse Cellular States 
PLoS ONE  2013;8(12):e80999.
Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that “paints the cell” with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar  annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.
doi:10.1371/journal.pone.0080999
PMCID: PMC3847047  PMID: 24312513
2.  Synthesis and Biological Evaluation of Non-Hydrolizable 1,2,3-Triazole Linked Sialic Acid Derivatives as Neuraminidase Inhibitors 
European journal of organic chemistry  2009;2009(16):10.1002/ejoc.200900117.
α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition (“click chemistry”). Our approach is to generate non-natural N-glycosides of sialic acid that are resistant to neuraminidase catalyzed hydrolysis as opposed to the natural O-glycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71-89% yield. A disaccharide mimic of sialic acid has also been prepared using the α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor in 68% yield. A model sialic acid coated dendrimer was also synthesized from a per-propargylated pentaerythritol acceptor. These novel sialic acid derivatives were then evaluated as potential neuraminidase inhibitors using a 96-well plate fluorescence assay; micromolar IC50 values were observed, comparable to the known sialidase inhibitor Neu5Ac2en.
doi:10.1002/ejoc.200900117
PMCID: PMC3818918  PMID: 24223493
Sialic acids; Cycloaddition; N-glycosides; Neuraminidase inhibitors; Influenza virus
3.  A Novel HDAC Inhibitor with a Hydroxy-Pyrimidine Scaffold 
Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.
doi:10.1016/j.bmcl.2011.05.098
PMCID: PMC3248787  PMID: 21696956
Histone deacetylase; non-selective inhibitor; hydroxy-pyrimidine; SAR studies
4.  Host and Pathogen Glycosaminoglycan-Binding Proteins Modulate Antimicrobial Peptide Responses in Drosophila melanogaster▿ †  
Infection and Immunity  2010;79(2):606-616.
During group B streptococcal infection, the alpha C protein (ACP) on the bacterial surface binds to host cell surface heparan sulfate proteoglycans (HSPGs) and facilitates entry of bacteria into human epithelial cells. Previous studies in a Drosophila melanogaster model showed that binding of ACP to the sulfated polysaccharide chains (glycosaminoglycans) of HSPGs promotes host death and is associated with higher bacterial burdens. We hypothesized that ACP-glycosaminoglycan binding might determine infection outcome by altering host responses to infection, such as expression of antimicrobial peptides. As glycosaminoglycans/HSPGs also interact with a number of endogenous secreted signaling molecules in Drosophila, we examined the effects of host and pathogen glycosaminoglycan/HSPG-binding structures in host survival of infection and antimicrobial peptide expression. Strikingly, host survival after infection with wild-type streptococci was enhanced among flies overexpressing the endogenous glycosaminoglycan/HSPG-binding morphogen Decapentaplegic—a transforming growth factor β-like Drosophila homolog of mammalian bone morphogenetic proteins—but not by flies overexpressing a mutant, non-glycosaminoglycan-binding Decapentaplegic, or the other endogenous glycosaminoglycan/HSPG-binding morphogens, Hedgehog and Wingless. While ACP-glycosaminoglycan binding was associated with enhanced transcription of peptidoglycan recognition proteins and antimicrobial peptides, Decapentaplegic overexpression suppressed transcription of these genes during streptococcal infection. Further, the glycosaminoglycan-binding domain of ACP competed with Decapentaplegic for binding to the soluble glycosaminoglycan heparin in an in vitro assay. These data suggest that, in addition to promoting bacterial entry into host cells, ACP competes with Decapentaplegic for binding to glycosaminoglycans/HSPGs during infection and that these bacterial and endogenous glycosaminoglycan-binding structures determine host survival and regulate antimicrobial peptide transcription.
doi:10.1128/IAI.00254-10
PMCID: PMC3028840  PMID: 21078848
5.  Hyaluronan- and heparin-reduced silver nanoparticles with antimicrobial properties 
Nanomedicine (London, England)  2009;4(4):421-429.
Aims
Silver nanoparticles exhibit unique antibacterial properties that make these ideal candidates for biological and medical applications. We utilized a clean method involving a single synthetic step to prepare silver nanoparticles that exhibit antimicrobial activity.
Materials & methods
These nanoparticles were prepared by reducing silver nitrate with diaminopyridinylated heparin (DAPHP) and hyaluronan (HA) polysaccharides and tested for their efficacy in inhibiting microbial growth.
Results & discussion
The resulting silver nanoparticles exhibit potent antimicrobial activity against Staphylococcus aureus and modest activity against Escherichia coli. Silver–HA showed greater antimicrobial activity than silver–DAPHP, while silver–glucose nanoparticles exhibited very weak antimicrobial activity. Neither HA nor DAPHP showed activity against S. aureus or E. coli.
Conclusion
These results suggest that DAPHP and HA silver nanoparticles have potential in antimicrobial therapeutic applications.
doi:10.2217/nnm.09.24
PMCID: PMC2717895  PMID: 19505245
antimicrobial; Escherichia coli; silver heparin nanoparticles; silver hyaluronan nanoparticles; silver nanoparticles; Staphylococcus aureus
6.  Synthesis of Gold and Silver Nanoparticles Stabilized with Glycosaminoglycans having Distinctive Biological Activities 
Biomacromolecules  2009;10(3):589-595.
Metal nanoparticles have been studied for their anticoagulant and anti-inflammatory efficacy in various models. Specifically, gold and silver nanoparticles exhibit properties that make these ideal candidates for biological applications. The typical synthesis of gold and silver nanoparticles incorporates contaminants that could pose further problems. Here we demonstrate a clean method of synthesizing gold and silver nanoparticles that exhibit biological functions. These nanoparticles were prepared by reducing AuCl4 and AgNO3 using heparin and hyaluronan, as both reducing and stabilizing agents. The particles show stability under physiological conditions, and narrow size distributions for heparin particles and wider distribution for hyaluronan particles. Studies show that the heparin nanoparticles exhibit anticoagulant properties. Additionally, either gold- or silver- heparin nanoparticles exhibit local anti-inflammatory properties without any significant effect on systemic hemostasis upon administration in carrageenan-induced paw edema models. In conclusion, gold and silver nanoparticles complexed with heparin demonstrated effective anticoagulant and anti-inflammatory efficacy, having potential in various local applications.
doi:10.1021/bm801266t
PMCID: PMC2765565  PMID: 19226107
7.  LC-MS to study chondroitin lyase action pattern 
Analytical biochemistry  2008;385(1):57-64.
Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes (chondroitinase ABC, Proteus vulgaris and chondroitinase ACII, Arthrobacter aurescens) having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host) were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparimum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.
doi:10.1016/j.ab.2008.10.014
PMCID: PMC2636793  PMID: 18992215
Liquid chromatography-mass spectrometry (LC-MS); Chondrontin lyase; Action pattern
8.  Oversulfated Chondroitin Sulfate: Impact of a Heparin Impurity, Associated with Adverse Clinical Events, on Low-Molecular-Weight Heparin Preparation 
Journal of medicinal chemistry  2008;51(18):5498-5501.
Heparin, a widely used anticoagulant, is being rapidly displaced by low-molecular-weight heparins. Recently, certain lots of heparin have been associated with anaphylactoid-type reactions resulting from contamination with oversulfated chondroitin sulfate. This impurity has also contaminated low-molecular-weight heparins obtained by chemical and enzymatic depolymerization of heparin. The sensitivity of oversulfated chondroitin sulfate to five different depolymerization processes similar to ones used in preparing low-molecular-weight heparins is reported.
doi:10.1021/jm800785t
PMCID: PMC2630253  PMID: 18754653
9.  Mosquito Heparan Sulfate and Its Potential Role in Malaria Infection and Transmission* 
The Journal of biological chemistry  2007;282(35):25376-25384.
Heparan sulfate has been isolated for the first time from the mosquito Anopheles stephensi, a known vector for Plasmodium parasites, the causative agents of malaria. Chondroitin sulfate, but not dermatan sulfate or hyaluronan, was also present in the mosquito. The glycosaminoglycans were isolated, from salivary glands and midguts of the mosquito in quantities sufficient for disaccharide microanalysis. Both of these organs are invaded at different stages of the Plasmodium life cycle. Mosquito heparan sulfate was found to contain the critical trisulfated disaccharide sequence, →4)β-D-GlcNS6S(1 → 4)-α-L-IdoA2S(1→, that is commonly found in human liver heparan sulfate, which serves as the receptor for apolipoprotein E and is also believed to be responsible for binding to the circumsporozoite protein found on the surface of the Plasmodium sporozoite. The heparan sulfate isolated from the whole mosquito binds to circumsporozoite protein, suggesting a role within the mosquito for infection and transmission of the Plasmodium parasite.
doi:10.1074/jbc.M704698200
PMCID: PMC2121605  PMID: 17597060
10.  Investigation of the Mechanism of Binding between Internalin B and Heparin using Surface Plasmon Resonance 
Biochemistry  2007;46(10):2697-2706.
Listeria monocytogenes, a food-borne pathogen that infects immunocompromised patients, enters and proliferates within mammalian cells by taking advantage of host cell machinery. While entry into macrophages and other phagocytic cells occurs constitutively, intracellular invasion of nonphagocytic cells, such as epithelial and endothelial cells, occurs through induced phagocytosis. Invasion of these nonphagocytic cell types is under the control of the secreted L. monocytogenes protein internalin B (InlB), which directly associates with and activates the receptor tyrosine kinase Met. Activation of Met by InlB has previously been shown to be potentiated by binding of glycosaminoglycans to the GW domains of this protein. We studied the interaction between heparin and full-length InlB as well as a truncated, functional form of InlB to understand the mode of interaction between these two molecules. InlB preferred long-chain (≥dp14) heparin oligosaccharides, and the interaction with heparin fit a complicated binding model with a dissociation constant in the nanomolar range. While there are various explanations for this complicated binding model, one supported by our data involves binding and rebinding of InlB to multiple binding sites on heparin in a positive and weakly cooperative manner. This mode is consistent with enhancement of interaction of InlB with glycosaminoglycans for activation of Met.
doi:10.1021/bi062021x
PMCID: PMC2034450  PMID: 17305366
11.  A Small-Molecule Probe of the Histone Methyltransferase G9a Induces Cellular Senescence in Pancreatic Adenocarcinoma 
ACS Chemical Biology  2012;7(7):1152-1157.
Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor genes, among others. Here, we report the discovery of a novel histone methyltransferase inhibitor, BRD4770. This compound reduced cellular levels of di- and trimethylated H3K9 without inducing apoptosis, induced senescence, and inhibited both anchorage-dependent and -independent proliferation in the pancreatic cancer cell line PANC-1. ATM-pathway activation, caused by either genetic or small-molecule inhibition of G9a, may mediate BRD4770-induced cell senescence. BRD4770 may be a useful tool to study G9a and its role in senescence and cancer cell biology.
doi:10.1021/cb300139y
PMCID: PMC3401036  PMID: 22536950

Results 1-11 (11)