chemical biology progress & challenges...2013/05/06 · sixth annual frontiers at the...

Chemical Biology Progress & Challenges

May 4th, 2013 College Park, Maryland

Sixth Annual Frontiers at the Chemistry‐Biology Interface Symposium University of Maryland College Park, May 4th, 2013

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Organizers

Herman O. Sintim and Shuwei Li (University of Maryland, College Park) Jim Fishbein (University of Maryland, Baltimore County) John Koh and Millie Sullivan (University of Delaware) Steve Rokita and Jin Zhang (Johns Hopkins University) E. James Petersson (University of Pennsylvania) Ronen Marmorstein (The Wistar Institute)

Sponsors Department of Chemistry The Wistar Institute University of Pennsylvania

Department of Chemistry and Biochemistry University of Delaware

School of Medicine, Department of Pharmacology Johns Hopkins University

Department of Chemistry Chemistry-Biology Interface Training Program John Hopkins University Johns Hopkins University

Department of Chemistry and Biochemistry University of Maryland, Baltimore County Department of Chemistry and Biochemistry University of Maryland, College Park

ACS Chemical Biology


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Program Schedule 8:00-9:00 Pick up name tag, breakfast and set up posters

9:00-9:05 am Introduction by UMD Department Chair, Michael Doyle

9:00-9:05 Welcome Message by UMD Department Chair, Michael Doyle

9:05-9:10 Introduction by Chair of the Organizing Committee, Herman Sintim

9:10-9:35 Ben L. Feringa (University of Groningen) Exploring Chiral Space in Asymmetric Catalysis 9:35-10:00 Alex Deiters (North Carolina State University) Synthetic Chemical Tools for the Regulation of Cellular Processes 10:00-10:20 Jason Kahn (Poster speaker, University of Maryland College Park) Rationally Designed Coiled‐coil DNA Looping Peptides Control DNA Topology

10:20-10:45 Barbara Gerratana (National Institute of Health) Navigating the NIH Funding Process

11:00-11:25 David Chenoweth (University of Pennsylvania) Structure Specific Nucleic Acid Targeting 11:25-11:50 Bruce Yu (University of Maryland Baltimore) Develop Fluorinated Dendrimers for 19F MRI 11:50-12:15 Tanakari Inoue (John Hopkins University) Total Synthesis of Chemotaxis

1:50-2:15 Cynthia Dowd (George Washington University) Dxr Inhibitors to Combat Mycobacterium tuberculosis 2:15-2:40 Catherine Grimes (University of Delaware) Chemical Tools to Study the Activation of the Intracellular Innate Immune Protein Nod2 2:40-3:00 Meiyao Wang (Poster speaker, National Institute of Standards and Technology)

Absolute Quantification and Isoforms Differentiation of Membrane‐Associated Proteins Using Multiple Reaction Monitoring Mass Spectrometry and Isotope Labeled Full‐Length Protein Standards

3:00-3:25 Christian Melander (North Carolina State University) Small Molecule Suppression of Antibiotic Resistance

9:00-10:45 AM, Session I Chair: James Petersson (University of Pennsylvania)

10:45-11:00 AM, Coffee Break

11:00-12:15 PM, Session II Chair: Catherine Grimes (University of Delaware)

12:15-1:50 PM, Lunch and Poster

1:50-3:25 PM, Session III Chair: Herman Sintim (University of Maryland, College Park)


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3:40-4:05 Hui Zhang (Johns Hopkins University) Glycomic Analysis Using Solid-Phase Glycan Extraction and Mass Spectrometry 4:05-4:30 Jeff Gildersleeve (National Cancer Institute) Carbohydrate-Binding Serum Antibodies as Biomarkers for Personalized Medicine 4:30-5:30 Gerald Hart (Keynote Speaker, John Hopkins University)

Glycomics Reveals Extensive Crosstalk Between O-GlcNAcylation and Other PTMs: Roles in Signaling, Transcription and Chronic Disease

3:25-3:40 PM, Coffee Break

3:40-5:30 PM, Session IV Chair: Shuwei Li (University of Maryland, College Park)

5:30-5:40 PM, Presentation of Poster Prizes and Closing Remarks Herman Sintim (University of Maryland, College Park)


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Posters (in alphabetic order by the presenter's last name)

1. Presenter: Ara Abramyan, University of the Sciences Complete List of Authors: Ara Abramyan, Zhiwei Liu, Vojislava Pophristic Title: "Molecular Dynamics Investigation of DNA-binding Foldamers" "Foldamers are synthetically derived oligomers inspired by the structures of biopolymers. Some of

these oligomers have been programmed to selectively recognize specific sequences in DNA. A number of human cancers are caused by dysregulation of transcription factors leading to an anomalous gene expression. Therefore ligands that are capable of binding DNA sequences and interrupting transcription factor-DNA interactions are of a great interest. We apply molecular dynamics methods to study oligoamides experimentally shown to bind to the DNA minor groove. We analyze structural changes in DNA upon oligoamide binding as well as the influence of various chemical and structural changes in a series of oligoamides. Particularly, we discuss how the presence and position of positively charged amino group on oligoamides' turn unit perturbs the DNA sequence of interest. Our aim is to design an oligoamide with improved affinity and selectivity for further experimental studies. "

2. Presenter: Smriti Agrawal, University of Delaware Complete List of Authors: "Smriti A. Agrawal, Atul Kakrana, David Scheiblin, Christine A. Dang,

Stephanie M. Waters, Abhyudai Singh, Hozumi Motohashi, Salil A. Lachke" Title: "Deficiency of small Maf family transcription factors MafG and MafK disrupts gene

regulation in the lens and causes cataract" "The ocular lens is a transparent tissue that functions to focus light on the retina and is critical for

providing us with high-resolution vision. Loss of lens transparency, which occurs due to genetic changes or aging, results in a disease called “cataract” – the leading cause of blindness worldwide. To devise strategies to delay or prevent cataract formation, it is critical to first understand how the lens develops and maintains transparency. We used a bioinformatics approach, iSyTE (integrated System Tool for Eye gene discovery) to identify the small Maf (musculoaponeurotic fibrosarcoma) family gene MafG as a potential regulator in the lens. The Maf gene family encodes basic leucine zipper transcription regulators that are classified into “large” and “small” MAF subgroups. Although mutations in the large Maf family gene Maf (c-Maf) are associated with human congenital cataracts, the function of the small Maf subgroup proteins MafG, MafK, and MafF in the lens remains uncharacterized. Our expression analysis confirmed iSyTE’s prediction that MafG is highly enriched in both embryonic and adult lens tissue. We also detected MafK expression in the lens although at significantly lower levels. To test the function of these small Maf genes in the lens, we generated and characterized mouse mutants that carry various combinations of MafG and MafK null alleles. These analyses demonstrate that Mafg-/-:Mafk+/- compound mutants exhibit severe lens defects starting at 2 months and develop distinct pre-senile cataract at age 6 months. To identify the target genes of these transcriptional regulators, we performed whole genome transcript profiling by microarrays on lenses from control and Mafg-/-:Mafk+/- mutant mice. These analyses indicate that expression of the heat shock protein gene Hspb1(Hsp27), a heme oxygenase gene Hmox1(Hsp32) and a novel gene Ttc27 are differentially regulated in Mafg-/-:Mafk+/- compound mutant lenses. Interestingly, Hspb1 is found to directly interact with and stabilize lens crystallin proteins Crystallin alpha A and Crystallin alpha B, which are necessary for lens transparency. Hspb1 is also found to be downregulated in mouse lenses carrying homozygous null mutations of the RNA granule component gene Tdrd7, deficiency of which in mouse or human causes cataract. Collectively, our data have identified and characterized a new role for MafG and MafK in controlling lens fiber cell gene expression, and demonstrated that deficiency of these proteins in mouse results in cataract. "

3. Presenter: Solongo Batjargal, University of Pennsylvania Complete List of Authors: "Solongo Batjargal, E. James Petersson" Title: "Labeling Proteins with Fluorophore/Thioamide FRET Pairs"


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"A thioamide, the substitution of a carbonyl oxygen with a sulfur in the peptide backbone, is capable of quenching the fluorescence of p-cyanophenylalanine (Cnf) in a distant dependent fashion. The combination of native chemical ligation and unnatural amino acid (UAA) mutagenesis can be used to generate a thioamide/Cnf FRET pair in a protein. We recently have shown that a thioamide containing full-sized protein can be generated by ligating N-terminal Cys protein expressed in E. Coli and thioamide-containing peptide thioester. This method locates the thioamide in the N-terminal region of the protein. To insert thioamides at the C-terminus, we demonstrate that the intein fusion constructs can be employed to generate a protein thioester. The use of UAA mutagenesis and intein fusion permits us to obtain a Cnf-labeled protein thioester fragment which then can be ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for quick access to double-labeled proteins with site-specificity."

4. Presenter: Petrina Boucher, University of Maryland Complete List of Authors: Steven Rokita Title: "Flavin Electron Transfer During the Reductive Dehalogenation of Iodotyrosine

Deiodinase" "Iodotyrosine deiodinase (IYD) is the flavoprotein responsible for iodide salvage from the mono- and

diiodotyrosine byproducts of thyroxine biosynthesis in the thyroid. It is one of the few known mammalian enzymes capable of reductive dehalogenation. Crystal structures of IYD have shown aromatic stacking between the substrate and FMN, which aligns the C-I bond over the C4a position of FMN. Still, the exact mechanism of reduction and deiodination is not clear. As a flavoprotein, IYD possesses the ability to reduce its substrate by either a single two electron transfer or by two sequential one electron transfers with a radical intermediate at the C4a position that is known to be quite stable. Distinction between the radical and non-radical processes will focus subsequent mechanistic studies. Flavin analogs provide a convenient approach to differentiate between these alternatives. 5-Deazariboflavin contains a carbon atom in place of nitrogen at the 5-position of the isoalloxazine ring rendering it incapable of stabilizing radical formation at the C4a position, allowing only transfer of electron pairs. 1-Deazariboflavin retains the ability to undergo both types of electron transfer processes, as the central pyrazine ring remains intact. However, replacement of the nitrogen at 1- position of the isoalloxazine ring provides a control for how general changes to the redox potential affects activity. Organic synthesis and enzymatic phosphorylation of the above mentioned flavin analogs will result in the formation of both the 1-deaza- and 5-deazaFMN products. Bacterial expression of the human IYD enzyme in E. coli has been carried out for in vitro studies to probe the redox chemistry, substrate recognition and activation parameters necessary for dehalogenation. Reconstitution of the native enzyme with these deaza-analogs will provide rate constants and substrate binding affinities for the deazaFMN substituted enzyme. Expectations suggest the reductive dehalogenation catalyzed by IYD occurs via two single electron transfer steps. "

5. Presenter: Michael Bruist, University of teh Sciences Complete List of Authors: Michael F. Bruist, Cassandre Cavanaugh Title: "Molecular dynamics study of the stability of the sarcin/ricin domain of RNA" "The importance of noncoding RNAs is being appreciated more. These participate in regulation of all

aspects of nucleic acids biology and also have structural and catalytic roles. Noncoding RNAs are assembled from motifs, A-form helices and unstructured single strands. I am using molecular dynamics (MD) to understand the basis of stability of RNA motifs, the dynamics of these motifs and their junctions to other motifs and helices. The sarcin/ricin domain is one of the most common motifs in RNA. It is used by RNAs to interact with proteins and may also provide a separator of variable stiffness. The stability of the SRD motif is being studied in an RNA ""dumbbell"". Such dumbbells are formed from circular RNAs in which opposite strands in the circle form a double helix capped by a hairpin loop on each end. In this structure the SRD is flanked by a G/C-rich and an A/U-rich helix each capped with a closing tetraloop. The AU- and GC-rich sequences provide examples of known RNA. Adaptively


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biased molecular dynamics and replica exchange methods are being used to explore the free energy profile as the dumbbell begins to melt. I have found that more conformations can be explored rapidly when the negative charges of the RNA are not completely neutralized by the cations. Artificial cations with less than unit charge allow multiple runs with different degree of neutralization. I am now comparing the conformations explored under these conditions with those seen in a standard net neutralized system. This research is supported in part by a gift from the H. O. West Foundation, and grants from the University of the Sciences in Philadelphia and the National Science Foundation (CHE-1229564). "

6. Presenter: Evan Cameron, "University of Maryland, Baltimore County" Complete List of Authors: "Evan Cameron, Phyllis Robinson" Title: "Exploring Arrestin Function in the Melanopsin Signaling Pathway" "The unique photopigment, melanopsin, is restricted to a small subset of retinal ganglion cells (RGCs)

within the mammalian retina. These intrinsically photosensitive cells (ipRGCs) regulate numerous non-visual functions including sleep, circadian photoentrainment and pupil constriction. IpRGCs exhibit attenuated response characteristics following intense and prolonged light exposures indicative of an adaptational response. This notion is supported by the phosphorylation dependent reduction of melanopsin signaling in vitro and ubiquitous expression of β-arrestin in the retina. These observations along with the conspicuous absence of visual arrestin in ipRGCs suggest melanopsin is deactivated in a β-arrestin dependent manner. The overall goal of this research is to identify the role of βarr1 and βarr2 in the melanopsin signaling pathway and elucidate the molecular mechanisms that contribute to ipRGC adaptation. Double labeling experiments utilizing melanopsin and β-arrestin antibodies confirm co-expression of these proteins in mouse ipRGCs, supporting their putative interaction. Using an in vitro calcium imaging assay we can monitor the affect of βarr1 and βarr2 on melanopsin signaling. Our studies demonstrate increasing concentrations of βarr1 and βarr2 significantly inhibit the melanopsin calcium response. Furthermore, we show that this effect is absolutely dependent on melanopsin C-tail phosphorylation. Co-IP experiments confirm βarr1 and βarr2 binding with melanopsin in a light dependent and phosphorylation dependent manner. These data are further supported by proximity ligation assays (PLA) which demonstrate a melanopsin/β-arrestin interaction within fixed HEK293A cells. In the future, in situ PLAs will be attempted to show the melanopsin/β-arrestin interaction within ipRGCs. Overall, this evidence supports a β-arrestin dependent deactivation of melanopsin and suggests melanopsin is internalized following deactivation. "

7. Presenter: Michael Cavalier, University of Maryland Baltimore Complete List of Authors: "Michael C. Cavalier, Laura E. McKnight, E. Prabhu Raman, Padmavani

Bezawada, Sucheta Kudrimoti, Paul T. Wilder, Kira G. Hartman, Eric A. Toth, Andrew Coop, Alexander D. MacKerell Jr. , and David J. Weber†"

Title: "Structure/Function Characterization of Five Inhibitors of the Calcium-Binding Protein S100B"

"The S100 family of proteins displays no enzymatic activity but has been shown to play roles in an array of biological functions such as calcium homeostasis, cellular growth, inflammation, and metabolism. The influence of these homodimeric proteins is mediated through protein-protein interactions in a typically calcium dependent manner and aberrant expression of S100 proteins has been linked to a variety of cancers, cardiomyopathies, and neurological disorders. Of particular interest, S100B is highly over-expressed in many cancers, including malignant melanoma (MM). In MM, S100B is used as prognostic indicator in evaluating treatment success and in predicting relapse. Studies now show that S100B is more than a clinical marker, and that it binds wild-type p53 in a calcium-dependent manner, sequestering it, and promoting its degradation. This results in the loss of p53-dependent tumor suppression activities. As inhibition of S100B expression by RNA interference restores both wild-type p53 protein levels and normal transcriptional activation/apoptosis activities, we have made it our long-term goal is to specifically inhibit the S100B-p53 interaction in order to restore the p53 tumor


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suppression function as a potential therapy for malignant melanoma. Many S100B inhibitors (also known as SBiXs) have been identified, and studies of structure-activity relationships (SAR) are under way to aid in the development and optimization of novel SBiXs. As described within, the binding of SBiXs to S100B has been investigated using X-ray crystallography. Additionally, NMR data including chemical shift perturbations of isotopically labeled protein amides were consistent with the X-ray crystal structures and demonstrated the relevance of these SBiX-S100B complexes in solution. This new understanding of the SBiX structure/function relationship will impact the development and optimization of future SBiXs and potential therapeutics in the treatment of malignant melanoma. "

8. Presenter: Xing Chen, University of Pennsylvania Complete List of Authors: "Xing S. Chen, Jocob M. Goldberg, E. James Petersson" Title: "Thioamides as Fluorescence Quenching Probes to Study Proteases" "Thioamides can be paired with a variety of fluorophores to design “turn-on fluorescence” protease

substrates. These peptides or proteins contain a thioamide and a selectively-excitable fluorophore on opposite ends of a target recognition sequence. Incubation with a proteolytic enzyme leads to cleavage, which can be monitored in real time as an increase in fluorescence. Although there are many conventional labeling strategies that operate on the same premise, we believe that the small size of the thioamide affords investigators the ability to scan candidate substrate sequences more thoroughly and accurately than other methods would allow. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases including chymotrypsin, trypsin, papain, pepsin, and thermolysin. Additionally, we are conducting experiments with calpain, a calcium-dependent cysteine protease implicated in many disease pathologies. Since thioamides quench bright fluorophores, such as fluorescein, we expect to be able to apply this technique to site-specifically monitoring protease activity in vivo or in cell lysate."

9. Presenter: Kenneth Childers, "University of Maryland, Baltimore County" Complete List of Authors: Kenneth Childers and Elsa Garcin Title: "Structural studies of β-HNOX-HNOXA construct of soluble guanylate cyclase" "Soluble guanylate cyclase (sGC) is a heterodimeric enzyme that catalyzes the conversion of GTP into

cGMP for cardiovascular homeostasis by inducing vasodilation and inhibiting platelet aggregation. The enzyme uses a heme prosthetic group to bind nitric oxide (NO) and stimulate its activity. Dysfunctions of the NO-sGC-cGMP pathway have been linked to many cardiovascular diseases including hypertension and atherosclerosis. Thus there is huge interest in discovering small molecules that can activate sGC independently of NO. sGC consists of an α and β chain with four domains- a heme nitric oxide oxygen (HNOX) domain, HNOX-A, a coiled-coiled domain (CC), and a guanylate cyclase catalytic domain (GC). The β chain is the only chain with a heme group located in the HNOX domain. The unligated sGC binds its heme group through a histidine bond. Once NO binds to the heme, this histidine bond is broken and the cGMP output from the enzyme increases anywhere between 50 and 200 fold. However, how the enzyme activity is increased via NO binding to the HNOX domain remains largely unknown. To date, there is no x-ray crystal structure of a mammalian sGC HNOX domain. It is the goal of this research to elucidate that mechanism of NO-induced sGC activation at the molecular level. To achieve this goal, we will (1) express and purify a mammalian sGC construct encompassing the HNOX domain with proper heme incorporation and (2) crystallize and solve the x-ray structure of this construct. These results should provide a better understanding of the mechanisms responsible for sGC activation and guide the design of better sGC activation to treat cardiovascular diseases. "

10. Presenter: Szu-Ting Chou, Univeristy of Maryland Complete List of Authors: "Szu-Ting Chou, Kellie Hom, Lucas Tricoli, Jason Hustedt, Amy Lee, Joonil

Seog, Jason Kahn, Qixin Leng, Daoning Zhang, Michael Shapiro, Archibald J. Mixson" Title: "Silencing Activity of Imidazole-containing Peptide siRNA Polyplexes Are Dependent on

Hydrogen Bonding"


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"Branched peptides containing lysines and histidines (HK) have been determined to be effective carriers for DNA or siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the polyplex. In siRNA silencing experiments with mammalian cells, a four branched HK peptide siRNA polyplex maintained silencing activity even with prolonged pre-incubation with serum. In contrast, siRNA in complex with four-branched N3K4b peptide, in which histidines were substituted with asparagines, showed a marked decreased in silencing activity with pre-exposure to serum. Consequently, we explored the hypothesis that histidine might form non-covalent bonds with nucleic acids to enhance the stability of siRNA polyplexes. To accomplish this, we initially compared the biophysical properties of H3K(+H)4b with N3K4b and polylysine. Consistent with siRNA silencing experiments, gel electrophoresis analysis demonstrated that the HK siRNA polyplex maintained its integrity for more than 24 h incubation in 50% serum, whereas siRNA in complex to N3K4b or polylysine was degraded in a time-dependent manner. We next studied the thermodynamic profiles of various peptides binding to siRNA at pH 7.3 with isothermal titration calorimetry. While polylysine and linear lysine-alanine peptide (A2K) interacted with siRNA resulting in an endothermic reaction, branched and linear lysine-histidine peptides (H3K(+H)4b and H2K) both exhibited an exothermic reaction. This indicates that an important part of the exothermic interaction was based on non-ionic bond formation of histidines with siRNA. To investigate the type of non-ionic bond, we studied the protonation state of the imidazole ring of a selectively N15 labeled H3K(+H)4b upon siRNA binding with heteronuclear single quantum coherence (HSQC) NMR. The peak of Nδ1-protonated tautomers of imidazole (type α, neutral form) shifted downfield (in the direction of deprotonation, represented by the green arrows in the figure) by 0.5 to 1.0 ppm with addition of siRNA at pH 7.3, providing direct evidence that uncharged histidines formed hydrogen bonds with siRNA at physiological pH. Moreover, unshifted Nε1 tautomers (black boxes) described the directionality of hydrogen bonds. Taking together, these results indicate that histidine-rich peptides form non-ionic bonds, and in particular hydrogen bonds, with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo. "

11. Presenter: Gail V. Clements, The George Washington University Complete List of Authors: Gail V. Clements Alex S. Yepikhin Helena I. Boshoff Cynthia S. Dowd Title: "Mycobacterium tuberculosis Proteasome Inhibitors" "The World Health Organization estimates that in 2012 over 2 million people will die from TB, caused

by the bacteria Mycobacterium tuberculosis (Mtb). Mtb is difficult to eradicate because the bacteria are often in a latent state and unresponsive to current drugs. Inhibition of cellular components that are required for the bacteria’s survival in the latent phase might help to eliminate this disease. One such target is the proteasome, the largest protein complex involved in protein degradation. The proteasome has been shown to be essential for TB in its latent phase. Genomic and crystallographic studies show that the Mtb proteasome is similar to, but not exactly like, the human proteasome. Ligands specifically designed to have greater affinity for the Mtb proteasome over the human proteasome were modeled in silico, synthesized and assayed for their potency on Mtb cells. The modeling, synthetic methods and biological assay results will be discussed. "

12. Presenter: Nicholas C. Corsepius, University of Maryland College Park Complete List of Authors: Nicholas C. Corsepius1,2,3 and George H. Lorimer1,2,3,4* 1) Center for Biological

Structure and Organization, University of Maryland, College Park, MD20742. 2) Biochemistry Graduate Program, University of Maryland, College Park, MD20742. 3) Department of Chemistry and Biochemistry, University of Maryland, College Park, MD20742. 4) Institute for Physical Science and Technology, University of Maryland, College Park, MD20742.

Title: "How much work can GroEL do?" "Non-covalently “stacked” tetramethylrhodamine (TMR) dimers have been used to both report and

perturb the allosteric equilibrium in GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to the peptide-binding site in the apical domain, such that TMR molecules on adjacent subunits are able


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to form dimers in the T allosteric state. Addition of ATP induces the transition to the R state, the dispersion of the peptide-binding sites, and the un-stacking of the TMR dimers. A statistical analysis of the spectra, assuming a binomial distribution, allowed us to compute the number and orientation of TMR dimers per ring as a function of the average number of TMR molecules per ring. The TMR dimers thus serve as quantitative reporter of the allosteric state of the system. Additionally the TMR dimers also serve as a surrogate for substrate protein substituting in a more homogeneous, quantifiable manner for the heterogeneous inter-subunit, intra-ring, non-covalent cross-links provided by the substrate protein. The characteristic stimulation of the ATPase activity by substrate protein is also mimicked by the TMR dimers. Using an expanded version of the nested cooperativity model we determine values for the free energy of the TT to TR and TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, respectively. The free energy of unstacking of the TMR dimers was estimated at 2.6 ± 1.0 kJ/mole dimer. These results demonstrate that GroEL performs work during the T to R transition, supporting the iterative annealing model of chaperonin function."

13. Presenter: Di Cui, University of Delaware Complete List of Authors: Di Cui Shuching Ou Yuan Hu Sandeep Patel Title: "Study of spanning water networks of hydration water in crowded protein solutions" "Living cells contain high concentrations of macromolecules such as proteins, resulting in a crowded

environment. The crowded environment significantly affects the state of hydration water around proteins. For isolated proteins, using molecular dynamics simulations, we study the transition between two distinct states of hydration water, states with small, disconnected clusters and states with a spanning hydrogen-bonded water network. With increasing hydration shell water molecules, a spanning H-bonded network was formed around protein surfaces via a percolation transition. We consider the nature of the surface water network for the ubiquitin system. We attempt to connect the protein-protein dimerization interface with the dynamical characteristics of the enveloping hydration layer of water molecules. We will provide further analysis of interactions of ubiquitin with binding motifs in model crowded cellular environments. "

14. Presenter: Vidyadhar Daithankar, University of Delaware Complete List of Authors: "Vidyadhar Daithankar, Fei Li, Sharon Rozovsy" Title: "Redox characterization of human selenoprotein M and its putative role in oxidative

stress" "The health benefits of essential trace element selenium have been attributed to selenoproteins, a class

of proteins containing genetically encoded 21st amino acid selenocysteine. Besides thioredoxin reductase and glutathione peroxidases, two well-studied examples of the selenoproteome family, the enzymatic functions of other family members remain elusive. About one third of these selenoproteins possess a minimal thioredoxin fold with the reactive selenocysteine positioned on a flexible loop where it is easily accessible to substrates. This study focuses on characterization of a representative member of the family – the human selenoprotein M (SelM). SelM is localized to endoplasmic reticulum and is highly expressed in brain where it protects the neurons from oxidative stress. Herein, we have identified selenocysteine dependent peroxidase and reductase activities of SelM. In addition we present measurements of the redox potential of SelM. Since the redox potentials of selenoproteins are by large unknown, the determination of redox potentials of selenoproteins with minimal thioredoxin fold will help us understand their physiological functions."

15. Presenter: Blessing Deeyaa, Johns Hopkins University Complete List of Authors: "Blessing D. Deeyaa, Steven Rokita" Title: "Reversible DNA Alkylation by Pyrrolizidine Alkaloids: Monocrotaline" "Pyrrolizidine alkaloids (PAs) represent a class of plant derived natural products that disrupt cellular

function by alkylating DNA and proteins. Monocrotaline, a compound belonging to the PA family, is dehydrogenated by cytochrome P450 oxidases (CYP 450) to generate the cytotoxic


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dehydromonocrotaline (DHM). Loss of the macrocyclic ester leads to the formation of two electrophilic carbons. This is similar to a process well studied in the Rokita lab, in which an ortho-quinone methide (QM) intermediate is generated and used to crosslink DNA reversibly. Nucleophilic DNA bases and amino acid residues have been shown to alkylate each electrophilic carbon of QM and DHM, forming DNA-DNA and DNA-protein crosslinks. The electron rich nature of the DHM ring and the good leaving ability of the most nucleophilic DNA sites lead us to believe that DHM may also be capable of forming reversible DNA crosslinks. Current studies are testing the reversibility of DHM-DNA adducts using dsDNA. The reversibility of alkylation in DHM could serve as a template for optimizing selective degradation of diseased cells in anti-cancer therapy."

16. Presenter: Xue Fei, University of Maryland College Park Complete List of Authors: Xue Fei1,2 , Dong Yang2,3,4, Nicole LaRonde-LeBlanc2,3,4 and George H.

Lorimer1,2,3,4,5*, 1) Biophysics Graduate Program, University of Maryland, College Park, MD20742. 2) Center for Biological Structure and Organization, University of Maryland, College Park, MD20742. 3) Biochemistry Graduate Program, University of Maryland, College Park, MD20742. 4) Department of Chemistry and Biochemistry, University of Maryland, College Park, MD20742. 5) Institute for Physical Science and Technology, University of Maryland, College Park, MD 20742.

Title: "Crystal Structure of a GroEL-ADP complex in the Relaxed Allosteric State" "In assisting other proteins to fold, the chaperonin GroEL/ES cycles through a series of conformational

states, from T ⇔ R ⇔ R” ⇔ T. We report the first crystal structure of GroEL in the R state, D83A/R197A GroEL-ADP14 at 2.7Å resolution. To achieve, this two (X14) salt-bridges, known to break during the T ⇔ R transition, have been removed. The intermediate and apical domains of the R state adopt a distinctly asymmetric conformation compared with that in the T or R” states. This asymmetry, caused by the loss of inter-subunit contacts, was overlooked by the imposition of symmetry in a cryo-EM study of GroEL-ATP. Biochemical analyses of the D83A/R197A are consistent with the destabilization of the T state, in accordance with the crystal structure. The structural changes during the T ⇔ R ⇔ R” transitions suggest a structural basis for substrate-protein accelerated nucleotide exchange."

17. Presenter: Madison Fletcher, Temple University Complete List of Authors: Madison Fletcher, Megan Jennings, Bulan Wu, Lifan He, Stacy Passciola,

William Wuest* Title: "Synthesis of Non-Hydrolyzable pGpG Analogs" "The necessity of developing novel antibiotics as well as novel modes of inhibition for bacteria is

abundant. This is especially true for biofilm producing bacteria such as P. aeruginosa, P. fluorescens, and P. syringae. Drug resistance is at an all-time high and through the investigation and development of new compounds and molecular pathways we endeavor to provide biologically active and interesting molecules to the scientific community. We, like others, are interested in the elaboration of the biofilm propagation mechanism that is instigated by the small molecule cyclic-di-GMP. In the pathway of synthesizing these analogs, we will develop preliminary linear pGpG compounds. By generating these analogues we hope to be able to probe the molecules biological targets and discover novel and inhibitory molecules that can allow for better disbursement of biofilms and these harmful bacteria. Here, we report the synthesis of a biologically active pGpG analog, as well as the synthetic pathways of other analogs currently under development."

18. Presenter: Christina Forbes, University of Delaware Complete List of Authors: Neal J. Zondlo Title: "Thiophenylalanine as a new tool for biological chemistry of thiols and synthetic

proteins" "Cysteine is a uniquely reactive residue that has critical roles in redox chemistry, post-translational

modifications, cell signaling, protein folding, and is synthetically modified for protein tagging and


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modification. Aryl thiolates represent an intriguing functional hybrid of tyrosine and cysteine, and would serve as versatile tools for understanding the sulfur redox proteome and would function as a site for ready modification. We have developed a practical synthesis for thiophenylalanine from iodophenylalanine in high yield in peptides on solid phase using a novel copper-mediated cross-coupling strategy. This practically accessible amino acid is readily functionalized due to its increased nucleophilicity and acidity (pKa = 6.4 as 4-thiophenylalanine) and can tune protein structure via an aromatic-prolyl CH/� interaction. This electronic structural control of the cis-trans isomerism of the aromatic-prolyl amide bond was further examined in the context of 57 TXPN model peptides. 4-Thiophenylalanine was also readily modified in solution phase peptides to form versatile thioethers (i.e. S-allyl for cross-metathesis conjugations, 2-nitrobenzyl thioether as a photocleavable protecting group), the S-glutathionylated disulfide, sulfoxide, sulfone, sulfinic, and sulfonic acids, and S-nitrosyl, and was demonstrated as a spectroscopic probe for sulfur oxidation state. This cross-coupling approach was also applied to synthesize the redox-active thiocatechol amino acid 3-mercaptotyrosine. 4-Thiophenylalanine was incorporated as a tyrosine analogue into the trp cage miniprotein as a switch for structural modulation. 2-Thiophenylalanine at the N-terminus of a peptide has increased acidity (pKa = 5.1), and was used as a rapid, practical approach to native chemical ligation at phenylalanine. Herein, potential applications of this versatile amino acid in chemical biology are presented."

19. Presenter: Ian Glassford, Temple University Complete List of Authors: "Ian Glassford, Miseon Lee, Bharat Wagh and Rodrigo B. Andrade" Title: "Discovery of Novel Macrolide Antibiotics: The Total Synthesis and Biological Evaluation

of 4-desmethyl Telithromycin" "The rapid emergence of antibiotic-resistant bacteria due in part to overuse and misuse of current

antibiotics presents a serious health threat that shows no signs of abatement. A sharp decline in the number of pharmaceutical companies with active antimicrobial research programs underscores the need for new sources of antibiotics. As a part of a rational structure-based drug design program, we apply the paradigm of natural product structural simplification (i.e., desmethylation) to the 3rd-generation macrolide antibiotic telithromycin (2), which is an FDA-approved semisynthetic analogue of erythromycin (1) in clinical use since 2004. The rationale behind desmethylation comes from crystallographic studies of 1 and 2 bound to ribosomal subunits, which corroborate biochemical mechanisms of antibiotic resistance (e.g., ribosomal modification and mutation). From his original work co-crystalizing telithromycin with the ribosome of Haloarcula marismortui, Steitz has put forth a ‘steric clash’ hypothesis to rationalize how A2058G ribosomal mutations confer bacterial resistance; namely, the change from an adenine (A) to guanine (G) results in the replacement of a proximal hydrogen in adenine to an amine group in guanine. As a consequence, a steric clash between the C4 methyl and the amino group of guanine would result. In turn, we hypothesize that the replacement of the C4 methyl group of 2 with hydrogen should relieve this ‘steric clash’ and thus offer a means of addressing antibiotic resistance arising from mutation by desmethylation. As there is no facile way to synthesize desmethyl telithromycin analogues from 1 or 2, total synthesis was employed to synthesize the desired analogues. To date our lab has accomplished the total synthesis and bilological evaluation of 4-desmethyl telithromycin (3), 4,8,10-tridesmethyl telithromycin (4), 4,8-didesmethyl telithromycin (5), and 4,10 didesmethyl telithromycin (6) which all displayed biological activity against both wild type and mutant bacterial strains and comparable to 2 in potency. This work will highlight our effort of the total synthesis and biological evaluation of 3. "

20. Presenter: Brandy Haines-Menges, University of Delaware Complete List of Authors: B.L. Haines-Menges E.F. Boyd Title: "The Discoordinated Role of Sigma factor, RpoE and Outer Membrane Protein, OmpU in

the Stress Response of Vibrio parahaemolyticus" "Vibrio parahaemolyticus is a moderate halophile with an absolute requirement for salt and inhabits

brackish waters, such as marine and estuarine environments. V. parahaemolyticus is ubiquitous within


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its environment, having the ability to colonize a wide range of host including crustaceans, fish, mollusks, and humans, and can also be found as a free living organism, or in association with zooplankton. In humans it is the leading cause of bacterial seafood borne gastroenteritis. Due to its diversity of niches, V. parahaemolyticus must adapt to a number of environmental and host stresses. The focus of this work is to investigate the role of alternative sigma-factors in the stress response of V. parahaemolyticus. Alternative sigma-factors are global regulators that provide a mechanism for redirecting transcription initiation allowing the bacteria to respond to various stimuli. We show here that the number of sigma factors varies among Vibrio species, with the greatest diversity seen among extracytoplasmic factors (ECF) and RpoS-like sigma factors. We demonstrate that V. parahaemolyticus has 11 sigma factors, including 4 putative ECF-factors, the function of which is unknown. We also demonstrate that one such sigma-factor, RpoE (σE) is essential for survival under a number of cell envelope stress conditions and is important in gastrointestinal colonization of a streptomycin-treated adult murine model. In addition, we examined the role of the outer membrane protein, OmpU, in cell membrane stress response and as a potential signal for the release and increased expression of RpoE. In V. parahaemolyticus RIMD2210622 we suggest OmpU does not play a role in RpoE function under the conditions studied."

21. Presenter: Romina R. Heymann, University of Maryland College Park Complete List of Authors: Romina R. Heymann and Daniel Falvey Title: "New light triggered calcium release system that creates biologically relevant hydrogels" "Calcium is an important second messenger in biological systems controlling many aspects of cellular

function and is used to produce biocompatible alginate hydrogels. The release of calcium with light has several distinct advantages including that light can be directed making the area of release highly specific, as well as being a rapid and easily controlled trigger. Previous photorelease mechanisms include the photochemical scission of a nitrophenyl derivatized calcium chelators such as EGTA. Our study involves the release of calcium through an electron abstraction mechanism using an electron accepting sensitizer and a calcium chelator, EDTA. The sensitizers that were investigated consisted of riboflavin, riboflavin tetraacetate,and several anthraquinone sulfonic acid derivatives. The excited state sensitizers accepts an electron from EDTA causing the degradation of EDTA and subsequent calcium release at the biologically relevant pH range of 6-8. Experimental evidence of calcium release and its mechanism include: UV-VIS assays, NMR spectroscopy and gelation studies."

22. Presenter: JIMIN HU, University of Maryland-College Park Complete List of Authors: "Authors: Jimin Hu1, Watchalee Chuenchor1,2 and Steven E Rokita1,3 1)

Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, United States; 2) National Institutes of Health, Bethesda, MD, 20892, United States; 3) Department of Chemistry, Johns Hopkins University, Baltimore, MD, 21218, United States "

Title: "Structural, Kinetic and Redox Characteristics of Human Iodotyrosine Deiodinase" "Iodotyrosine deiodinase (IYD) is critical for maintaining the iodide level of mammals by recycling iodide

from mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) with assistance of its cofactor, FMN. IYD is a unique flavoprotein since it catalyzes reductive dehalogenation under aerobic conditions. While previous studies investigated the enzyme from mouse, the human enzyme is now available by heterologous expression in E.coli and purification by a Ni2+ column. Steady-state kinetics and binding parameters of the human IYD are similar to those reported for mouse IYD. The crystal structure of human IYD has also been solved, providing invaluable information on the interactions between the zwitterion region of the substrate MIT and the pyrimidine ring of FMN. This interaction influences the redox potential of FMN and likely controls the reductive dehalogation process. The redox potential of FMN within IYD has been examined by the xanthine-xanthine oxidase method. Formation of the neutral flavin semiquinone is only detected in the presence of a substrate analogue, mono-fluorotyrosine."

23. Presenter: YUAN HU, UNIVERSITY OF DELAWARE Complete List of Authors: "Yuan Hu, Shuching Ou, Sandeep Patel*"


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Title: "Free Energetics of Polyarginine Permeation into Model Lipid Bilayers" "Cationic arginine-rich peptides, such as Tat and nona-arginine, are known to transfer into cells via

membrane permeation pathways. Their fast membrane penetrating ability is promising as a vector to deliver therapeutic molecules into cells, both in vitro and in vivo. The exact, general mechanism of translocation is still not fully understood or agreed upon. Two possible mechanisms are proposed: endocytotic and non-endocytic pathways. Using molecular dynamics simulations we study the free energetics of polyarginine permeation into model DMPC bilayers. The potential of mean force (PMF) for various polyarginines from bulk water to the center of lipid bilayer are computed using adaptive biasing force (ABF) methods. We will discuss aspects of the relative stability of peptides of varying length and factors stabilizing (destabilizing) various peptides. Moreover, we will comment on non-additive aspects of membrane permeation of polyarginines."

24. Presenter: Benjamin Israel, University of Delaware Complete List of Authors: Benjamin A. Israel, Vamsi K. Kodali, and Colin Thorpe Title: "Dissecting catalysis: probing the redox balance of the Quiescin sulfhydryl oxidase from

Trypanosoma brucei" "The Quiescin sulfhydryl oxidase (QSOX) enzymes are flavoproteins capable of efficiently generating

disulfide bonds in protein substrates. During catalysis, electrons are transferred through three successive redox centers to reach molecular oxygen. Here we explore the role of redox potentials on individual catalytic steps in the QSOX mechanism. We determined the redox potentials of the thioredoxin CxxC motif (CxxCTrx), the proximal CxxC (CxxCprox), and the flavin prosthetic group of QSOX. To explore the importance of thermodynamic balance between the redox centers in QSOX, mutations were introduced in the intervening xx residues of the CxxCTrx motif. We confirmed that these mutations generate the expected changes in redox potential of the CxxCTrx center, and found that they drastically modulate the steady-state parameters for QSOX. Further, rapid-reaction kinetics revealed strongly perturbed rates of the internal redox steps, allowing the visualization of intermediates that are only weakly populated during turnover of the wild-type enzyme."

25. Presenter: Jason Kahn, University of Maryland Complete List of Authors: "Daniel B. Gowetski, Erin J. Kodis" Title: "Rationally Designed Coiled-coil DNA Looping Peptides Control DNA Topology" "Rationally designed small artificial DNA looping peptides were developed to address longstanding

questions about DNA flexibility and its role in DNA looping. Topological methods and models presented here enable definitive measurements of DNA flexibility and looping that are essential for modeling gene regulation and for systems biology models. The results suggest that a rigid protein requires much longer DNA loops than natural proteins, which are flexible, suggesting that unusual DNA flexibility does not need to be invoked to explain short-range looping. The looping peptides should also be useful in constructing novel protein-DNA nanostructures and for genome engineering or targeted repression."

26. Presenter: Justin Kaplan, Temple Complete List of Authors: "Justin M. Kaplan, Vijay Chatare, Jing Shang, Conrad Pfeiffer, Dr. Christian E.

Schafmeister, Dr. Daniel M. Ratner, Dr. Flavio Maran, Dr. Rodrigo B. Andrade" Title: "Investigation of the Surface Chemistry of Carbohydrate Microarrays" "Carbohydrate microarrays are at the forefront of new biophysical methods used to study

glycoconjugates and their many roles in biology. Performance of these microarrays is contingent on presentation and accessibility of the immobilized glycan on the surface, yet little is known about the effects of these surface chemistries. Herein, we discuss the exploration of these surface chemistries by developing a modular synthetic approach to functionalize the surface and utilizing modern analytical techniques to study the interfacial chemistry and bioactivity of our gold-plated arrays. Our synthetic approach employs the facile and robust copper mediated azide-alkyne cycloaddition to attach glycans


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to our scaffolds. In tandem, use of techniques such as surface plasmon resonance were implemented as a label-free method to characterize the kinetics of biological interactions in real-time. "

27. Presenter: Gizem Keceli, Johns Hopkins University Complete List of Authors: "Gizem Keceli, Cathy D. Moore and John P. Toscano" Title: "Reactivity of HNO-Derived Modifications of Peptides and Proteins" "HNO, a potential heart failure therapeutic, is known to result in post-translational modifications

involving cysteine and tryptophan residues. We have applied ESI-MS and 15N-edited 1H-NMR techniques to the detection of HNO-induced thiol to sulfinamide modifications in several small organic molecules, peptides, and cysteine containing-proteins. Major reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids. We have extended our previous work on sulfinamide reactivity to investigate sulfinamide hydrolysis at physiological pH and temperature. These studies with the cysteine protease, papain and a related model peptide containing the active site thiol suggest that sulfinamide hydrolysis can be enhanced in a protein environment. Moreover, we have begun to study the reactivity of the HNO-derived N-nitrosotryptophan. "

28. Presenter: Mohd M. Khan, University of Maryland, Baltimore County Complete List of Authors: "Mohd M. Khan, and Elsa D. Garcin" Title: "Understanding the role of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the

post-transcriptional regulation of Endothelin-1 mRNA stability" "Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also possesses non-glycolytic functions and

can regulate gene expression at the level of mRNA stability by binding to the adenine-uridine rich (ARE) areas in the 3’-untranslated region (UTR) of Endothelin-1 (ET-1) mRNA. Dysregulation of the vasoconstrictor: ET-1 expression is involved in the pathophysiology of many diseases: pulmonary diseases (asthma and hypertension), central nervous system diseases (Hirschprung’s disease, stroke and subarachnoid hemorrhage) and cardiac and vascular diseases (congestive heart failure, hypertension, coronary heart diseases, and atherosclerosis). So far research has focused on targeting ET-1 receptors or ET-1 processing proteases to control the pathophysiological effects of ET-1. Targeting ET-1 expression via modulation of its mRNA stability by GAPDH offers a promising alternative strategy. However, clear understanding of GAPDH:ET-1 mRNA specific interactions is lacking and needs to be investigated as this information can be used to rationally design small molecules that can modulate the ET-1 mRNA stability, thereby treating vascular dysfunction, pulmonary, renal, and heart diseases. Our goal is to elucidate the molecular and structural aspects of GAPDH interaction with ET-1 mRNA and determine the specific interactions between GAPDH and ET-1 mRNA, as well as roles of GAPDH structural elements in binding and destabilizing ET-1 mRNA using size-exclusion chromatography, RNA electrophoretic mobility shift assay, CD, Fluorescence anisotropy, molecular modeling, and crystallography."

29. Presenter: Minjoung Kyoung, UMBC Complete List of Authors: "Minjoung Kyoung,* Sarah J. Russell, Nopondo N. Esemoto, Songon An" Title: "Kinetic Organization of a Transient Metabolic Complex, the Purinosome, in Cancer

Cells" "Enzymes in human de novo purine biosynthesis have been demonstrated to form a transient

multienzyme complex, the purinosome, in live cells upon purine depletion (1). However, our current knowledge of the purinosome has limited understanding its regulatory mechanism inside a cell (2-4). In this work we carried out a fluorescence recovery after photobleaching technique to characterize dynamic organization of the purinosome in human breast carcinoma cell line, Hs 578T. Comprehensive analyses of the diffusion coefficients of all the enzymes in purine biosynthesis provide compelling evidence for the sequential order of the six enzymes to assemble and disassemble the purinosome in live cells and further the identity of a scaffolder protein of the purinosome. In addition, we reveal that


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salvage enzyme for purine biosynthesis, hypoxantine phosphoribosyltransferase, do not participate in the purinosome and indeed its diffusion coefficient in cells was not influenced by culture conditions. Given that our current understanding of metabolic pathways is solely based on individual enzyme activities per se, our kinetic model of the purinosome organization proposed here provides unprecedented insights how a cell operates sequential metabolic enzymes to regulate metabolic pathways upon cellular demands. "

30. Presenter: YI LI, University of Delaware Complete List of Authors: Yi Li and Joseph M. Fox* Title: "Selective Syntheses of ∆α,β and ∆β,γ Butenolides from Allylic Cyclopropene

Carboxylates, Scope and Applications" "Allylic cyclopropene carboxylates undergo ring expansion reactions to give 2-allyloxyfuran

intermediates, which subsequently rearrange to ∆β,γ-butenolides via a Claisen rearrangement or to the corresponding ∆α,β-butenolides via further Cope rearrangement. A Chirality Transfer was also observed from non-racemic allylic esters. Additional efforts was made towards the total synthesis of Sessilifoliamide alkaloids. "

31. Presenter: QIN LIANG, UNIVERSITY OF DELAWARE Complete List of Authors: "Qin Liang, Thomas S. Dexheimer, Andrew S. Rosenthal, Ping Zhang, Mark

A. Villamil, Junjun Chen, Edward H. Kerns, Anton Simeonov, Ajit Jadhav, David J. Maloney, Zhihao Zhuang"

Title: "Discovery of Small-Molecule Inhibitors of the USP1/UAF1 Deubiquitinase Complex in DNA Damage Response"

"Deubiquitinases (DUBs) have in recent years emerged as a promising therapeutic target class. The functions of ubiquitination and deubiquitination have been implicated in a wide range of critical biological processes. Ubiquitin-specific protease 1 (USP1) in association with its WD40-repeat protein partner, UAF1 (USP1-associated factor 1), is known as a regulator in translesion synthesis and Fanconi anemia pathway in response to DNA damage and has recently been suggested as a promising target for drug intervention. Herein, to better evaluate the therapeutic potential of the USP1/UAF1 target, a quantitative high-throughput screen and a subsequent medicinal chemistry optimization have been conducted to pursuit small molecules inhibitors against USP1/UAF1 complex. Using biochemical assay and activity-based DUB profiling in live cells, we identified a noncompetitive inhibitor that displays nanomolar inhibition (Ki of 68 nM) and excellent selectivity toward USP1/UAF1. In addition, the USP1/UAF1 inhibitor acts synergistically with cisplatin in potentiating non-small cell lung cancer (NSCLC) cells to cisplatin killing. Furthermore, it inhibits USP1/UAF1 activity in cells, resulting in increased level of monoubiquitinated PCNA and FANCD2, two known cellular targets of USP1/UAF1. Lastly, the USP1/UAF1 inhibitor possesses a promising in vitro ADME profile, suggesting its suitability for further testing in PK/PD studies. "

32. Presenter: Venkata M.Yenugonda, Center for Drug Discovery, Georgetown University Medical Center. Complete List of Authors: "Tushar B. Deb, Scott C. Grindrod, Sivanesan Dakshanamurthy, Milton

L.Brown " Title: "Development of Novel CDK1 Small Molecule ‘VMY-1-103’ for Inhibition of Triple Negative

Breast Cancer Cell Growth" "Triple negative breast cancer (TNBC), defined as tumors that are lacking estrogen, progesterone

receptors (ER/PR) expression, as well as human epidermal growth factor receptor-2 (HER2) amplification. Approximately 15% of all breast cancers are triple negative and more frequently affect younger women and African-American women. Due to heterogeneity and lack of well-defined molecular targets, TNBC patients could be not be treated by treatment options available to ER positive cancers (hormonal therapy) or anti-HER2 therapies (monoclonal antibody and kinase inhibitors). In this respect, a synthetic-lethality approach could provide selective response in killing triple negative breast tumors as


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recently shown that MYC onco-protein overexpression in TNBC cells rendered the tumors susceptible to novel specific cell division kinase-1 (Cdk-1) inhibitors. Mitotic phase of cell cycle is often deregulated in TNBC cells, and Cyclin-dependent kinase (Cdk)-1 is being the only rate limiting kinase of this phase of cell cycle. Therefore, specific therapeutic inhibition Cdk-1 is considered as a viable approach for developing chemotherapy for treatment of TNBC patients. We have recently developed a novel small molecule inhibitor of Cdk1 called as “VMY-1-103” which showed greater growth inhibitory effect in TNBC cells as compared to known Cdk1 inhibitors Roscovitine and Purvalanol B. In addition, the fluorescence emitting property of VMY-1-103 allows identification intracellular targets and bio distribution at cellular and tissue level. The unique characteristics of VMY-1-103, demands further evaluation of this molecule alone or in combination of chemotherapeutic drugs in Myc-driven triple negative breast cancer cells and study their in vitro mechanism.Our work is directed towards the goal of developing VMY-1-103 as future potential preclinical drug candidate for testing triple negative breast cancer animal models and finally treatment of TNBC patients who ""unfortunately"" have limited options for treatment"

33. Presenter: Pavan Kumar Mantravadi, University of Delaware Complete List of Authors: "Pavan Kumar Mantravadi, Yuchen Zhang and John T. Koh" Title: "Artificial Acetyltransferases" "Post translational modifications represent an important mechanism to regulate protein function in cells.

We have developed a general platform to artificially acetylate target proteins of interest using small molecule acyltransferase mimics. These compounds are metabolically acetylated in cells and can transfer acetate to proteins of interest through ligand mediated proximity directed acyl transfer. The acyltrasferase mimc can be metabolically reacetylated to generate a catalytic cycle. Using analogs of the androgen receptor (AR) ligand tolfenamic acid, we designed ligand conjugates that acetylate androgen receptor through proximity directed acyl transfer. Proximity directed acetylation represents a new paradigm in protein modulation by small molecules that can have broad applicability as biological probes and potential small molecule therapeutics. "

34. Presenter: vishnu mohanan, university of delaware Complete List of Authors: Catherine L. Grimes Title: "Regulation of NOD2 activity by HSP70" "In animals, microbes are detected by the pathogen-associated molecular patterns (PAMPs) by specific

host pattern recognition receptors (PRR). Nucleotide binding oligomerization domain-containing protein 2 (NOD2, CARD15, IBD1) is an intracellular PRR that recognizes molecules containing muramyl dipeptide (MDP) that are found on bacterial cell walls. Its expression is restricted within a limited number of cells including intestinal epithelial cells (Paneth cells) and monocyte derived cells of the immune system. NOD2 mutations are linked with Crohn’s disease. Our objective is to discover the interacting proteins of NOD2 pre- and post-MDP stimulation and determine the associated signaling pathway. HSP70 has been widely studied and has been linked with inflammation especially in the anti-inflammatory regulation. Co-immunoprecipitaion followed by co-immunostaining confirmed NOD2-HSP70 interaction. Induced HSP70 expression in cells increased the activation of NOD2. HSP70 inhibitor KNK437, when incubated with the cells showed an decrease in NOD2 activation. Our study shows that HSP70 is involved in the regulation of NOD2 activation. "

35. Presenter: Justin Northrup, Temple University Complete List of Authors: "Dr. Christian E. Schafmeister, Temple University" Title: "Spiroligomer talons as new moities for potential protein ligands" "Mimicking protein-protein interactions with synthetically based molecules can be difficult since many

interactions involve large surface areas. On top of that, the use of synthetic proteins as drugs is challenging due to proteases that will destroy the protein before it has a chance to interact. Peptoids have been shown to interact with proteins, and combinatorial libraries of peptoids have been proven to


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be effective in discovering new ligands for protein binding. However, peptoids are flexible and can lack the surface area needed to compete with larger protein interactions. To combat these problems, we have created spiroligomers that have a rigid backbone, exhibit functionality comparable to proteins, and are completely resistant to proteases. We then tethered spiroligomers together utilizing a peptoid backbone to create ‘talon’ molecules that have a potential interacting surface area of 2000 angstroms. These talon molecules can be synthesized in a modular fashion to create a combinatorial library, which will be used to discover spiroligomers ligands that bind proteins of interest."

36. Presenter: SHUCHING OU, UNIV OF DELAWARE Complete List of Authors: "Shuching Ou, Di Cui, Sandeep Patel" Title: "Free Energetics of Guanidinium and Methyl Guanidinium Cations at Aqueous Liquid-

Vapor Interface" "The guanidinium cation (C(NH2)3+) is a highly stable cation in aqueous solution due to the efficient

resonance stabilization of the charge and efficient solvation by water molecules. Its salt increase the solubility of nonpolar molecules (“salting-in”) and decrease the order of water, therefore is one of the strongest denaturants used in physiochemical studies of protein folding. We investigate the behavior of guanidinium and its derivative, methyl guanidinium (an amino acid analogue) at the air-water surface, using atomistic molecular dynamics (MD) simulations. Methyl guanidinium cation is less excluded from the air-water surface than guanidinium cation, but both cations show orientational dependence of the surface affinity. Calculations of surface fluctuations demonstrate that near the air-water surface, the parallel-oriented cations generate significantly greater interfacial fluctuations compared to other orientations, which induces more long-ranged perturbations and solvent density redistribution. Our results suggest a strong correlation with induced interfacial fluctuations and the ion surface stability."

37. Presenter: Anil Kumar Pandey, University of Delaware Complete List of Authors: "Anil K. Pandey, Devan Naduthambi,Krista M. Thomas, Neal J. Zondlo" Title: "Proline Editing: A General and Practical Approach to the Synthesis of Functionally and

Structurally Diverse Peptides. Analysis of Steric versus Stereoelectronic Effects of 4-Substituted Prolines on Conformation within Peptides."

"Proline residues are unique structural loci in proteins, with special identity in turns, loops, secondary structure, polyproline helices, and biomolecular recognition. Proline residues are also unique in their propensity for cis amide bonds. There has been considerable effort in the development of functionalized proline residues, for applications in medicinal chemistry, protein engineering, structural biology, molecular probes, and materials science. However, practical access is limited by the need for multistep synthesis of proline amino acids, including protection of the amine and carboxylic acid. Herein we describe a general and highly practical approach, termed proline editing, for the synthesis of peptides with stereospecifically modified proline residues. In this approach, peptides are synthesized by standard solid-phase peptide synthesis to incorporate commercially available and inexpensive Fmoc-Hydroxyproline ((4R)-Hyp). In a fully automated manner, the Hyp hydroxyl is protected, as a trityl ether, a TBS silyl ether, a nitrobenzoate ester, or an Alloc carbonate, and the remainder of the peptide synthesized. After peptide synthesis, the Hyp protecting group is orthogonally removed and Hyp selectively modified to generate the substituted proline amino acids, with the peptide main chain functioning to “protect” the proline amino and carboxyl groups. In the context of a model tetrapeptide (Ac-TYPN-NH2), the (4R)-Hyp residue was stereospecifically converted to 123 different 4-substituted prolyl amino acids, with 4R or 4S stereochemistry, via Mitsunobu, oxidation, reduction, acylation, and substitution reactions. 4-Substituted proline derivatives synthesized via proline editing include incorporated structured amino acid mimetics (Cys, Asp/Glu, Phe, Lys, Arg, pSer/pThr), recognition motifs (biotin, RGD), electron-withdrawing groups to induce stereoelectronic effects (fluoro, hydroxyl, benzoate), handles for heteronuclear NMR (19F: 4R- and 4S- fluoro; pentafluorophenyl or perfluoro-tert-butyl ether; 4,4-difluoro; 77SePh) and other spectroscopies (fluorescence and IR via cyanophenyl ether), leaving groups (mesylate, tosylate, nosylate, alkyl halide, NHS ester, bromoacetate), and other


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reactive handles (amine, thiol, thioester, ketone, hydroxylamine, maleimide, dienophile, acrylate, azide, alkene, alkyne, aryl halide, tetrazine, and 1,2-aminothiol). Proline editing provides access to these diverse proline derivatives with no solution phase synthesis, and is thus broadly accessible and highly practical. In addition, proline editing provides the ability to readily modify a single peptide on solid phase to generate multiple peptides with diverse substitution. All proline derivatives were analyzed by NMR in the model tetrapeptide context for their structural effects, via their Ktrans/cis and 3JαN. The largest stereoelectronic effects were observed for protonated-aminoxy, fluoro, trifluoromethylbenzoate, nitrobenzoate, and O-phthalimide substitutions. In contrast, thiol, thioether, sulfoxide, sulfone, phenylselenyl, iodo, and triazolyl groups exhibited structural effects that were less dependent on the stereochemistry of substitution, suggestive of steric effects negating or dominating over stereoelectronic effects. The disubstituted derivatives 4-oxoproline, 4,4-difluoroproline, and 4,4-dithiolane exhibited unique structural signatures by circular dichroism. Proline derivatives were synthesized to permit bioorthogonal conjugation reactions, including Huisgen (azide-alkyne/""click"" chemistry), tetrazine-trans-cyclooctene cycloaddition, oxime formation, reductive amination, native chemical ligation, Suzuki, Sonogashira, Grubbs cross-metathesis, and Diels-Alder reactions. The proline derivatives synthesized allowed three parallel bioorthogonal reactions (tetrazine-trans-cyclooctene, oxime, and Diels-Alder) to be conducted in a single solution, demonstrating the potential applications of structured and functionalized proline derivatives."

38. Presenter: Matthew Parker, Temple University Complete List of Authors: "M. F. L. Parker, C. E. Schafmeister" Title: "Design and Synthesis of Highly Functionalized and Complex Bis-Peptide Bundles:

Progress Toward Artificial “Tertiary Structure”" "Bis-peptides are shape-programmable macromolecules assembled from a collection of building blocks

called bis-amino acids that are connected to one another through pairs of amide bonds. They serve as water-soluble, rigidified scaffolds capable of presenting collections of functional groups in different spatial orientations by virtue of the sequence, shape and stereochemistry inherent in each chiral building block. We have previously demonstrated short sequences of bis-amino acids that create “secondary structures” and locally present functional groups for applications of catalysis, binding protein surfaces, and control of election transfer in water, three essential processes of native proteins. In order to expand on this, we have set our sights on developing a strategy for introducing “tertiary structure” into our bis-peptide oligomers. We have synthesized linear sequences of two and three bis-peptide oligomers spaced by short, flexible linkers and cross-linked them into large macrocycles and covalently linked bundles. With these macromolecules, we can now position multiple functional groups with larger surface areas for mimicry of protein-protein interactions, recreate complex active sites of enzymes, and form unique, chiral pockets for host-guest molecular recognition. "

39. Presenter: Joanna Maria N. San Pedro, Johns Hopkins University Complete List of Authors: Marc M. Greenberg Title: "Pyrimidine Radical Reactivity" "Ionizing radiation damages DNA by direct ionization of the nucleobases or indirectly, through the

production of hydroxyl radical, that then reacts with DNA. The direct effect ionizes the biopolymer, creating cation radicals that can react further to cause deleterious lesions. Our goals are to study the role of the major hydroxyl radical adduct formed with thymidine in tandem lesion formation and electron transfer in DNA. We synthesized a precursor for the radical, and present studies on its reactivity. We detected the formation of 8-oxodG at the 5'-adjacent and distal dGs to where the radical precursor is positioned, presumably due to peroxyl radical addition to the nucleotide. A very low yield of electron transfer originating from the pyrimidine peroxyl radical was observed. Cation radical intermediates have never been independently generated from synthetic precursors. We also describe attempts to detect the formation of thymidine radical cation from an independently synthesized precursor. "


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40. Presenter: Matthew Rehmann, University of Delaware Complete List of Authors: "Matthew S. Rehmann, April M. Kloxin" Title: "Biochemical Cues for Directing Mesenchymal Stem Cell Function for Ligament Repair" "Mesenchymal stem cells (MSCs) are multipotent cells present in the bone marrow that show promise

for ligament repair. MSCs are attractive for improved ligament repair as they proliferate rapidly, produce large amounts of extracellular matrix proteins, and can differentiate into ligament cells. However, controlled conditions promoting ligamentogenic differentiation of MSCs are not yet well-established. In this study, we aim to elucidate the effects of ligament development-related biochemical signals on directing MSC function for ligament repair. MSCs are grown in the presence of growth factors and ascorbic acid, and the synergistic effects of these soluble factors are assessed by measuring collagen production and the expression of ligament-related markers, including scleraxis and tenascin-C, by immunocytochemical staining and RT-PCR."

41. Presenter: Benjamin T Roembke, "University of Maryland, College Park" Complete List of Authors: "Jinxin Wang, Shizuka Nakayama, Jie Zhou, Herman Sintim" Title: "New Insights into G-Quadruplex Enhanced Hemin Peroxidation" "The G-Quadruplex enhanced peroxidation reaction by hemin has been heavily utilized in a plethora of

biotechnological applications. Until recently, oligonucleotides (DNA/RNA) have proven to be the best nucleotide-based peroxidases. These DNA/RNA peroxidases contain several G-tetrad ""floors"" as well as loop sequences connecting them. In this work, we present a new, ""loopless,"" small molecule-based peroxidase comprised of a bis-(3′,5′)-cyclic dimeric guanosine monophosphate octamer. Using chemical modification, we have also shown that the carboxylate moieties are important for hemin catalysis to occur."

42. Presenter: Meghan Roig, University of the Sciences Complete List of Authors: Jessica Zinskie Michael Bruist Title: "T7 Transcription of RNA" "Transcription produces ribonucleic acid (RNA) in cells. It is done by the enzyme RNA polymerase

(RNAP), using DNA as a template. The bacteriophage T7 RNA polymerase is well studied and has some very practical characteristics as a molecular biology tool. The DNA sequence recognized by T7 RNAP as the start point for transcription (the promoter) has a strict specificity and few of these promoters occur randomly in E. coli DNA. The T7 RNAP has only one subunit and is quite stable, simplifying its cloning and overproduction. The T7 RNAP gene originally used has been modified so that six residues of the amino acid histidine (a His tag) have been added to the protein on the N-terminus. The original objective of this project was to express and isolate T7 RNAP from Escherichia coli using Western blotting techniques. Due to unforeseen causes in the laboratory, Western blot analysis was unsuccessful in identifying the His6X tagged T7 RNAP; however the protein was able to be visualized on a denaturing polyacrylamide gel stained with Ponceau S. This project has been shifted to do an in vitro transcription of small, satellite (sat) RNAs using commercial T7 RNAP. The plasmid containing the gene for the sat RNAs will be created. The DNA template for in vitro transcription has been produced by polymerase chain reaction (PCR). Using a commercially available T7 RNAP, RNA transcripts has been produced."

43. Presenter: David Rushmore, University of the Sciences Complete List of Authors: David Rushmore Daniel Olea Melissa Neavear Dr. John Tomsho Title: "Construction of a Reverse-Two Hybrid System to Probe Influenza NS1A Dimerization" "Influenza A has a component known as non-structural protein 1 (NS1A). NS1A is vital to the life cycle

of influenza. Mutated viruses lacking the NS1A gene are unable to readily spread the infection. NS1A represses the host innate immune response and controls expression and replication of the viral genome. Two different domains within the protein carry out these two functions of NS1A. The Effector Domain (ED), which is the C-terminal end, is responsible for limiting the host innate immune response.


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The RNA Binding Domain’s (RBD), which is the N-terminal, is responsible for regulating the expression and replication of viral genome. Currently, there are no drugs that target the NS1A protein. Since this protein has no catalytic activity, the development of small molecule inhibitors has proven difficult. In this work, we take advantage of the fact that NS1A is active as a dimer with either itself or select host proteins. Disruption of this dimerization will lead to the loss of activity. These protein-protein complexes have much larger interaction sites and therefore require a different approach to discover inhibitors. Our research focuses on the construction of fusion complexes between the NS1A protein and 434 DNA Binding Domain (DBD) proteins. These complexes will be used to monitor the dimerization of NS1A in a Reverse Two Hybrid System (RTHS), in order to determine interaction strength and to test for the 434 DBD association to the target sequence. Future research will include the screening of cyclic peptide libraries for inhibitors of these protein-protein interactions. "

44. Presenter: Renee M. Salvo, University of the Sciences Complete List of Authors: "Dillon Friday, Michael F. Bruist, Tilman Baumstark" Title: "Conformational Stability of Loop E PSTVd" "Viroids are small autonomously replicating plant pathogens consisting of non-encapsidated, circular,

single-stranded RNA. Viroid RNA does not code for proteins; therefore their primary function is to interact with host proteins or other RNAs to carry out replication and pathogenesis. The potato spindle tuber viroid (PSTVd) is composed of 359 nucleotides and adopts a rod-like secondary structure. The viroid has a central conserved region that contains a loop E motif. During various stages of viroid replication, the loop E motif takes on different folds, even though the sequence of the monomer remains unchanged. The fold of the loop E sequence dictates the cleavage and ligation that create the final viroid circle. Little direct evidence of what stabilizes these folds has been found. We show the contribution of component base pairs to stability using susceptibility to chemical probes of wild-type and mutated loop E sequences. "

45. Presenter: Aparna Sapra, University of Delaware Complete List of Authors: Aparna Sapra & Colin Thorpe Title: "An Arsenical-Maleimide for the generation of new targeted biochemical reagents" "The surprising efficacy of arsenic trioxide in the treatment of acute promyelocytic leukemia has

renewed interest in the synthesis and testing of arsenicals as chemotherapeutic agents for solid tumors. The biological effects of arsenicals largely reflect the coordination of As (III) species to vicinal thiols. Several enzymes of oxidative protein folding are up-regulated in certain solid tumors, and contain catalytically essential vicinal thiols. Here, we sought to develop arsenical-based inhibitors that would capture these redox-active motifs. We synthesized an As (III) containing maleimide (As-Mal) in high yield and showed that the reagent can be readily conjugated to exposed cysteine residues in peptides and proteins. This Conjugation provides a scaffold for directing the As (III) species to the target proteins for their inhibition. Experiments with thioredoxin reductase and protein disulfide isomerase show that their CxxC motifs can be efficiently captured by their cognate arsenical-carrying substrates. Not only can As-Mal be used to generate multiply-labeled protein derivatives, but the reagent provides a facile route to new arsenic-based resins for the affinity purification of proteins and peptides containing vicinal thiols. Strategies for the regeneration of these resins will be discussed. In summary, As-Mal provides a prototype for thiol-directed reagents that allow the facile installation of arsenical in a wide range of thiol-containing proteins, biomaterials and surfaces "

46. Presenter: Christian Schafmeister, Temple University Complete List of Authors: "Christian Schafmeister, Kavitha Akula, Matthew Parker, Qingquan Zhao,

Mahboubeh Kheirabadi" Title: "Developing synthetic, shape-programmable macromolecules to bind protein surfaces

and accelerate chemical reactions."


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"My group is developing large, shape-programmable macromolecules (1,000 to 4,000 Daltons) to bind protein surfaces and to mimic enzyme active sites. We will demonstrate an example of a macromolecule that binds and stabilizes the protein MDM2 and two other spiroligomers that accelerate organic reactions including a transesterification reaction and an aromatic Claisen rearrangement. Background: organic synthesis is excellent for creating small molecules


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"Hepatitis C virus (HCV) is a wide spread health concern and, although there are treatments available, these cause many adverse side effects and are not completely effective. HCV contains a positive sense single-stranded RNA genome and replicates with the aid of the NS5B enzyme which is an RNA-dependent RNA polymerase. This polymerase is known to have at least two different conformations: open and closed. Transitions between these two conformations are thought to play a crucial role in NS5B function. Our goal is to understand how the transition between these two states occurs and how this conformational change impacts enzyme activity. This knowledge may be useful in identifying novel and more effective ways to inhibit the enzyme. To accomplish this goal we employ molecular dynamics (MD) simulations in conjunction with several enhanced sampling methods including Dynamic Importance Sampling (DIMS), Temperature accelerated Molecular Dynamics (TAMD) and implicit solvent methods. DIMS is a pathway finding algorithm that provides information about the intermediate states between defined starting and ending points. TAMD enhances exploration of phase space by propagating collective variables at an elevated temperature. The implicit solvent methods represent the solvent as a continuous medium instead of individual molecules, which speeds up the simulation remarkably. This novel combination of approaches will allow exhaustive sampling in order to obtain a comprehensive description of the enzyme conformational transition"

51. Presenter: ARCHANA SIDDAM, University of Delaware Complete List of Authors: "Christine Dang, Stephanie M. Waters, Luc Paillard, Salil A. Lachke" Title: "Role of CELF1 in lens development and disease" "The lens of the eye is a transparent tissue that focuses light on the retina and is critical for providing us

with high-resolution vision. Loss of lens transparency, which occurs due to genetic changes or aging, results in a disease called “cataract”. Cataract is the leading cause of blindness affecting ~80 million individuals worldwide. To devise strategies to delay or prevent cataract formation, it is critical to first understand how the lens develops and maintains transparency. We have developed a bioinformatics approach, iSyTE (integrated System Tool for Eye gene discovery), which has allowed us to identify several new genes associated with cataract. Using iSyTE, we predicted that the RNA binding protein CELF1 (CUGBP, Elav-like family member 1) plays an important function in lens biology. We generated and analyzed mouse mutants wherein both copies of the Celf1 gene were deleted either in all tissues or specifically in the lens. We find that both mutant models exhibit severe lens defects including cataract at birth. CELF1 controls expression of genes by regulating molecular events such as alternative splicing of pre-mRNA, stability of specific mRNAs and/or their translation into proteins. We are presently characterizing the molecular function of CELF1 in the lens. Our findings are supported by data that suggest altered CELF1 expression to be associated with the underlying pathogenic mechanism in human myotonic dystrophy patients who also exhibit cataracts. In sum, our results provide the first evidence of a protein implicated in RNA metabolism that is associated with cataract in mammals."

52. Presenter: Jessica M Smith, "Johns Hopkins University, School of Medicine" Complete List of Authors: "Jessica M. Smith, Ryan J. Vierling, Caren Freel Meyers" Title: "Development and evaluation of butylacetylphosphonate as a selective inhibitor of DXP

synthase." "The unique methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most

human pathogens, including Mycobacterium tuberculosis and the malaria parasite Plasmodium falciparum. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes the thiaminediphosphate (ThDP)-dependent formation of DXP from D-glyceraldehyde 3-phosphate (GAP) and pyruvate. Results of our studies support the unique requirement for ternary complex formation in DXP synthase catalysis and highlight the flexibility of this enzyme for aliphatic acceptor substrates. Taken together, these observations have guided the design of a new class of DXP synthase inhibitors. An unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhibits selective inhibition of DXP synthase over ThDP-dependent pyruvate dehydrogenase E1 and transketolase. Further, BAP displays antimicrobial activity and is shown to act in synergy with other antimicrobial


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agents. These results suggest a new approach for selective inhibition of early stage isoprenoid biosynthesis, toward development of new anti-infective agents."

53. Presenter: Lee Colyer Speight, University of Pennsylvania Complete List of Authors: "Speight, Lee C.; Goldberg, Jacob M. Warner, John B.; Willi, Taylor;

Woodman, Bradley, F.; Mehl, Ryan A.; Petersson, E. James." Title: "Polysubstituted Acridonyl-alanines: Fluorescent Amino Acid Synthesis, Photophysical

Characterization, and Biophysical Studies" "Acridonyl-2-alanine(Acd) is a ribosomally permissible fluorescent amino acid that posses a long

lifetime, resilience to photobleaching, a near unity quantum yield (φ = 0.95), and visible wavelength emissions. Recently, we have shown that it can be incorporated into proteins in vivo and can be a valuable probe of protein conformational change due to its red-shifted excitation wavelength, unique solvatochromic properties, and ability to participate in energy transfer with endogenous amino acids (Trp and Tyr) as well as exogenous fluorophores such as methoxycoumarin (Mcm). While many of th

chemical biology progress & challenges...2013/05/06 · sixth annual frontiers at the...

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