Papers by Carston R Wagner
Journal of Chemical Theory and Computation, Mar 10, 2009
We have tested a variety of approximate methods for modeling 30 systems containing mixtures of ni... more We have tested a variety of approximate methods for modeling 30 systems containing mixtures of nitrogen heterocycles and exocyclic amines, each of which is studied with up to 31 methods in one or two phases (gaseous and aqueous). Fifteen of the systems are protonated, and 15 are not. We consider a data set consisting of geometric parameters, partial atomic charges, and water binding energies for the methotrexate fragments 2-(aminomethyl)pyrazine and 2,4-diaminopyrimidine, as well as their cationic forms 1H-2-(aminomethyl)pyrazine and 1H-2,4-diaminopyrimidine. We first evaluated the suitability of several density functionals with the 6-31+G(d,p) basis set to serve as a benchmark by comparing calculated molecular geometries to results obtained from coupledcluster [CCSD/6-31+G(d,p)] wave function theory (WFT). We found that the M05-2X density functional can be used to obtain reliable geometries for our data set. To accurately model partial charges in our molecules, we elected to utilize the well-validated Charge Model 4 (CM4). In the process of establishing benchmark values, we consider gas-phase coupled cluster and density functional theory (DFT) calculations followed by aqueous-phase DFT calculations, where the effect of solvent is treated by the SM6 quantum mechanical implicit solvation model. The resulting benchmarks were used to test several widely available and economical semiempirical molecular orbital (SE-MO) methods and molecular mechanical (MM) force fields for their ability to accurately predict the partial charges, binding energies to a water molecule, and molecular geometries of representative fragments of methotrexate in the gaseous and aqueous phases, where effects of water were simulated by the SM5.4 and SM5.42 quantum mechanical implicit solvation models for SE-MO and explicit solvation used for MM. In addition, we substituted CM4 charges into the MM force fields tested to observe the effect of improved charge assignment on geometric and energetic modeling. The most accurate MM force fields (with or without CM4 charges substituted) were validated against gas-phase and aqueous-phase geometries and charge distributions of a larger set of 16 drug-like ligands, both neutral and cationic. This process showed that the Merck Molecular Force Field (MMFF94) with or without CM4 charges substituted, is, on average, the most accurate force field for geometries of molecules containing nitrogen
Nucleosides, Nucleotides & Nucleic Acids, Oct 1, 1997
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ACS Combinatorial Science, Mar 31, 2017
Yeast surface display selections against mammalian cell monolayers have proven effective in isola... more Yeast surface display selections against mammalian cell monolayers have proven effective in isolating proteins with novel binding activity. Recent advances in this technique allow for recovery of clones with even micromolar binding affinity. However, no efficient method has been shown for affinity based selection in this context. This study demonstrates the effectiveness of titratable avidity reduction using dithiothreitol (DTT) to achieve this goal. A series of epidermal growth factor receptor binding fibronectin domains with a range of affinities are used to quantitatively identify the number of ligands per yeast cell that yield the strongest selectivity between strong, moderate, and weak affinities. Notably, reduction of ligand display to 3,000 -6,000 ligands per yeast cell yields 16-fold selectivity of a 2 nM binder relative to a 17 nM binder. These lessons are applied to affinity maturation of an EpCAM-binding fibronectin population, yielding an enriched pool of ligands with significantly stronger affinity than an analogous pool sorted by standard cellular selection methods. Collectively, this study offers a facile approach for affinity selection of yeast displayed ligands against full length cellular targets and demonstrates the effectiveness of this method by generating EpCAM-binding ligands that are promising for further applications.
Journal of the American Chemical Society, Feb 1, 2012
Dihydrofolate reductase-single-chain variable fragment (scFv) fusion proteins can be used for the... more Dihydrofolate reductase-single-chain variable fragment (scFv) fusion proteins can be used for the targeted cellular delivery of oligonucleotides, conjugated small molecules and proteins, via labeling of oligonucleotides by bis-methotrexate.
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Acta Crystallographica Section A Foundations and Advances, 2017
Translation initiation is often attributed as the rate determining step of eukaryotic protein syn... more Translation initiation is often attributed as the rate determining step of eukaryotic protein synthesis and key to gene expression control1. Despite this centrality the series of steps involved in this process are poorly understood2,3. Here we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5’ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features regulating the recruitment and processivity of scanning ribosomes, redefine optimal initiation contexts and demonstrate endoplasmic reticulum specific regulation of initiation. Our approach enables deconvoluting translation initiation into separate stages and identifying the regulators at each step.
Acta Neuropathologica Communications, 2019
Recently, moderate prenatal alcohol exposure (PAE) was shown to be a risk factor for peripheral n... more Recently, moderate prenatal alcohol exposure (PAE) was shown to be a risk factor for peripheral neuropathy following minor nerve injury. This effect coincides with elevated spinal cord astrocyte activation and ex vivo immune cell reactivity assessed by proinflammatory cytokine interleukin (IL) -1β protein expression. Additionally, the β2-integrin adhesion molecule, lymphocyte function-associated antigen-1 (LFA-1), a factor that influences the expression of the proinflammatory/anti-inflammatory cytokine network is upregulated. Here, we examine whether PAE increases the proinflammatory immune environment at specific anatomical sites critical in the pain pathway of chronic sciatic neuropathy; the damaged sciatic nerve (SCN), the dorsal root ganglia (DRG), and the spinal cord. Additionally, we examine whether inhibiting LFA-1 or IL-1β actions in the spinal cord (intrathecal; i.t., route) could alleviate chronic neuropathic pain and reduce spinal and DRG glial activation markers, proinflammatory cytokines, and elevate anti-inflammatory cytokines. Results show that blocking the actions of spinal LFA-1 using BIRT-377 abolishes allodynia in PAE rats with sciatic neuropathy (CCI) of a 10 or 28-day duration. This effect is observed (utilizing immunohistochemistry; IHC, with microscopy analysis and protein quantification) in parallel with reduced spinal glial activation, IL-1β and TNFα expression. DRG from PAE rats with neuropathy reveal significant increases in satellite glial activation and IL-1β, while IL-10 immunoreactivity is reduced by half in PAE rats under basal and neuropathic conditions. Further, blocking spinal IL-1β with i.t. IL-1RA transiently abolishes allodynia in PAE rats, suggesting that IL-1β is in part, necessary for the susceptibility of adult-onset peripheral neuropathy caused by PAE. Chemokine mRNA analyses from SCN, DRG and spinal cord reveal that increased CCL2 occurs following CCI injury regardless of PAE and BIRT-377 treatment. These data demonstrate that PAE creates dysregulated proinflammatory IL-1β and TNFα /IL-10 responses to minor injury in the sciatic-DRG-spinal pain pathway. PAE creates a risk for developing peripheral neuropathies, and LFA-1 may be a novel therapeutic target for controlling dysregulated neuroimmune actions as a consequence of PAE.
Molecular Pharmaceutics, 2018
Contrast Media & Molecular Imaging, 2018
Atherosclerosis-related morbidity and mortality remain a global concern. Atherosclerotic disease ... more Atherosclerosis-related morbidity and mortality remain a global concern. Atherosclerotic disease follows a slow and silent progression, and the transition from early-stage lesions to vulnerable plaques remains difficult to diagnose. Inflammation is a key component of the development of atherosclerotic plaque and consequent life-threatening complications. This study assessed 111In-DANBIRT as an in vivo, noninvasive SPECT/CT imaging probe targeting an inflammatory marker, Lymphocyte Function Associated Antigen-1 (LFA-1), in atherosclerotic plaques. Methods. Selective binding of 111In-DANBIRT was assessed using Sprague-Dawley rats exposed to filtered air and ozone (1 ppm) by inhalation for 4 hours to induce a circulating leukocytosis and neutrophilia in peripheral blood. After 24 hours, whole blood was collected and incubated with radiolabeled DANBIRT (68Ga-DANBIRT and 111In-DANBIRT). Isolated cell component smeared slides using cytospin technique were stained with Wright-Giemsa stain....
Molecular pharmaceutics, Jan 6, 2017
Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTid... more Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural ...
Biochemistry, Jul 18, 2017
Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleos... more Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES(1)) and product (EP(2)) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observa...
British journal of cancer, Jan 7, 2009
The type I insulin-like growth factor receptor (IGF1R) is a transmembrane tyrosine kinase involve... more The type I insulin-like growth factor receptor (IGF1R) is a transmembrane tyrosine kinase involved in cancer proliferation, survival, and metastasis. In this study, we used two different fluorescent technologies (small-molecule fluorophores and quantum dot (QD) nanoparticles) to detect receptor expression and its downregulation by antibodies in vivo. After conjugation with AVE-1642, a humanised anti-IGF1R monoclonal antibody, both QDs (705 nm) or Alexa 680 (small-molecule fluorophore) detected expression and downregulation of IGF1R in vitro. To examine their utility in vivo, either AVE-1642 conjugates were intravenously delivered to mice bearing xenograft tumours of mouse embryo fibroblasts expressing human IGF1R or MCF-7 human breast cancer cells. Quantum dot fluorescence was mainly localised to the reticuloendothelial system in several organs and engulfed by macrophages, with only very small amount of QDs detected in the xenograft tumours. Depletion of macrophages by clodronate li...
Cancer research, Jan 15, 1997
Originally designed as an antitumor agent, zidovudine (AZT) has exhibited only marginal tumor gro... more Originally designed as an antitumor agent, zidovudine (AZT) has exhibited only marginal tumor growth inhibitory activity. Recently, three abstracts have described positive clinical outcomes for a small number of patients with advanced breast cancer treated with weekly infusions of either methotrexate or cisplatin and AZT. Consequently, we conducted a preclinical study of the anti-breast cancer and anti-mammary tumor activity of AZT. Here we have demonstrated that AZT, alone, has a preferential in vitro and in vivo effect on breast and mammary cancer cells. It is 1000 times as potent as an inhibitor of the in vitro growth of the human breast cancer cell line MCF-7 (IC50 = 10 +/- 5 nM) than of the growth of the T-cell leukemia cell line CEM (IC50 = 14 +/- 2 microM). A novel mechanism for this preferential effect on growth is indicated by the 3-4-fold increase in production of phosphorylated AZT (mono-, di-, and triphosphate) in MCF-7 relative to CEM. We extended these in vitro observa...
Proceedings of the National Academy of Sciences, 2015
Significance Cancer is a leading cause of mortality worldwide, with the identification of novel d... more Significance Cancer is a leading cause of mortality worldwide, with the identification of novel drug targets and chemotherapeutic agents being a high priority in the fight against it. The NEET proteins mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) were recently shown to be required for cancer cell proliferation. Utilizing a combination of experimental and computational techniques, we identified a derivative of the mitocan cluvenone that binds to NEET proteins at the vicinity of their 2Fe-2S clusters and facilitates their destabilization. The new drug displays a high specificity in the selective killing of human epithelial breast cancer cells, without any apparent effects on normal breast cells. Our results identify the 2Fe-2S clusters of NEET proteins as a novel target in the chemotherapeutic treatment of breast cancer.
Molecular Pharmaceutics, 2007
Molecular Pharmaceutics, 2004
Journal of the American Chemical Society, 2012
Dihydrofolate reductase-single-chain variable fragment (scFv) fusion proteins can be used for the... more Dihydrofolate reductase-single-chain variable fragment (scFv) fusion proteins can be used for the targeted cellular delivery of oligonucleotides, conjugated small molecules and proteins, via labeling of oligonucleotides by bis-methotrexate.
Journal of the American Chemical Society, 2006
The exploitation of biological macromolecules, such as nucleic acids, for the fabrication of adva... more The exploitation of biological macromolecules, such as nucleic acids, for the fabrication of advanced materials is a promising area of research. Although a greater variety of structural and functional uses can be envisioned for protein-based materials, systematic approaches for their construction have yet to emerge. Consistent with theoretical models of polymer macrocyclization, we have demonstrated that, in the presence of dimeric methotrexate (bisMTX), wild-type Escherichia coli dihydrofolate reductase (DHFR) molecules tethered together by a flexible peptide linker (ecDHFR2) are capable of spontaneously forming highly stable cyclic structures with diameters ranging from 8 to 20 nm. The nanoring size is dependent on the length and composition of the peptide linker, on the affinity and conformational state of the dimerizer, and on induced protein-protein interactions. Delineation of these and other rules for the control of protein oligomer assembly by chemical induction provides an avenue to the future design of protein-based materials and nanostructures.
Journal of Controlled Release, 2001
Formulations of antisense oligonucleotides (asODNs) against c-myb or c-myc protooncogenes have be... more Formulations of antisense oligonucleotides (asODNs) against c-myb or c-myc protooncogenes have been prepared by a new technique that sequesters cationic lipid in the interior of a lipid particle. This technique results in high loading efficiency for the asODNs, small particle size and good stability. When targeted against melanoma cells or neuroblastoma cells via anti-GD2 coupled at the particle surface, increased
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Papers by Carston R Wagner