Biomedical subjects

Eric Oldfield

Publications and source records attributed to Eric Oldfield.

At least 19 recordsLinked to original sources

PubMed2006

Isoprenoid biosynthesis as a drug target: bisphosphonate inhibition of Escherichia coli K12 growth and synergistic effects of fosmidomycin.

We screened a library of 117 bisphosphonates for antibacterial activity against Escherichia coli. The most potent growth inhibitors where N-[methyl(4-phenylalkyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonates, known potent bone resorption inhibitors, and there was a generally good correlation between cell growth inhibition and E. coli farnesyl diphosphate synthase (FPPS) inhibition. However, some potent FPPS inhibitors had no activity in cell growth inhibition, and based on the result of Catalyst pharmacophore modeling, this could be attributed to the requirement of a large hydrophobic feature for cellular activity (due most likely to transport). The activity of the most potent compound, N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (13), was strongly potentiated by the drug fosmidomycin. The transcription profiles for 13 or fosmidomycin alone were different from those found with carbenicillin or ciprofloxacin alone, but there were many similarities between the combination (13-fosmidomycin) and carbenicillin or ciprofloxacin, reflecting the more potent bactericidal activity of the drug combination on bacterial growth.

Anti-Bacterial Agents↗

PubMed2006

Bisphosphonate inhibition of phosphoglycerate kinase: quantitative structure-activity relationship and pharmacophore modeling investigation.

We report the results of a three-dimensional quantitative structure-activity relationship (3D-QSAR) and pharmacophore modeling investigation of the interaction of the enzyme 3-phosphoglycerate kinase (PGK) with aryl and alkyl bisphosphonates. For the human enzyme, the IC50 values are predicted within a factor of 2 over the 240x experimental range in activity, while for the yeast enzyme, binding of the more flexible alkyl bisphosphonates is predicted within a factor of approximately 4 (over a 2500x range in activity). Pharmacophore models indicate the importance of two negative ionizable features, one hydrophobic feature, and one halogen feature, and docking studies indicate that bisphosphonates bind in a manner similar to the 3-phosphoglycerate molecule identified crystallographically. The results give a good account of the activities of a diverse range of bisphosphonate inhibitors and are of interest in the context of developing inhibitors of glycolysis in organisms that are totally reliant on glycolysis for ATP production, such as trypanosomatid parasites.

Amino Acid Sequence↗

PubMed2006

Solid-state NMR, crystallographic, and computational investigation of bisphosphonates and farnesyl diphosphate synthase-bisphosphonate complexes.

Bisphosphonates are a class of molecules in widespread use in treating bone resorption diseases and are also of interest as immunomodulators and anti-infectives. They function by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), but the details of how these molecules bind are not fully understood. Here, we report the results of a solid-state (13)C, (15)N, and (31)P magic-angle sample spinning (MAS) NMR and quantum chemical investigation of several bisphosphonates, both as pure compounds and when bound to FPPS, to provide information about side-chain and phosphonate backbone protonation states when bound to the enzyme. We then used computational docking methods (with the charges assigned by NMR) to predict how several bisphosphonates bind to FPPS. Finally, we used X-ray crystallography to determine the structures of two potent bisphosphonate inhibitors, finding good agreement with the computational results, opening up the possibility of using the combination of NMR, quantum chemistry and molecular docking to facilitate the design of other, novel prenytransferase inhibitors.

Crystallography, X-Ray↗

PubMed2006

Solid-state NMR of a paramagnetic DIAD-FeII catalyst: sensitivity, resolution enhancement, and structure-based assignments.

A general protocol for the structural characterization of paramagnetic molecular solids using solid-state NMR is provided and illustrated by the characterization of a high-spin Fe(II) catalyst precursor. We show how good NMR performance can be obtained on a molecular powder sample at natural abundance by using very fast (>30 kHz) magic angle spinning (MAS), even though the individual NMR resonances have highly anisotropic shifts and very short relaxation times. The results include the optimization of broadband heteronuclear (proton-carbon) recoupling sequences for polarization transfer; the observation of single or multiple quantum correlation spectra between coupled spins as a tool for removing the inhomogeneous bulk magnetic susceptibility (BMS) broadening; and the combination of NMR experiments and density functional theory calculations, to yield assignments.

Algorithms↗

PubMed2006

Particle-size effect of nanoscale platinum catalysts in oxygen reduction reaction: an electrochemical and 195Pt EC-NMR study.

Oxygen reduction reaction (ORR) measurements and (195)Pt electrochemical nuclear magnetic resonance (EC-NMR) spectroscopy were combined to study a series of carbon-supported platinum nanoparticle electrocatalysts (Pt/CB) with average diameters in the range of roughly 1-5 nm. ORR rate constants and H(2)O(2) yields evaluated from hydrodynamic voltammograms did not show any particle size dependency. The apparent activation energy of 37 kJ mol(-1), obtained for the ORR rate constant, was identical to that obtained for bulk platinum electrodes. Pt/CB catalysts on Nafion produced only 0.7-1% of H(2)O(2), confirming that the direct four-electron reduction of O(2) to H(2)O is the predominant reaction. NMR spectral features showed characteristic size dependence, and the line shapes were reproduced by using the layer-deconvolution model. Namely, the variations in the NMR spectra with particle size can be explained as due to the combined effect of the layer-by-layer variation of the s-type and d-type local density of states. However, the surface peak position of (195)Pt NMR spectra and the spin-lattice relaxation time of surface platinum atoms showed practically no change with the particle size variation. We conclude that there is a negligible difference in the surface electronic properties of these Pt/CB catalysts due to size variations and therefore, the ORR activities are not affected by the differences in the particle size.

Catalysis↗

PubMed2006

Activity of nitrogen-containing and non-nitrogen-containing bisphosphonates on tumor cell lines.

We synthesized and tested three series of bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines: MCF-7 (breast), NCI-H460 (lung), and SF-268 (CNS). The first series of compounds consisted of 49 nitrogen-containing bisphosphonates, the most active species being a tetrakispivaloyloxymethyl (POM) ester, having an (average) IC(50) of 6.8 microM. The second series of compounds consisted of nine terphenylbisphosphonates, the most active species also being a POM ester, having an IC(50) of 2.2 microM. The third series of compounds consisted of seven halogen or cyanophenylbisphosphonates, the most active species again being a POM ester, having an IC(50) of 500 nM. Taken together, these results are of interest because they show that bisphosphonate esters can have potent activity against a variety of tumor cell lines, with the most active terphenyl- and halophenyl-containing species having IC(50) values approximately 10-40x lower than the most potent commercially available bisphosphonates.

Antineoplastic Agents↗

PubMed2006

Enthalpy versus entropy-driven binding of bisphosphonates to farnesyl diphosphate synthase.

We report the results of an ITC (isothermal titration calorimetry) investigation of the binding of six bisphosphonates to the enzyme farnesyl diphosphate synthase (FPPS; EC 2.5.1.10) from Trypanosoma brucei. The bisphosphonates investigated were zoledronate, risedronate, ibandronate, pamidronate, 2-phenyl-1-hydroxyethane-1,1-bisphosphonate, and 1-(2,2-bisphosphonoethyl)-3-iodo pyridinium. At pH = 7.4, both risedronate and the phenylethane bisphosphonate bind in an enthalpy-driven manner (DeltaH approximately -9 to 10 kcal mol-1), but the other four bisphosphonates bind in an entropy-driven manner (DeltaS varying from 31.2 to 55.1 cal K-1 mol-1). However, at pH = 8.5, zoledronate binding switches from entropy to enthalpy-driven. The DeltaG results are highly correlated with FPPS inhibition results obtained using a radiochemical assay (R2 = 0.85, N = 11, P < 0.001). The DeltaH and DeltaS results are interpreted in terms of a model in which bisphosphonates with charged side chains have positive DeltaH values, due to the enthalpic cost of desolvation (due to strong ion-dipole interactions) and, likewise, a positive DeltaS, due to an increase in water entropy (both ligand and protein associated) on ligand binding to FPPS: the hydrophobic effect. For the neutral side chains (risedronate at pH 7.4, 8.5 and zoledronate at pH 8.5, as well as the phenylethane bisphosphonate), binding is overwhelmingly enthalpy-driven, with the enhanced activity of the basic side chain containing species being attributable to their becoming protonated in the active site. Given the large size of the bisphosphonate market and the potential importance of the development of these compounds for cancer immunotherapy and anti-parasitic chemotherapy, these results are of broad general interest in the context of the development of new, potent, and selective FPPS inhibitors.

Animals↗

PubMed2006

Amiodarone has intrinsic anti-Trypanosoma cruzi activity and acts synergistically with posaconazole.

There is no effective treatment for the prevalent chronic form of Chagas' disease in Latin America. Its causative agent, the protozoan parasite Trypanosoma cruzi, has an essential requirement for ergosterol, and ergosterol biosynthesis inhibitors, such as the antifungal drug posaconazole, have potent trypanocidal activity. The antiarrhythmic compound amiodarone, frequently prescribed for the symptomatic treatment of Chagas' disease patients, has also recently been shown to have antifungal activity. We now show here for the first time that amiodarone has direct activity against T. cruzi, both in vitro and in vivo, and that it acts synergistically with posaconazole. We found that amiodarone, in addition to disrupting the parasites' Ca(2+) homeostasis, also blocks ergosterol biosynthesis, and that posaconazole also affects Ca(2+) homeostasis. These results provide logical explanations for the synergistic activity of amiodarone with azoles against T. cruzi and open up the possibility of novel, combination therapy approaches to the treatment of Chagas' disease using currently approved drugs.

Acute Disease↗

PubMed2006

Inhibition of Trypanosoma cruzi hexokinase by bisphosphonates.

Hexokinase is the first enzyme involved in glycolysis in most organisms, including the etiological agents of Chagas disease (Trypanosoma cruzi) and African sleeping sickness (Trypanosoma brucei). The T. cruzi enzyme is unusual since, unlike the human enzyme, it is inhibited by inorganic diphosphate (PPi). Here, we show that non-hydrolyzable analogues of PPi, bisphosphonates, are potent inhibitors of T. cruzi hexokinase (TcHK). We determined the activity of 42 bisphosphonates against TcHK, and the IC(50) values were used to construct pharmacophore and comparative molecular similarity indices analysis (CoMSIA) models for enzyme inhibition. Both models revealed the importance of electrostatic, hydrophobic, and steric interactions, and the IC(50) values for 17 active compounds were predicted with an average error of 2.4x by using the CoMSIA models. The compound most active against T. cruzi hexokinase was found to have a 2.2 microM IC(50) versus the clinically relevant intracellular amastigote form of T. cruzi, but only a approximately 1-2 mM IC(50) versus Dictyostelium discoideum and a human cell line, indicating selective activity versus T. cruzi.

Animals↗

PubMed2006

31P NMR chemical shifts in hypervalent oxyphosphoranes and polymeric orthophosphates.

We report the first quantum chemical investigation of the solid- and solution-state 31P NMR chemical shifts in models for phosphoryl transfer enzyme reaction intermediates and in polymeric inorganic phosphates. The 31P NMR chemical shifts of five- and six-coordinate oxyphosphoranes containing a variety of substitutions at phosphorus, as well as four-coordinate polymeric orthophosphates and four-coordinate phosphonates, are predicted with a slope of 1.00 and an R2= 0.993 (N = 34), corresponding to a 3.8 ppm (or 2.1%) error over the entire 178.3 ppm experimental chemical shift range, using Hartree-Fock methods. For the oxyphosphoranes, we used either experimental crystallographic structures or, when these were not available, fully geometry optimized molecular structures. For the four-coordinate phosphonates we used X-ray structures together with charge field perturbation, to represent lattice interactions. For the three-dimensional orthophosphates (BPO4, AlPO4, GaPO4 we again used X-ray structures, but for these inorganic systems we employed a self-consistent charge field perturbation approach on large clusters, to deduce peripheral atom charges. For pentaoxyphosphoranes, the solvent effect on 31P NMR chemical shieldings was found to be very small (<0.5 ppm). The 31P NMR chemical shielding tensors in the pentaoxyphosphoranes were in most cases found to be close to axially symmetric and were dominated by changes in the shielding tensor components in the equatorial plane (sigma22 and sigma33). The isotropic shifts were highly correlated (R2= 0.923) with phosphorus natural bonding orbital charges, with the larger charges being associated with shorter axial P-O bond lengths and, hence, more shielding. Overall, these results should facilitate the use of 31P NMR techniques in investigating the structures of more complex systems, such as phosphoryl transfer enzymes, as well as in investigating other, complex oxide structures.

Magnetic Resonance Spectroscopy↗

PubMed2006

Structure and mechanism of the farnesyl diphosphate synthase from Trypanosoma cruzi: implications for drug design.

Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C5 alcohols (isopentenyl and dimethylallyl) to form C10 and C15 diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

Alendronate↗

PubMed2006

Structural studies of Vgamma2Vdelta2 T cell phosphoantigens.

Human gammadelta T cells containing the Vgamma2Vdelta2 (Vgamma9Vdelta2) T cell receptor are stimulated by a broad variety of small, phosphorus-containing antigenic molecules called phosphoantigens. The structures of several species present in both Mycobacteria (TUBags1-4) and in Escherichia coli have been reported to contain a formyl-alkyl diphosphate core. Here we report the synthesis of the lead member of the series, 3-formyl-1-butyl diphosphate. This compound has low activity for gammadelta T cell stimulation, unlike its highly active isomer (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate, necessitating a revision of the structure of TUBag1. Likewise, the structure of the species identified as the pentyl analog (TUBag 2) is revised to 6-phosphogluconate. These results indicate that neither TUBag1 nor the m/e 275 species proposed for TUBag2 are 3-formyl-1-alkyl diphosphates, leading to the conclusion that none of the natural phosphoantigens (TUBags1-4) possess the structures reported previously.

Antigens, Bacterial↗

PubMed2005

An NMR determination of CO diffusion on platinum electrocatalysts.

We report the first direct measurement of CO diffusion on nanoparticle Pt electrocatalysts at the solid/liquid interface, carried out using 13C nuclear magnetic resonance (NMR) with a spin-labeling pulse sequence. Diffusion parameters were measured in the temperature range of 253-293 K for CO adsorbed on commercial Pt-black under saturation coverage. 2H NMR of the same system indicates that the electrolyte remains in the liquid state at temperatures where the CO diffusion experiments were performed. The CO diffusion parameters follow typical Arrhenius behavior with an activation energy of 6.0 +/- 0.4 kcal/mol and a pre-exponential factor of (1.1 +/- 0.6) x 10-8 cm2/s. Exchange between different CO populations, driven by a chemical potential gradient, is suggested to be the main mechanism for CO diffusion. The presence of the electrolyte medium considerably slows down the diffusion of CO as compared to that seen on surfaces of bulk metals under UHV conditions. This work opens up a new approach to the study of surface diffusion of adsorbed molecules on nanoparticle electrode catalysts, including the possibility of correlating diffusion parameters to catalytic activity in real world applications of broad general interest.

Carbon Monoxide↗

PubMed2005

Bisphosphonate inhibition of the exopolyphosphatase activity of the Trypanosoma brucei soluble vacuolar pyrophosphatase.

Trypanosoma brucei, the causative agent of African trypanosomiasis, contains a soluble, vacuolar pyrophosphatase, TbVSP1, not present in humans, which is essential for the growth of bloodstream forms in their mammalian host. Here, we report the inhibition of a recombinant TbVSP1 expressed in Escherichia coli by a panel of 81 bisphosphonates. The IC50 values were found to vary from approximately 2 to 850 microM. We then used 3D QSAR (comparative molecular field and comparative molecular similarity index; CoMFA and CoMSIA) methods to analyze the enzyme inhibition results. The R2 values for the experimental versus the QSAR-predicted activities were 0.78 or 0.61 for CoMFA and 0.79 or 0.68 for CoMSIA, for two different alignments. The root-mean-square (rms) pIC50 error for the best CoMFA model was 0.41 for five test sets of five activity predictions, which translates to a factor of approximately 2.6 error in IC50 prediction. For CoMSIA, the rms pIC50 error and error factors were 0.35 and 2.2, respectively. In general, the most active compounds contained both a single aromatic ring and a hydrogen bond donor feature. Thirteen of the more potent compounds were then tested in vivo in a mouse model of T. brucei infection. The most active compound in vivo provided a 40% protection from death with no apparent side effects, suggesting that further development of such compounds may be of interest.

Acid Anhydride Hydrolases↗

PubMed2005

Activation of nanoparticle Pt-Ru fuel cell catalysts by heat treatment: a 195Pt NMR and electrochemical study.

(195)Pt NMR spectroscopic and electrochemical measurements were carried out on commercial Pt-Ru alloy nanoparticle samples to investigate the effect of high-temperature annealing in different vacuum/gas-phase environments. Samples annealed at 220 degrees C in Ar gas, or in a vacuum, did not show any demonstrable change in catalytic activity vs electrochemically reduced, room-temperature samples. In contrast, annealing at 220 degrees C in H(2) gas led to a 3-fold increase in reactivity toward methanol oxidation (per surface site). NMR experiments show that annealing at 220 degrees C (in both Ar and H(2)) leads to a slight reduction in the Fermi level local density of states (E(F)-LDOS) at the Pt sites, which we attribute to surface enrichment of Ru. This electronic effect alone, however, appears to be too small to account for the increase in the catalytic activity observed for the H-treated catalyst. By comparing the electrochemical and NMR data of the H- and Ar-treated samples, we conclude that annealing at 220 degrees C in the hydrogen atmosphere reduces surface Ru oxides into metallic Ru, and consequently, the presence of metallic Ru and its enrichment on the surface are essential for the enhanced catalytic activity. In contrast, heat treatment at 600 degrees C in both vacuum and argon atmosphere increases the particle size and reduces the amount of platinum on the nanoparticle surface, thereby increasing the surface Ru content beyond the optimum surface composition values. This causes a large reduction in catalytic activity. Our results suggest that optimizing the amount of surface Ru by heat treatment at temperatures near 200 degrees C, in a hydrogen atmosphere, can be utilized to produce Pt-Ru alloy nanoparticles with high methanol oxidation activity. Finally, our NMR and electrochemical data, taken together with the lattice parameter measurements, lead to a novel model of Pt-Ru alloy nanoparticles having a Ru-rich core and a Pt-Ru alloy overlayer.

Journal Article↗

PubMed2005

A solid state 13C NMR, crystallographic, and quantum chemical investigation of chemical shifts and hydrogen bonding in histidine dipeptides.

We report the first solid-state NMR, crystallographic, and quantum chemical investigation of the origins of the 13C NMR chemical shifts of the imidazole group in histidine-containing dipeptides. The chemical shift ranges for Cgamma and Cdelta2 seen in eight crystalline dipeptides were very large (12.7-13.8 ppm); the shifts were highly correlated (R2= 0.90) and were dominated by ring tautomer effects and intermolecular interactions. A similar correlation was found in proteins, but only for buried residues. The imidazole 13C NMR chemical shifts were predicted with an overall rms error of 1.6-1.9 ppm over a 26 ppm range, by using quantum chemical methods. Incorporation of hydrogen bond partner molecules was found to be essential in order to reproduce the chemical shifts seen experimentally. Using AIM (atoms in molecules) theory we found that essentially all interactions were of a closed shell nature and the hydrogen bond critical point properties were highly correlated with the N...H...O (average R2= 0.93) and Nepsilon2...H...N (average R2= 0.98) hydrogen bond lengths. For Cepsilon1, the 13C chemical shifts were also highly correlated with each of these properties (at the Nepsilon2 site), indicating the dominance of intermolecular interactions for Cepsilon1. These results open up the way to analyzing 13C NMR chemical shifts, tautomer states (from Cdelta2, Cepsilon1 shifts), and hydrogen bond properties (from Cepsilon1 shifts) of histidine residue in proteins and should be applicable to imidazole-containing drug molecules bound to proteins, as well.

Carbon Isotopes↗

PubMed2005

Quantum chemical studies of protein structure.

Quantum chemical methods now permit the prediction of many spectroscopic observables in proteins and related model systems, in addition to electrostatic properties, which are found to be in excellent accord with those determined from experiment. I discuss the developments over the past decade in these areas, including predictions of nuclear magnetic resonance chemical shifts, chemical shielding tensors, scalar couplings and hyperfine (contact) shifts, the isomer shifts and quadrupole splittings in Mössbauer spectroscopy, molecular energies and conformations, as well as a range of electrostatic properties, such as charge densities, the curvatures, Laplacians and Hessians of the charge density, electrostatic potentials, electric field gradients and electrostatic field effects. The availability of structure/spectroscopic correlations from quantum chemistry provides a basis for using numerous spectroscopic observables in determining aspects of protein structure, in determining electrostatic properties which are not readily accessible from experiment, as well as giving additional confidence in the use of these techniques to investigate questions about chemical bonding and chemical reactions.

Models, Molecular↗

PubMed2005

Bisphosphonate inhibitors of Toxoplasma gondi growth: in vitro, QSAR, and in vivo investigations.

We have investigated the activity of 60 bisphosphonates against the replication of Toxoplasma gondii in vitro and of three of the most active compounds, in vivo. The two most active compounds found were n-alkyl bisphosphonates containing long (n = 9 or 10) hydrocarbon chains, not the nitrogen-containing species used in bone resorption therapy. The target of all of the most active bisphosphonates appears to be the isoprene biosynthesis pathway enzyme farnesyl pyrophosphate synthase (FPPS), as indicated by the correlations between T. gondii growth inhibition and FPPS (human and Leishmania major) enzyme inhibition and by the fact that a T. gondii strain engineered to overexpress FPPS required considerably higher levels of bisphosphonates to achieve 50% growth inhibition, while the IC(50) for atovaquone (which does not inhibit FPPS) remained the same in the overexpressing strain. The phosphonate inhibitor of the non-mevalonate pathway, fosmidomycin, which inhibits the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase, had no effect on T. gondii growth. To investigate structure-activity relationships (SARs) in more detail, we used two three-dimensional quantitative SAR methods: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), to investigate all 60 bisphosphonates. Both the CoMFA and CoMSIA models indicated a 60-70% contribution from steric interactions and a 30-40% contribution from electrostatic interactions and using four N = 55 training sets for each method, we found on average between a factor of 2 and 3 error in IC(50) prediction. The three most active compounds found in vitro were tested in vivo in a Smith-Webster mouse model and the two most active bisphosphonates were found to provide up to an 80% protection from death, a considerable improvement over that found previously with nitrogen-containing bisphosphonates. This effect may originate in the much higher therapeutic indices of these alkyl bisphosphonates, as deduced from in vitro assays using LD(50) values for growth inhibition of a human cell line. Overall, these results indicate that alkyl bisphosphonates are promising compounds for further development as agents against Toxoplasma gondii growth, in vivo.

Aldose-Ketose Isomerases↗