Systems-based design of bi-ligand inhibitors of oxidoreductases: filling the chemical proteomic toolbox

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases.     

Efficient plasmid DNA cleavage by copper(II) complexes of 1,4,7-triazacyclononane ligands featuring xylyl-linked guanidinium groups

Three new metal-coordinating ligands, L(1), L(2), and L(3), have been prepared by appending o-, m-, and p-xylylguanidine pendants, respectively, to one of the nitrogen atoms of 1,4,7-triazacyclononane (tacn). The copper(II) complexes of these ligands are able to accelerate cleavage of the P-O bonds within the model phosphodiesters bis(p-nitrophenyl)phosphate (BNPP) and [2-(hydroxypropyl)-p-nitrophenyl]phosphate (HPNPP), as well as supercoiled pBR 322 plasmid DNA. Their reactivity toward BNPP and HPNPP is not significantly different from that of the nonguanidinylated analogues, [Cu(tacn)(OH(2))(2)](2+) and [Cu(1-benzyl-tacn)(OH(2))(2)](2+), but they cleave plasmid DNA at considerably faster rates than either of these two complexes. The complex of L(1), [Cu(L(1)H(+))(OH(2))(2)](3+), is the most active of the series, cleaving the supercoiled plasmid DNA (form I) to the relaxed circular form (form II) with a k(obs) value of (2.7 ± 0.3) × 10(-4) s(-1), which corresponds to a rate enhancement of 22- and 12-fold compared to those of [Cu(tacn)(OH(2))(2)](2+) and [Cu(1-benzyl-tacn)(OH(2))(2)](2+), respectively. Because of the relatively fast rate of plasmid DNA cleavage, an observed rate constant of (1.2 ± 0.5) × 10(-5) s(-1) for cleavage of form II DNA to form III was also able to be determined. The X-ray crystal structures of the copper(II) complexes of L(1) and L(3) show that the distorted square-pyramidal copper(II) coordination sphere is occupied by three nitrogen atoms from the tacn ring and two chloride ions. In both complexes, the protonated guanidinium pendants extend away from the metal and form hydrogen bonds with solvent molecules and counterions present in the crystal lattice. In the complex of L(1), the distance between the guanidinium group and the copper(II) center is similar to that separating the adjacent phosphodiester groups in DNA (ca. 6 ?). The overall geometry of the complex is also such that if the guanidinium group were to form charge-assisted hydrogen-bonding interactions with a phosphodiester group, a metal-bound hydroxide would be well-positioned to affect the nucleophilic attack on the neighboring phosphodiester linkage. The enhanced reactivity of the complex of L(1) at neutral pH appears to also be, in part, due to the relatively low pK(a) of 6.4 for one of the coordinated water molecules.     

Using the atomic force microscope as a nanomechanical partner to support evanescent field imaging

Quantum Dot (QD)/microsphere structures supporting Whispering Gallery Modes (WGMs) are attached to Atomic Force Microscope (AFM) cantilevers for characterization of the evanescent field around the QD/microsphere and utilization of the evanescent field for sensing at the apical surface of live cells. Following laser excitation, QD emission couples to WGMs that circumnavigate the microsphere via total internal reflection at the internal surfaces of the microsphere. The resulting evanescent field is characterized utilizing the high spatial control of an AFM in approaching a dye monolayer on a test surface. The measured evanescent field extends approximately 50 nm from the microsphere surface, matching theoretical predictions. This system was then used to sense the accumulation of integrin and formation of focal adhesions at the apical surface of cells.     

Radial Spectroscopic Imaging

A family of fast chemical-shift imaging techniques, which takes advantage of the cylindrical symmetry found, for instance, in some plants, is introduced. Variants with one and two spectral dimensions allow rapid measurement of the radially dependant spatial distribution of metabolites in such objects. Compared to conventional chemical-shift imaging and correlation-peak imaging, the minimal experimental duration can be reduced substantially. The impact on the SNR is discussed. Radial spectroscopic¹H NMR images of metabolites inAncistrocladus heyneanus,a tropical liana plant, are shown, using both radial chemical-shift imaging and radial correlation-peak imaging. The spatial distribution of various metabolites in this plant have been measured. The reduction of experimental duration compared to the conventional techniques was by a factor of 63 and 31, respectively.     

Simple Liquid Chromatographic Determination of Minocycline in Brain and Plasma

A new high-performance liquid chromatography assay was developed for the determination of minocycline in plasma and brain. A solid–liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase consisting of acetonitrile:water:perchloric acid (26:74:0.25, v/v/v) adjusted to pH 2.5 with 5 M sodium hydroxide for elution through a RP8 column (250 × 3.0 mm, i.d.) with UV detection set at 350 nm. The method proved to be accurate, precise (RSD < 20%) and linear between 0.15–20 μg mL⁻¹ in plasma and 1–20 μg mg⁻¹ in brain. The method was successfully applied to a blood-brain barrier minocycline transport study.     

Column separation of tetravalent cerium fission products from trivalent rare earth radio-isotopes

Journal of Radioanalytical and Nuclear Chemistry - Rapid and efficient isolation of individual rare earth element (REE) radioisotopes from complex mixtures is necessary to support the fields of...