Draft Genome Sequences of Four NDM-1-Producing Klebsiella pneumoniae Strains from a Health Care Facility in Northern California.

We report the draft genome sequences of Klebsiella pneumoniae strains from four patients at a northern California health care facility. All strains contained the New Delhi metallo-β-lactamase (NDM1) carbapenemase with extended antibiotic resistance, including resistance to expanded-spectrum cephalosporins, imipenem, ertapenem, and meropenem. NDM gene alignments revealed that the resistance was plasmid encoded

Novel Human Lipoxygenase Inhibitors Discovered Using Virtual Screening with Homology Models

We report the discovery of new, low micromolar, small molecule inhibitors of human platelet-type 12- and reticulocyte 15-lipoxygenase-1 (12-hLO and 15-hLO) using structure-based methods. Specifically, we created homology models of 12-hLO and 15-hLO, based on the structure of rabbit 15-lipoxygenase, for in silico screening of a large compound library followed by in vitro screening of 20 top scoring molecules. Eight of these compounds inhibited either 12- or 15-human lipoxygenase with lower than 100 microM affinity. Of these, we obtained IC50 values for the three best inhibitors, all of which displayed low micromolar inhibition. One compound showed specificity for 15-hLO versus 12-hLO; however, a selective inhibitor for 12-hLO was not identified. As a control we screened 20 randomly selected compounds, of which none showed low micromolar inhibition. The new low-micromolar inhibitors appear to be suitable as leads for further inhibitor development efforts against 12-hLO and 15-hLO, based on the fact their size and chemical properties are appropriate to classify them as drug-like compounds. The models of these protein-inhibitor complexes suggest strategies for future development of selective lipoxygenase inhibitors

De novo structure prediction and experimental characterization of folded peptoid oligomers

Peptoid molecules are biomimetic oligomers that can fold into unique three-dimensional structures. As part of an effort to advance computational design of folded oligomers, we present blind-structure predictions for three peptoid sequences using a combination of Replica Exchange Molecular Dynamics (REMD) simulation and Quantum Mechanical refinement. We correctly predicted the structure of a N-aryl peptoid trimer to within 0.2 Å rmsd-backbone and a cyclic peptoid nonamer to an accuracy of 1.0 Å rmsd-backbone. X-ray crystallographic structures are presented for a linear N-alkyl peptoid trimer and for the cyclic peptoid nonamer. The peptoid macrocycle structure features a combination of cis and trans backbone amides, significant nonplanarity of the amide bonds, and a unique "basket" arrangement of (S)-N(1-phenylethyl) side chains encompassing a bound ethanol molecule. REMD simulations of the peptoid trimers reveal that well folded peptoids can exhibit funnel-like conformational free energy landscapes similar to those for ordered polypeptides. These results indicate that physical modeling can successfully perform de novo structure prediction for small peptoid molecules

Tryptophan 500 and Arginine 707 Define Product and Substrate Active Site Binding in Soybean Lipoxygenase-1 †

ABSTRACT: There is much debate whether the fatty acid substrate of lipoxygenase binds "carboxylate-end first" or "methyl-end first" in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steadystate and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly π-π interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind "carboxylate-end first" with sLO-1 and not "methylend first" as has been proposed for human 15-lipoxygenase. Lipoxygenases (LO) 1 catalyze the peroxidation of dienecontaining fatty acids and belong to a class of non-heme iron metalloenzymes found in both plants and mammals (1-3). Mammalian lipoxygenases serve vital roles in the biosynthesis of lipoxins and leukotrienes, which are critical signaling molecules (4, 5). There are three major mammalian isozymes, 5-LO, 12-LO, and 15-LO, which oxygenate arachidonic acid (AA) at specific carbon centers (C5, C12, and C15, respectively) (6). These isozymes of human lipoxygenase have been shown to be involved in several human diseases: asthma (7) and prostate cancer (8) for human 5-LO (5-hLO), immune disorders (9) and breast cancer (10, 11) for human 12-LO (12-hLO), and atherosclerosis (12) and colorectal cancer (13) for human 15-LO (15-hLO). To develop effective therapeutic agents against these diseases, an intimate knowledge of their active sites is needed so that specific inhibitors of a particular lipoxygenase isozyme can be designed. Sloane and co-workers made significant progress in this regard when they converted reticulocyte 15-hLO-1 into a "12-hLO" by increasing the active site volume; they proposed that the substrate sat deeper in the active site, with the hydrophobic methyl end inserted first (14). This hypothesis was supported by site-directed mutagenesis investigations of Gan et al., which suggested that Phe414 of 15-hLO-1 π-π-stacked with the C11-C12 double bond of the substrate and that the positively charged residue Arg402 interacted with the negatively charged carboxylate of the substrate (15). Because Arg402 is located close to the surface of 15-hLO-1, this study supported a "methyl-end first" binding of the substrate. In addition, mutagenesis experiments on Phe353 and Ile593 of rabbit 15-LO (15-rLO) also supported the hypothesis that the size and shape of the active site defined the specificity and were consistent with a "methyl-end first" binding for mammalian Nevertheless, when the rabbit 15-LO (15-rLO) structure was compared with soybean lipoxygenase-1 (sLO-1), a debate concerning the manner in which the substrate bound to the active site developed. Amzel and co-workers proposed that, to obtain the known stereochemistry of the product, only a "carboxylate-end first" insertion of linoleic acid (LA) into the active site was plausible for sLO-

Tryptophan 500 and Arginine 707 Define Product and Substrate Active Site Binding in Soybean Lipoxygenase-1 †

There is much debate whether the fatty acid substrate of lipoxygenase binds "carboxylate-end first" or "methyl-end first" in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steady-state and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly pi-pi interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind "carboxylate-end first" with sLO-1 and not "methyl-end first" as has been proposed for human 15-lipoxygenase

In silico selection of therapeutic antibodies for development: Viscosity, clearance, and chemical stability

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery

In silico selection of therapeutic antibodies for development: Viscosity, clearance, and chemical stability

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery