No abstract

Many drug candidates fail in clinical trials due to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug:target complex has a longer half-life than off-target:drug complexes. However, while slow-binding inhibition kinetics are a key feature of many marketed drugs1,2, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.

In this article we introduce a new scoring system for doing operational code analysis and test its reliability and validity by measuring and modeling President Jimmy Carter's operational code. Using speeches from the public record, we construct indices for the elements of the operational code construct. Based upon the valences and scaled intensities of verbs uttered in the speeches, President Jimmy Carter's views of the political universe and approaches to political action in different issue areas are identified and compared. The results of the analysis provide reasonable support for the face, construct, and content validity of the operational code indices. We find that Carter's view of the political universe and approach to political action were consistent across issue areas during the first three years of his term as president. Following the Soviet invasion of Afghanistan President Carter's support for human rights remained steadfast. Statistically significant shifts occurred in his views of the Soviet Union and others in the political universe and in his approach to political action regarding the conduct of U.S.-Soviet relations and other issues.An important set of variables in the decision-making approach to foreign policy analysis is the decision maker's worldview and predispositions toward political action (Snyder, Bruck, and Sapin, 1962;Herrmann, 1988;Kaplowitz, 1990). A variety of methods exist for evaluating these elements of a leader's definition of the International Studies Quarterly (1998) 42, [175][176][177][178][179][180][181][182][183][184][185][186][187][188][189][190] We would like to thank Alexander George, Joe Hagan, and the editors and anonymous reviewers of ISQ for their helpful questions, comments, and suggestions.

Antibacterial polymers have potential as pharmaceuticals and as coatings for implantation devices. The design of these materials will be optimized when we have a complete understanding of the structural features that impart activity toward target organisms and those that are benign with respect to the mammalian host. In this work, four series of polymers in which cationic and hydrophobic groups were distributed along the backbone were tested against six different bacterial species (both Gram positive and Gram negative) and for host cytotoxicities (red blood cell lysis). The most effective of the polymers studied are regularly spaced, featuring a 6-8 carbon stretch along the backbone between side chains that present positively charged groups. They cause potassium efflux, disorder the bacterial cytoplasmic membrane, and disrupt the membrane potential. These polymers, available from alternating ring opening metathesis polymerization (AROMP), offer proof of principle for the importance of regular spacing in antibacterial polymers and for the synthesis of additional functional materials based on regularly spaced scaffolds.In the more than 80 years since Fleming discovered penicillin, humans have expended a great deal of effort in the search for, optimization of, and testing of new antibiotics. During the same period, new pathogens have appeared and antibiotic-resistant strains have evolved. The war against the microbes is far from over (1).Of particular concern in the western world is the incidence of hospital-acquired infections and the drug resistance of many of the causative phenotypes (2,3). The appearance of methicillin-resistant Staphylococcus aureus (4) and resistant strains of pneumonia and tuberculosis (5,6) as community-acquired infections have served to focus public attention on this growing health problem, leading The Infectious Diseases Society of America to call for renewed efforts to develop antimicrobial therapies (7).One attractive avenue for new antibiotic development is the exploitation or development of "host-defense" antimicrobial peptides (AMPs). Eukaryotes produce these small peptides (about 12 to 80 amino acid residues) as part of their innate immune response against pathogen infection (8-10). Some AMPs are preorganized so that they are amphipathic; i.e. cationic residues are segregated from hydrophobic residues onto opposing faces of the peptide (11). Others, it appears, are induced to adopt an amphipathic topology by contact with cell membranes (or, in laboratory experiments, with micellar surfaces). The inherent or induced amphipathic conformation of an AMP facilitates binding to and insertion into lipid bilayers. Subsequent disruption of the cytoplasmic membrane (8) can lead to bacterial death. Alternatively, some antimicrobial peptides are thought to cause cell death by additional mechanisms (11) including membrane depolarization, binding to cytoplasmic components (12,13), and inhibition of cell wall synthesis (14). Regardless of the killing mechanism, the ability to interact with...

©Operative Dentistry, 2008, 33-1, 72-78 SUMMARYImproving the adaptation of resin composites during placement is necessary to increase durability and reduce microleakage. Flowable resin liners have been introduced to improve adaptation in composite restorations. In addition, a device that lowers the viscosity of regular dental composites has been introduced (Calset, AdDent Inc, Danbury, CT, USA). This device lowers the viscosity of composites by preheating them to 54.4°C, which should lead to improved adaptation. This study compared microleakage in Class II composite restorations prepared using: 1) preheated resin composite, 2) unheated composite and 3) a flowable liner followed by unheated composite. Class II cavities were prepared on the mesial and distal surfaces of extracted third molars. Ten preparations were restored with resin composite (Esthet-X, Dentsply, York, PA, USA) for each of the following four techniques: Control (Esthet-X with Prime & Bond NT, Dentsply), Flowable (f) (as Control but used Esthet-X Flow liner), Preheated (p) (as Control but with preheating composite to 54.4°C) and Delay (d) (as Preheated but followed by a 15-second delay before curing). The teeth were restored, finished, stored in distilled water for 24 hours, then thermocycled between water bath temperatures of 5°C and 55°C with a one-minute dwell time for 1000 cycles. Tooth apices were sealed with epoxy and varnish was applied to within 1 mm of the restoration margins. The teeth were placed in 0.5% basic fuschin dye for 24 hours, rinsed, then embedded in self-curing resin. The embedded teeth were sectioned mesiodistally with a slow-speed diamond saw, providing multiple sections per restoration. Microleakage was rated by two evaluators using Clinical RelevanceThe results of this study indicate that preheating composites can improve adaptation of resin composites to tooth structure. This technique significantly reduced microleakage. However, delaying light curing of the preheated composite after placement appears to be counterproductive and diminishes the positive effects from the preheating treatment. Flowable liner was less effective than preheating the composite in reducing microleakage.

Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol using 2-monoacylglycerol and fatty acyl coenzyme A. This enzymatic reaction is believed to be an essential and ratelimiting step for the absorption of fat in the small intestine. Although the first MGAT-encoding cDNA, designated MGAT1, has been recently isolated, it is not expressed in the small intestine and hence cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption. In the current study, we report the identification of a novel MGAT, designated MGAT3, and present evidence that it fulfills the criteria to be the elusive intestinal MGAT. MGAT3 encodes a ϳ36-kDa transmembrane protein that is highly homologous to MGAT1 and -2. In humans, expression of MGAT3 is restricted to gastrointestinal tract with the highest level found in the ileum. At the cellular level, recombinant MGAT3 is localized to the endoplasmic reticulum. Recombinant MGAT3 enzyme activity produced in insect Sf9 cells selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers. The molecular identification of MGAT3 will facilitate the evaluation of using intestinal MGAT as a potential point of intervention for antiobesity therapies.

Strains of Caulobacter crescentus express a paracrystalline surface layer (S-layer) consisting of the protein RsaA. Mutants of C. crescentus NA1000 and CB2, isolated for their ability to grow in the absence of calcium ions, uniformly no longer had the S-layer attached to the cell surface. However, RsaA was still produced, and when colonies grown on calcium-sufficient medium were examined, large two-dimensional arrays of S-layer were found intermixed with the cells. Such arrays were not found in calcium-deficient medium even when high levels of magnesium ions were provided. The arrays could be disrupted with divalent ion chelators and more readily with the calcium-selective ethylene glycol-bis(P-aminoethyl ether)NNN',N'-tetraacetic acid (EGTA).Thus, the outer membrane surface was not needed as a template for self-assembly, but calcium likely was. The cell surface and S-layer gene of assembly-defective mutants of NA1000 were examined to determine the basis of the S-layer surface attachment defect. Mutants had no detectable alteration in the rough lipopolysaccharide (LPS) or a characterized capsular polysaccharide, but another polysaccharide molecule was greatly reduced or absent in all calcium-independent mutants. The molecule was shown to be a smooth LPS with a core sugar and fatty acid complement identical to those of the rough LPS and an 0 polysaccharide of homogeneous length, tentatively considered to be composed of 4,6-dideoxy-4-amino hexose, 3,6-dideoxy-3-amino hexose, and glycerol in equal proportions. This molecule (termed SLPS) was detectable by surface labeling with a specific antiserum only when the S-layer was not present. The rsaA genes from three calcium-independent mutants were cloned and expressed in an S-layer-negative, SLPS-positive strain. A normal S-layer was produced, ruling out defects in rsaA4 in these cases. It is proposed that SLPS is required for S-layer surface attachment, possibly via calcium bridging. The data support the possibility that calcium binding is required to prevent an otherwise lethal effect of SLPS. If true, mutations that eliminate the 0 polysaccharide of SLPS eliminate the lethal effects of calcium-deprived SLPS, at the expense of S-layer attachment.Surface layers (S-layers) are a common feature on the surface of eubacteria and archaebacteria (34). The structures are generally composed of repeated units of a single protein or glycoprotein (44). It is likely that in many bacteria the layer is the outermost layer, directly exposed to the environment (44). In such a position, S-layers may well have roles of protection for the cell or be involved with pathogenesis in the case of pathogenic bacteria (34). Many aspects of this general bacterial structure were summarized in a recent international meeting devoted to such structures (5).Most often, S-layer proteins are assembled on (rather than within) the underlying layer, whether it is a bilayer membrane or a complex of peptidoglycan or pseudomurein (34). Thus, S-layer structural subunits are secreted proteins and must retain b...

Approximately one-third of the world's population carries Staphylococcus aureus. The recent emergence of extreme drug resistant strains that are resistant to the "antibiotic of last resort", vancomycin, has caused a further increase in the pressing need to discover new drugs against this organism. The S. aureus enoyl reductase, saFabI, is a validated target for drug discovery. To drive the development of potent and selective saFabI inhibitors, we have studied the mechanism of the enzyme and analyzed the interaction of saFabI with triclosan and two related diphenyl ether inhibitors. Results from kinetic assays reveal that saFabI is NADPH-dependent, and prefers acyl carrier protein substrates carrying fatty acids with long acyl chains. On the basis of product inhibition studies, we propose that the reaction proceeds via an ordered sequential ternary complex, with the ACP substrate binding first, followed by NADPH. The interaction of NADPH with the enzyme has been further explored by site-directed mutagenesis, and residues R40 and K41 have been shown to be involved in determining the specificity of the enzyme for NADPH compared to NADH. Finally, in preliminary inhibition studies, we have shown that triclosan, 5-ethyl-2-phenoxyphenol (EPP), and 5-chloro-2-phenoxyphenol (CPP) are all nanomolar slow-onset inhibitors of saFabI. These compounds inhibit the growth of S. aureus with MIC values of 0.03-0.06 microg/mL. Upon selection for resistance, three novel safabI mutations, A95V, I193S, and F204S, were identified. Strains containing these mutations had MIC values approximately 100-fold larger than that of the wild-type strain, whereas the purified mutant enzymes had K i values 5-3000-fold larger than that of wild-type saFabI. The increase in both MIC and K i values caused by the mutations supports the proposal that saFabI is the intracellular target for the diphenyl ether-based inhibitors.