P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position.

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.

Characterization of the cell surface glycolipid from Spirochaeta aurantia.

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia.

Interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product, or procedure?

It is not clear whether improvement in environmental decontamination is more efficiently achieved through changes in cleaning products, cleaning procedures, or performance of cleaning personnel. To assess the impact of cleaning performance on environmental contamination with vancomycin-resistant enterococci (VRE), we conducted a sequential trial in which a multifaceted environmental cleaning improvement intervention was introduced in a medical intensive care unit and respiratory step-down unit. The intervention included educational lectures for housekeepers and an observational programme of their activities without changes in cleaning products or written procedures.

Structure-based design of novel groups for use in the P1 position of thrombin inhibitor scaffolds. Part 2: N-acetamidoimidazoles.

Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.

Risk of hand or glove contamination after contact with patients colonized with vancomycin-resistant enterococcus or the colonized patients' environment.

Objective: To estimate the level of hand or glove contamination with vancomycin-resistant enterococci (VRE) among healthcare workers (HCWs) who touch a patient colonized with VRE and/or the colonized patient's environment during routine care.

Design: Structured observational study.

Setting: Medical intensive care unit of a 700-bed, tertiary-care teaching hospital.

Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy.

TLRs constitute an essential family of pattern recognition molecules that, through direct recognition of conserved microbial components, initiate inflammatory responses following infection. In this role, TLR1 enables host responses to a variety of bacteria, including pathogenic species of mycobacteria. In this study, we report that I602S, a common single nucleotide polymorphism within TLR1, is associated with aberrant trafficking of the receptor to the cell surface and diminished responses of blood monocytes to bacterial agonists.

The polymorphism P315L of human toll-like receptor 1 impairs innate immune sensing of microbial cell wall components.

As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9-12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function.

Serum leptin levels, hepatic leptin receptor transcription, and clinical predictors of non-alcoholic steatohepatitis in obese bariatric surgery patients.

Background: Non-alcoholic steatohepatitis (NASH) is a major cause of liver disease in morbidly obese patients. Clinical predictors of NASH remain elusive, as do molecular mechanisms of pathogenesis.

Methods: A series of 35 morbidly obese patients undergoing bariatric surgery had a liver biopsy performed for standard histologic analysis.

Identification and characterization of 4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate, an orally bioavailable, brain penetrant NR2B selective N-methyl-D-aspartate receptor antagonist.

The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure-activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration.

Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus.

The lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory pattern-recognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts.

Alterations in peripheral blood lymphocyte cytokine expression in obesity.

Obesity is characterized by alterations in immune and inflammatory function. In order to evaluate the potential role of cytokine expression by peripheral blood mononuclear cells (PBMC) in obesity-associated inflammation, we studied serum protein levels and mRNA levels in PBMC of interleukin (IL)-6, IL-1beta, tumour necrosis factor (TNF)-alpha and IL-1Ra in nine lean and 10 obese subjects. Serum IL-1beta was undetectable, IL-1Ra serum levels were elevated, serum levels of TNF-alpha were decreased and serum levels of IL-6 were similar in obese subjects compared to lean subjects, while transcript levels of IL-6, IL-1beta and TNF-alpha, but not IL-1Ra, were decreased in PBMC from obese subjects.

Conformational chemistry of surface-attached calmodulin detected by acoustic shear wave propagation.

A thickness shear-mode acoustic wave device, operated in a flow-through format, was used to detect the binding of ions or peptides to surface-attached calmodulin. On-line surface attachment of the protein was achieved by immobilisation of the biotinylated molecule via a neutravidin-biotin linkage onto the surface of the gold electrode of the detector. The interaction between calmodulin, and calcium and magnesium ions induced an increase in resonant frequency and a decrease in motional resistance, which were reversible on washing with buffer.

Reduction in acquisition of vancomycin-resistant enterococcus after enforcement of routine environmental cleaning measures.

Background: The role of environmental contamination in nosocomial cross-transmission of antibiotic-resistant bacteria has been unresolved. Using vancomycin-resistant enterococci (VRE) as a marker organism, we investigated the effects of improved environmental cleaning with and without promotion of hand hygiene adherence on the spread of VRE in a medical intensive care unit.

Methods: The study comprised a baseline period (period 1), a period of educational intervention to improve environmental cleaning (period 2), a "washout" period without any specific intervention (period 3), and a period of multimodal hand hygiene intervention (period 4).

Domain exchange between human toll-like receptors 1 and 6 reveals a region required for lipopeptide discrimination.

Among the 10 human Toll-like receptors (TLRs), TLR2 appears to be unique in its requirement for cooperation with other TLRs, namely TLR1 and TLR6, to mediate cell signaling. Through reconstitution experiments, we have defined more precisely the function of these human TLRs. Human colonic epithelial cells cotransfected with TLR1 and -2 preferentially respond to a synthetic tripalmitoylated bacterial lipopeptide analogue (Pam(3)CSK(4)).

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs).

Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins.

The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2).

Transfer of vancomycin-resistant enterococci via health care worker hands.

Background: The roles of the contaminated hospital environment and of patient skin carriage in the spread of vancomycin-resistant enterococci (VRE) are uncertain. Transfer of VRE via health care worker (HCW) hands is assumed but unproved. We sought to determine the frequency of VRE transmission from sites in the environment or on patients' intact skin to clean environmental or skin sites via contaminated hands of HCWs during routine care.

The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGLB.

In an attempt to isolate lipopolysaccharide from Spirochaeta aurantia, Darveau-Hancock extraction of the cell mass was performed. While no lipopolysaccharide was found, two carbohydrate-containing compounds were detected. They were resolved by size-exclusion chromatography into high molecular mass (LGLA) and low molecular mass (LGLB) fractions.

Low molecular weight thrombin inhibitors with excellent potency, metabolic stability, and oral bioavailability.

Modification of lead compound 1 by reducing lipophilicity in the P3 group produced a series of low molecular weight thrombin inhibitors with excellent potency in functional assays, metabolic stability, and oral bioavailability. These modifications led to the identification of two optimized compounds, 14 and 16.

Differential effects of sodium nitroprusside and hydralazine in a rat model of topical FeCl3-induced carotid artery thrombosis.

Introduction: In the rat model of topical ferric chloride-induced carotid artery thrombosis, a transient blood flow velocity (VEL) increase is observed immediately following ferric chloride application. The immediacy of the response suggested vasoconstriction, as thrombotic narrowing of the vessel lumen was hypothesized to be too slow to account for the rapidity of the response.

Methods: To explore this phenomenon, the effects of two mechanistically distinct vasodilators, sodium nitroprusside (SNP) and hydralazine (HYD), on velocity increase, ex vivo platelet aggregation and thrombosis, were assessed in the rat ferric chloride-induced thrombosis model.

3-amino-4-sulfonylpyridinone acetamide and related pyridothiadiazine thrombin inhibitors.

We describe a series of highly potent and efficacious thrombin inhibitors based on a 3-amino-4-sulfonylpyridinone acetamide template. The functionally dense sulfonyl group stabilizes the aminopyridinone, conformationally constrains the 4-substituent, and forms a hydrogen bond to the insertion loop tyrosine OH. We also describe a related series of fused bicyclic dihydrothiadiazinedioxide derivatives, of which one had improved pharmacokinetics in dogs after oral dosing.

Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors.

Acoustic coupling of transverse waves as a mechanism for the label-free detection of protein-small molecule interactions.

An on-line acoustic transverse wave device has been used to study the binding interactions of human serum albumin with the small molecule drug, warfarin. Four linking systems for the covalent attachment of the protein to the surface of the gold electrode of the sensor were employed, namely thioctic acid, cysteamine, an N-hydroxysuccinimide ester and 11-mercaptoundecanoic acid. All the attachment protocols involve the ability of thiols to form gold-sulfur bonds at the metal surface.