No abstract

The interactions between a set of drugs, selected on the basis of reported human serum albumin (HSA) binding levels, and immobilized HSA were investigated using surface plasmon resonance technology. Major HSA binding sites were available after immobilization. The intensity of the signal obtained from the interaction of the drug with the HSA surface was correlated with the reported HSA binding level. Drugs were classified into groups corresponding to high, medium, or low HSA binding based on the injection of the drug at 80 microM concentration. A set of 10 drugs binding to alpha(1)-acid glycoprotein (AGP) was also investigated and correlated with reported AGP binding data. The throughput of the presented assay is 100 compounds/24 h, and the sample consumption is less than 100 microL (8 nmol).

The interaction between HIV-1 protease and 58 structurally diverse transition-state analogue inhibitors has been analyzed by a surface plasmon resonance based biosensor. Association and dissociation rate constants and affinities were determined and displayed as k(on)-k(off)-K(D) maps. It was shown that different classes of inhibitors fall into distinct clusters in these maps. Significant changes in association and dissociation rates were found as a result of modifying the P1/P1' or P2/P2' side chains of a linear lead compound. Similarly, cyclic urea and cyclic sulfamide inhibitors displayed different kinetic features and the affinities of both classes of cyclic compounds were limited by fast dissociation rates. These results confirm that association and dissociation rates are important features of drug-target interactions and indicate that optimization of inhibitor efficacy may be guided by aiming for high association and low dissociation rates rather than high affinity alone. The present approach thus provides a new tool for structure-interaction kinetic analysis and drug discovery.

Background-Passive smoking is associated with early arterial damage in adults, but its effect on endothelial function in children is unknown. Methods and Results-Serum cotinine concentration was measured annually in children between 8 and 11 years of age who had participated since infancy in a randomized, prospective atherosclerosis prevention trial (Special Turku Coronary Risk Factor Intervention Project for children [STRIP]). At age 11, endothelium-dependent flow-mediated vasodilatory responses of the brachial artery were examined with high-resolution ultrasound in 402 children. These children were divided into 3 groups according to serum cotinine concentrations: the noncotinine group (nondetectable cotinine, nϭ229), the low cotinine group (cotinine between 0.2 and 1.6 ng/mL, nϭ134), and the top decile cotinine group (cotinine Ն1.7 ng/mL, nϭ39). Longitudinal cotinine data in children aged 8 to 11 years and ultrasound studies were available in 327 children. At age 11, the increase in cotinine concentration was associated with attenuated peak flow-mediated dilation response (meanϮSD: the noncotinine group 9.10Ϯ3.88%, the low-cotinine group 8.57Ϯ3.78%, and the top-decile cotinine group 7.73Ϯ3.85%; Pϭ0.03 for trend). Similarly, total dilation response (the area under the dilation response versus time curve between 40 and 180 seconds after hyperemia) was affected by the cotinine level (Pϭ0.02 for trend). These trends were not explained by traditional atherosclerosis risk factors. Arterial measures and passive smoking showed even stronger associations when longitudinal cotinine data were used (peak flow-mediated dilation, Pϭ0.01 for trend; total dilation response, Pϭ0.008 for trend). Conclusions-Exposure

A general regeneration, identification, and optimization (RO) protocol for Biacore systems was developed. The RO protocol uses six multi-ingredient stock solutions that represent the six most common chemical properties employed as regeneration agents. The regeneration effect of different regeneration cocktails of these six stock solutions were tested iteratively until a satisfactory result was obtained. The RO protocol was designed with an ease-of-use and multivariate approach. The RO protocol was tested on 13 different antibody-antigen systems. For 10 of these, only the first screening session was tested. For 9 of the 13 systems, the RO-protocol screening session identified cocktails that removed more than 90% of the bound analyte in a 30 s pulse. For 5 systems, the RO protocol identified cocktails that regenerated the surface completely and that were more gentle than previously used regeneration conditions. Furthermore, the regeneration optimization results can be interpreted as a characterization of the interacting molecules. The relevance of testing cocktails was justified by the fact that at least one cocktail was significantly better than all diluted stock solutions for all tested model systems. By using the multivariate approach, the risk of missing relevant combinations of stock solutions was minimized. This resulted in an unexpected discovery of excellent properties of EDTA as an additive in regeneration cocktails containing chaotropic agents and ions in high concentration.

Background-Exposure to tobacco smoke is associated with markers of preclinical atherosclerosis in adults, but its effect on arterial structure in adolescents is unknown. Methods and Results-Healthy 13-year-old adolescents from the atherosclerosis prevention trial STRIP were studied.Maximum carotid and aortic intima-media thickness and brachial artery flow-mediated dilation were measured in 494 adolescents using high-resolution ultrasound. Serum lipid, lipoprotein, and apolipoprotein (Apo) A-I and B concentrations were determined using standard methods. Exposure to tobacco smoke was measured annually between ages 8 and 13 years using serum cotinine concentrations, analyzed with gas chromatography. To define longitudinal exposure, cotinine values of children having serum cotinine measured 2 to 6 times during follow-up were averaged and divided into tertiles (exposure groups): low (nϭ160), intermediate (nϭ171), and high (nϭ163 1 Passive smoking has been associated with attenuated endothelium-dependent dilation in young healthy adults. 2,3 We have previously shown in 11-year-old healthy children that tobacco smoke exposure is associated with endothelial dysfunction measured by flowmediated dilation (FMD) of the brachial artery. 4 In addition, both past and current passive smoking has been related with increased carotid intima-media thickness (cIMT) in adults. [5][6][7] Moreover, tobacco smoke exposure in pregnancy has recently been associated with increased aortic intima-media thickness (aIMT) in neonates 8 and increased cIMT in young adulthood. 9 Conventional cardiovascular risk factors have been related to early structural vascular wall changes in childhood, 10 -12 and exposure to cardiovascular risk factors in adolescence predicts increased adult cIMT. 13 However, none of the previous studies has examined the impact of exposure to tobacco smoke on IMT in healthy children or adolescents.Exposure to tobacco smoke may lead to alterations in serum lipid profile, especially to decrease in HDL cholesterol, in children 14,15 and adolescents. 16,17 In adults, heavy workplace exposure to tobacco smoke has been demonstrated to have an adverse influence on serum lipids. 18,19 Recently, it was indicated that maternal smoking in pregnancy is associ- 21 Exposure to tobacco smoke as indicated by objective measurement, serum cotinine concentration, has been frequently determined in school-aged children. The aim of the present study was to examine the effects of frequent exposure to tobacco smoke on vascular wall structure, endothelial function, and serum lipid profile in healthy 13-year-old adolescents. WHAT IS KNOWN• Exposure to environmental tobacco smoke is related to increased carotid intima-media thickness in adults, but this association has not been studied in children.• Exposure to environmental tobacco smoke may lead to alterations in serum lipid profile, but there are no data on the relations of tobacco smoke exposure and apolipoprotein levels in children. WHAT THE STUDY ADDS• This study shows that frequent exposure ...

No abstract

Interaction kinetic and thermodynamic analyses provide information beyond that obtained in general inhibition studies, and may contribute to the design of improved inhibitors and increased understanding of molecular interactions. Thus, a biosensor-based method was used to characterize the interactions between HIV-1 protease and seven inhibitors, revealing distinguishing kinetic and thermodynamic characteristics for the inhibitors. Lopinavir had fast association and the highest affinity of the tested compounds, and the interaction kinetics were less temperature-dependent as compared with the other inhibitors. Amprenavir, indinavir and ritonavir showed non-linear temperature dependencies of the kinetics. The free energy, enthalpy and entropy (DeltaG, DeltaH, DeltaS) were determined, and the energetics of complex association (DeltaG(on), DeltaH(on), DeltaS(on)) and dissociation (DeltaG(off), DeltaH(off), DeltaS(off)) were resolved. In general, the energetics for the studied inhibitors was in the same range, with the negative free energy change (DeltaG < 0) due primarily to increased entropy (DeltaS > 0). Thus, the driving force of the interaction was increased degrees of freedom in the system (entropy) rather than the formation of bonds between the enzyme and inhibitor (enthalpy). Although the DeltaG(on) and DeltaG(off) were in the same range for all inhibitors, the enthalpy and entropy terms contributed differently to association and dissociation, distinguishing these phases energetically. Dissociation was accompanied by positive enthalpy (DeltaH(off) > 0) and negative entropy (DeltaS(off) < 0) changes, whereas association for all inhibitors except lopinavir had positive entropy changes (DeltaS(on) > 0), demonstrating unique energetic characteristics for lopinavir. This study indicates that this type of data will be useful for the characterization of target-ligand interactions and the development of new inhibitors of HIV-1 protease.