Structural characterization and computer-aided optimization of a small-molecule inhibitor of the Arp2/3 complex, a key regulator of the actin cytoskeleton

CK-666 (1) is a recently discovered small-molecule inhibitor of the actin-related protein 2/3 (Arp2/3) complex, a key actin cytoskeleton regulator with roles in bacterial pathogenesis and cancer cell motility. Although 1 is commercially available, the crystal structure of Arp2/3 complex with 1 bound has not been reported, making its mechanism of action uncertain. Furthermore, its relatively low potency increases its potential for off-target effects in vivo, complicating interpretation of its influence in cell biological studies and precluding its clinical use. Herein we report the crystal structure of 1 bound to Arp2/3 complex, which reveals that 1 binds between the Arp2 and Arp3 subunits to stabilize the inactive conformation of the complex. Based on the crystal structure, we used computational docking and free-energy perturbation calculations of monosubstituted derivatives of 1 to guide optimization efforts. Biochemical assays of ten newly synthesized compounds led to the identification of compound 2, which exhibits a threefold increase in inhibitory activity in vitro relative to 1. In addition, our computational analyses unveiled a surface groove at the interface of the Arp2 and Arp3 subunits that can be exploited for additional structure-based optimization.     

Unifying model for associative thickener influences on waterborne coatings: II. Competitive adsorption of nonionic surfactants and HEUR thickeners on titanium dioxide pretreated with inorganic stabilizers and organic oligomeric dispersants

Size exclusion chromatography in tandem with UV absorbance of the surfactant is used to separate and quantify the amount of nonionic surfactant and model hydrophobically modified ethoxylated urethane (HEUR) thickeners adsorbed on different organic oligomeric polyacid, metal oxide-treated TiO₂ surfaces. The isoelectric point imposed on TiO₂ by the metal oxide surface treatment determines the amount of organic dispersant adsorbed. The size of the α-olefin on the maleic acid dispersant is also important in the adsorption of the dispersant. The size of the α-olefin on the organic acid dispersant, in turn, determines the amount of surfactant and HEUR thickener adsorbed. The surfactant, unlike the HEUR thickener, exhibits a dependence on the metal oxide treatment in neutral media, independent in several aspects of the amount of organic dispersant adsorbed. The adsorption dependence of HEUR thickeners and nonionic surfactant on the hydrophobe size of the HEUR is discussed. Presented at the 69th Annual Meeting of the Federation of Societies for Coatings Technology, on October 23, 1996 in Chicago, IL Polymers and Coatings Dept., Dunbar Hall, Fargo, ND 58105. Chestnut Run, Building 709, Wilmington, DE 19898.     

Surfactant behavior and its influence on the viscosity of associative thickeners solutions, thickened latex dispersions, and waterborne latex coatings

Surfactants, varying in their chemical composition and hydrophobic behavior, are used in the formulation of a waterborne coating. These differences influence their aggregation in micellar structures, their interaction with associative thickeners, and in particular, the synergies present in their competitive adsorptions on the disperse phases in a waterborne coating. Adsorption of HEUR thickeners on latexes and the ability of surfactants to displace them from those surfaces is an important variable in the dispersion’s viscosity. With large particle latexes, viscosity increases arise primarily from the network built through the interaction of HEURs with surfactants in the aqueous phase. Fluorescence is used to verify the mechanism by which surfactants enhance associative thickener viscosities. That is best achieved with nonionic surfactants, because of their synergies with large hydrophobe HEURs at low concentration. With decreasing latex particle size the adsorbed species is an important contributor to the dispersion’s viscosity through its contribution to the latex’s effective volume fraction increase and when the size of the adsorbed HEUR is matched to the separation distances of the latex at 0.25 volume fraction. Achieving controlled shear-thinning behavior in small particle size latex paints with the economic constraints on the amount of HEUR required to obtain 90 KU viscosities are discussed. Presented at the 80th Annual Meeting of the Federation of Societies for Coatings Technology, October 30–November 1, 2002, in New Orleans, LA. Polymer and Coatings Dept., Fargo, ND 58103.     

Effects of the Use of Pore Formers on Performance of an Anode Supported Solid Oxide Fuel Cell

The effects of amount of pore former used to produce porosity in the anode of an anode supported planar solid oxide fuel cell were examined. The pore-forming material utilized was rice starch. The reduction rate of the anode material was measured by Thermogravimetric analysis to qualitatively characterize the gas transport within the porous anode materials. Fuel cells with varying amounts of porosity produced by using rice starch as a pore former were tested. The performance of the fuel cell was the greatest with an optimum amount of pore former used to create porosity in the anode. This optimum is believed to be related to a trade off between increasing gas diffusion to the active three-phase boundary region of the anode and the loss of performance because of the replacement of active three-phase boundary regions of the anode with porosity.     

Chemical Ecology of the host searching behavior in an Egg Parasitoid: are Common Chemical Cues exploited to locate hosts in Taxonomically Distant Plant Species?

Parasitoids are known to exploit volatile cues emitted by plants after herbivore attack to locate their hosts. Feeding and oviposition of a polyphagous herbivore can induce the emission of odor blends that differ among distant plant species, and parasitoids have evolved an incredible ability to discriminate them and locate their hosts relying on olfactive cues. We evaluated the host searching behavior of the egg parasitoid Cosmocomoidea annulicornis (Ogloblin) (Hymenoptera: Mymaridae) in response to odors emitted by two taxonomically distant host plants, citrus and Johnson grass, after infestation by the sharpshooter Tapajosa rubromarginata (Signoret) (Hemiptera: Cicadellidae), vector of Citrus Variegated Chlorosis. Olfactory response of female parasitoids toward plants with no herbivore damage and plants with feeding damage, oviposition damage, and parasitized eggs was tested in a Y-tube olfactometer. In addition, volatiles released by the two host plant species constitutively and under herbivore attack were characterized. Females of C. annulicornis were able to detect and significantly preferred plants with host eggs, irrespectively of plant species. However, wasps were unable to discriminate between plants with healthy eggs and those with eggs previously parasitized by conspecifics. Analysis of plant volatiles induced after sharpshooter attack showed only two common volatiles between the two plant species, indole and β-caryophyllene. Our results suggest that this parasitoid wasp uses common chemical cues released by many different plants after herbivory at long range and, once on the plant, other more specific chemical cues could trigger the final decision to oviposit.

     

Rheology parameters controlling spray atomization and roll misting behavior of waterborne coatings

Formation of small coating droplets is a phenomenon common to spray and roll application of coatings. Generation of small droplets, referred to as atomization, is the mechanism of controlling the quality and thickness of the applied coating layer in spray technology. In roll coating application, small coating droplets are formed at the nip–exit region under certain conditions. Because of the high-shear rates at roll nips and spray nozzles, it is common to relate high-shear viscosity with atomization and roll misting behavior of coatings. This paper will review the relative importance of shear and extensional flows, and the response of coatings to such flows, in affecting misting and atomization behavior. Experimental efforts to quantify the effect of shear and extensional rheological parameters have clearly established a lack of correlation of high-shear viscosities with both spray atomization and roll misting behavior of coatings. However, extensional viscosity is shown to have a strong correlation. Finally, how rheology modifiers can be selected to circumvent misting problems in roll coating applications and atomization problems in spray applications is discussed.     

An investigation of particle behavior in gas–solid horizontal pipe flow by an extended LDA technique

An extended Laser Doppler Anemometry (LDA) technique has been developed to measure the distributions of particle velocities and particle number rates over a whole pipe cross-section in a dilute pneumatic conveying system. The first extension concentrates on the transform matrix for predicting the laser beams’ cross point in a pipe according to the shift coordinate of the 3D computer-controlled traverse system on which the probes of the LDA system were mounted. The second focuses on the proper LDA sample rate for the measurement of gas–solid pipe flow with polydisperse particles. A suitable LDA sample rate should ensure that enough data is recorded in the measurement interval to precisely calculate the particle mean velocity or other statistical values at every sample point. The present study explores the methodology as well as the fundamentals of measurements, using a laser facility, of the cross-sectional distributions of solid phase. In the horizontal gas–solid pipe flow (glass beads less than 110 μm), the experimental data show that the cross-sectional flow patterns of the solid phase can be classified by annulus-like flow describing the axial particle velocity contours and stratified flow characterising particle number rate distribution over a cross-section. Thus, the cross-sectional flow pattern of the solid phase in a horizontal pipe may be annular or stratified dependent on whether the axial particle velocity or particle number rate is the phenomenon studied.     

Long term assessment of a hybrid electrochemical treatment

Monitoring the performance of systems installed to protect against/halt the corrosion of reinforcing steel in concrete is essential in ensuring that the reinforcing bar (rebar) is adequately protected and remains in a non‐corroding state. A chloride contaminated concrete test block containing a hybrid anode system has been periodically monitored over the past 4.5 years to assess the anode current output and thus, the protection offered to the steel. De‐polarisation and impedance analysis data have been acquired to calculate steel corrosion rates. Both tests indicated that the steel was passive in the concrete environment and that the anode was able to protect all of the embedded steel despite the difficult geometry of the sample. It was also found that after 2 years it was still possible to drive a high current from the installed anode.     

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis,Characterization and Comparison

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.