Platelet GPIb-IX receptor complex has 3 subunits GPIb␣, GPIb␤, and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome. We have crystallized the GPIb␤ ectodomain (GPIb␤ E ) and determined the structure to show a single leucine-rich repeat with Nand C-terminal disulphide-bonded capping regions. The structure of a chimera of GPIb␤ E and 3 loops (a,b,c) taken from the GPIX ectodomain sequence was also determined. The chimera (GPIb␤ Eabc ), but not GPIb␤ E , forms a tetramer in the crystal, showing a quaternary interface between GPIb␤ and GPIX. Central to this interface is residue Tyr106 from GPIb␤, which inserts into a pocket generated by 2 loops (b,c) from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIb␤ and GPIX in the full-length complex. Eight GPIb␤ missense mutations identified from patients with BernardSoulier syndrome were examined for changes to GPIb-IX complex surface expression. Two mutations, A108P and P74R, were found to maintain normal secretion/folding of GPIb␤ E but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIb␤ E interface with GPIX indicates they disrupt the quaternary organization of the GPIb-IX complex. (Blood. 2011;118(19):5292-5301) IntroductionGPIb-IX-V complex is an abundant membrane receptor complex on the platelet surface that plays a critical role in mediating platelet adhesion to the damaged vessel wall under conditions of high shear stress. 1 Platelets adhere, and integrins are subsequently activated by interactions of GPIb-IX-V with VWF bound to the subendothelium. How GPIb-IX-V transmits the VWF-binding signal across the membrane is not clear, partly because the structure and organization of this complex receptor remain to be elucidated. Because GPV is only weakly associated with the receptor complex and is not essential for complex expression, assembly, VWF binding, or signal transduction, 2,3 we focus on the GPIb-IX complex here.The GPIb-IX complex contains 3 subunits, GPIb␣, GPIb␤, and GPIX, with a 1:2:1 stoichiometry. 4 Each subunit is a type I transmembrane (TM) protein, containing an ectodomain with leucine-rich repeats (LRRs), 5 a single TM helix, and a relatively short cytoplasmic tail. The GPIb␣ ectodomain contains binding sites for a growing list of hemostatically important ligands, including VWF and thrombin. 6-8 Covalent and noncovalent interactions are important to the quaternary stabilization of the receptor. GPIb␣ links to 2 GPIb␤ subunits through membrane-proximal disulfide bonds to constitute the GPIb complex. 4 GPIX tightly associates with GPIb through noncovalent interactions. 9 Assembly of these subunits into a tightly integrated complex is also supported by genetic evidence. Bernard-Soulier syndrome (BSS) is a hereditary bleeding disorder that is characterized in most cases by giant platelets, low platelet counts, and little or no expression of GPIb-IX on the p...

Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 μM; Nek2 IC50 = 0.62 μM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 μM; Nek2 IC50 = 0.27 μM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.

A new regulatory approach is needed to characterize peak pesticide concentrations in surface waters over a range of watershed scales. Methods now in common use rely upon idealized edge-of-field scenarios that ignore scaling effects. Although some watershed-scale regulatory models are available, their complexity generally prevents them from being used duringthe pesticide registration decision process, even though nearly all exposure to both humans and aquatic organisms can occur only at this scale. The theory of fractal geometry offers a simpler method for addressing this regulatory need. Mandelbrot described rivers as "space-filling curves" (Mandelbrot, B. B. The Fractal Geometry of Nature; Freeman: New York, 1983), a class of fractal objects implying two useful properties we exploit in this work. The first is a simple power-law relationship in which log-log plots of maximum daily concentrations as a function of watershed area tend to be linear with a negative slope. We demonstrate that the extrapolation of such plots down to smaller watersheds agrees with edge-of-field concentrations predicted using the Pesticide Root Zone Model, but only when the modeling results are properly adjusted for use intensity within the watershed. We also define a second useful property, "scale-invariant dispersion", in which concentrations are well described by a single analytical solution to the convective--dispersion equation, regardless of scale. Both of these findings make it possible to incorporate the effect of watershed scale directly into regulatory assessments.