Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine.

Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7-inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.

The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase.

Ascorbate peroxidase (APX) is a class I heme peroxidase. It has two sites for binding of substrates. One is close to the γ-heme edge and is used for oxidation of ascorbate; the other is at the δ-heme edge and is used for binding of aromatic substrates [Gumiero et al., (2010) Arch. Biochem. Biophys. 500, 13-20]. In this work, we have examined the structural factors that control binding at the δ-heme edge by replacement of Ala134 in APX with a proline residue that is more commonly found in other class II and III peroxidases. Kinetic data indicate that replacement of Ala134 by proline has only a small effect on the catalytic mechanism, or the oxidation of ascorbate or guaiacol. Chemical modification with phenylhydrazine indicates that heme accessibility close to the δ-heme edge is only minorly affected by the substitution. We conclude that the A134P mutation alone is not enough to substantially affect the reactivity of APX towards aromatic substrates bound at the δ-heme edge. The data are relevant to the recent application of APX (APEX) in cellular imaging.

Structure-guided design of purine-based probes for selective Nek2 inhibition.

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.

Quaternary organization of GPIb-IX complex and insights into Bernard-Soulier syndrome revealed by the structures of GPIbß and a GPIbß/GPIX chimera.

Platelet GPIb-IX receptor complex has 3 subunits GPIbα, GPIbß, 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 GPIbß ectodomain (GPIbß(E)) and determined the structure to show a single leucine-rich repeat with N- and C-terminal disulphide-bonded capping regions. The structure of a chimera of GPIbß(E) and 3 loops (a,b,c) taken from the GPIX ectodomain sequence was also determined. The chimera (GPIbß(Eabc)), but not GPIbß(E), forms a tetramer in the crystal, showing a quaternary interface between GPIbß and GPIX. Central to this interface is residue Tyr106 from GPIbß, 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 GPIbß and GPIX in the full-length complex. Eight GPIbß missense mutations identified from patients with Bernard-Soulier syndrome were examined for changes to GPIb-IX complex surface expression. Two mutations, A108P and P74R, were found to maintain normal secretion/folding of GPIbß(E) but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbß(E) interface with GPIX indicates they disrupt the quaternary organization of the GPIb-IX complex.

Fractal-based scaling and scale-invariant dispersion of peak concentrations of crop protection chemicals in rivers.

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.