Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands

Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential

Raf Kinase Inhibitory Protein Protects Cells against Locostatin-Mediated Inhibition of Migration

Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle.These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells

The granule size distribution influence in nanocomposites on optical and magnetooptical spectra

We have investigated the size effect (quasi-classical size effect) in nanocomposites. It was shown that the size effect can change the amplitude, form and sign of the optical and magnetooptical spectra. We have deduced formulas for size effect and discussed the applications of the distributions for corrected description of optical and magnetooptical properties with regard to the granule size effect. It is very important to consider the distribution on the granule size in size effect. This fact allows to describe optical and magnetooptical spectra of nanocomposites better, especially in near IR due to intraband electron transitions. We have deduced formulas for size effect and discussed applications of the distributions for corrected description of optical and magnetooptical properties with regard to the effect of the granule size

The granule size distribution influence in nanocomposites on optical and magnetooptical spectra

We have investigated the size effect (quasi-classical size effect) in nanocomposites. It was shown that the size effect can change the amplitude, form and sign of the optical and magnetooptical spectra. We have deduced formulas for size effect and discussed the applications of the distributions for corrected description of optical and magnetooptical properties with regard to the granule size effect. It is very important to consider the distribution on the granule size in size effect. This fact allows to describe optical and magnetooptical spectra of nanocomposites better, especially in near IR due to intraband electron transitions. We have deduced formulas for size effect and discussed applications of the distributions for corrected description of optical and magnetooptical properties with regard to the effect of the granule size

Solvable model of two-dimensional magnetophotonic crystal

We present theoretical investigation of two-dimensional (2D) magnetophotonic crystal (MPC) with square lattice constructed from two infinite arrays of magnetoactive dielectric sheets at right angle, in the limit of very small sheet thickness and very high dielectric constant, such that their product is constant. Alteration of band structure by external magnetic field is studied. We show that optical activity reduces symmetry of the system and removes degeneracy in the photonic band structure. Also, despite of natural weakness of the magnetooptic activity, dispersion near band edges is found to be strongly sensitive to external magnetic influence.6 page(s

Superprism effect in magneto-photonic crystals

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Potency and Mechanism of Action of E4021, a Type 5 Phosphodiesterase Isozyme-Selective Inhibitor, on the Photoreceptor Phosphodiesterase Depend on the State of Activation of the Enzyme

The ability of inhibitors selective for the type 5 phosphodiesterase isozyme (PDE5) to act on the photoreceptor PDE isozyme (PDE6, the central effector enzyme for visual transduction) is poorly understood. Because PDE5 inhibitors are currently used as therapeutic agents, it is important to assess the potency and mechanism of action of this class of PDE inhibitor on PDE6. We show that E4021 (sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-yl]piperidine-4-carboxylate sesquihydrate) inhibits activated PDE6 (KI = 1.7 nM) as potently as PDE5. This makes E4021 the most potent inhibitor of PDE6 discovered to date. The effectiveness of E4021 to inhibit nonactivated PDE6 (with bound inhibitory γ subunits) is reduced 40-fold compared with the activated enzyme. Furthermore, at intermediate E4021 concentrations and high cGMP concentrations, nonactivated PDE undergoes activation of cGMP hydrolysis rather than inhibition. We demonstrate direct competition of E4021 and the γ subunits for binding to the catalytic site. Measurements of cGMP binding to noncatalytic regulatory sites on the catalytic subunits of PDE6 rule out an allosteric effect of E4021 by direct binding to these noncatalytic sites. We conclude that E4021 is a competitive inhibitor of cGMP hydrolysis and that the γ subunit also competes with both E4021 and substrate for catalytic site binding. An understanding of the effects of PDE5-targeted drugs on retinal PDE6 requires a knowledge of the complex interactions among substrate, drug, and inhibitory γ subunit at the catalytic site of both nonactivated and activated forms of PDE6