AMP transfer catalyzed by Fic proteins and their inhibition-relieved variants.

<p>Autoradiography of VbhA/VbhT(FIC), SoFic and NmFic (wt, wild type; E/G, E->G mutant) after incubation with radioactively labeled α-³²P-ATP.</p

Sequence independent registration of peptide or target protein to the FIC flap.

<p>The bound peptide/protein segment (blue) and the target dock (brown) are shown in full. Main chain-main chain H-bonds are depicted as stippled lines. A, Product complex of IbpA(Fic2) with Cdc42 target <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064901#pone.0064901-Xiao1" target="_blank">[4]</a>. Tyrosine 32 from the switch1 region of Cdc42 is adenylylated. B, VbhA/VbhT(FIC) complexed with residues 203 to 206 of a symmetry related molecule. The 2Fo-Fc simulated annealing omit map covering the residues 203 to 206 is contoured at 1.1 σ. Note that the preceding 7 residues are disordered and not shown. C, SoFic complexed with residues 0 to 4 of a symmetry related molecule (PDB 3EQX) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064901#pone.0064901-Das1" target="_blank">[16]</a>. The side-chains of residues 0, 1 and 4 are disordered and not displayed for clarity reason. Note that Y32 in panel A, V203 in panel B and W3 in panel C are in equivalent positions.</p

Crystal structures of wild-type Fic proteins representing classes I to II in complex with ATP substrate.

<p>(A) VbhA/VbhT(FIC), (B) SoFic. Structures are shown in cartoon representation (red, FIC core as defined by PFAM; yellow, active site loop and N-terminal end of helix α5; dark-green, inhibitory helix α_inh). In (A), the fold of the antitoxin is shown in dark-green and steel-blue. Selected residues are shown in full with the inhibitory glutamate (E24 or E73, respectively) colored in dark. The 2Fo-Fc simulated annealing omit maps covering the ligand are contoured at 1.1 σ. In both structures, the orientation of the α-phosphate prevents nucleophilic attack of a putative target side-chain hydroxyl onto the ATP substrate, since the position inline with the scissile Pα-O3α bond (magenta star) is unattainable. C) Stereo view of the superposition of the ATP nucleotides shown in panel A and B with AMPPNP from the complex structure of the class III NmFic protein (PDB 3S6A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064901#pone.0064901-Engel1" target="_blank">[8]</a>) within the active site of the VbhA/VbhT(FIC) complex (same as in panel A). The nucleotides of the various complexes are distinguished by their colors (white for the ATP bound to VbhA/VbhT(FIC), green for the ATP bound to SoFic, and blue for the AMPPNP of the NmFic complex. Note that the AMPPNP γ-phosphate in NmFic is found disordered <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064901#pone.0064901-Engel1" target="_blank">[8]</a> and therefore not shown. The residues of the HxFx(D/E)GNGRxxR Fic signature motif are labeled, the two glycine and the two arginine residues are distinguished by a "1" or "2" in brackets. The phenylalanine (not shown) is part of the hydrophobic core. The inhibitory glutamate from α_inh is labeled as E_inh.</p

Comparison of triphosphate nucleotide structures as bound to wild-type and E->G mutated Fic proteins from class I to III.

<p>Stereo views of the ligand structures after superposition of the FIC domains (not shown). Also shown is the inhibitory glutamate of the wild-type structures. A, ATP as bound to VbhA/VbhT wild-type (white) and the E24G mutant (dark green). B, ATP and AMPPNP as bound to SoFic wild-type (green) and the E73G mutant (orange), respectively. C, AMPPNP as bound to NmFic (blue) and the E186G mutant (pink). Note that the AMPPNP γ-phosphate in NmFic is found disordered <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064901#pone.0064901-Engel1" target="_blank">[8]</a> and therefore not shown.</p

Crystal structures of E->G mutated Fic proteins representing classes I to III in complex with substrate or substrate analog.

<p>A, VbhA_E24G/VbhT(FIC) in complex with ATP/Mg²⁺; B, SoFic_E73G, C, NmFic_E186G, both in complex with AMPPNP/Mg²⁺. Representation as in Fig. 2 with magnesium ions shown as magenta spheres. The 2Fo-Fc simulated annealing omit maps covering the nucleotide/Mg²⁺ ligands are contoured at 1.1 σ. D, Stereo view of the superposition of the ligand structures shown in panels B and C onto the VbhA_E24G/VbhT(FIC) complex (same as in panel A). Note that the nucleotides of the various complexes are distinguished by their carbon color (VbhA_E24G/VbhT(FIC) ATP in green, SoFic_E73G AMPPNP in orange and NmFic_E186G AMPPNP in pink). The residues of the HxFx(D/E)GNGRxxR signature motif are labeled as in Fig. 2C with the phenylalanine not shown. Also shown is the modifiable hydroxyl side-chain Y32 of Cdc42 (blue) after superposition of the IbpA(FIC2)/Cdc42 complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064901#pone.0064901-Xiao1" target="_blank">[4]</a> onto VbhA_E24G/VbhT(FIC). For the superposition, only the Fic active site loops were used. The α-phosphate moieties appear well-suited for in-line attack of the target hydroxyl group (broken line in magenta).</p

Data collection and refinement statistics.

<p>Values for the highest resolution shell are shown in brackets.</p

A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role