The primary structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i> and its homology to other HPPK and DHPS enzymes.

<p>The organisms shown are <i>Francisella tularensis</i> (Ft), <i>Saccharomyces cerevisiae</i> (Sc), <i>Yersinia pestis</i> (Yp), <i>Escherichia coli</i> (Ec) and <i>Bacillus anthracis</i> (Ba), and numbering is with respect to the Ft enzyme. Secondary structure elements and key structural regions are labeled according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone-0014165-g003" target="_blank">Fig. 3A</a>. Strictly conserved regions are blocked in red, and conserved regions are boxed. Important loop regions are highlighted and labeled according to their domain association. (A) Multiple sequence alignment of the HPPK module. Residues that contribute to substrate binding are shown as blue triangles. The conserved motif that binds Mg²⁺ is shown as gray circles within blue triangles. (B) Alignment of the DHPS module. The inter-domain linker regions of <i>F. tularensis</i> and <i>S. cerevisiae</i> are highlighted in green and the corresponding β-hairpin of monofunctional DHPS is highlighted in orange. Residues that interact with substrates are indicated as purple triangles. Residues known to contribute to sulfonamide drug resistance are indicated by red circles. The missing Dα8 helix at the C-terminus is highlighted in purple. Sequence alignments were performed using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Thompson1" target="_blank">[39]</a> and analyzed using ESPript2.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Gouet1" target="_blank">[54]</a>.</p

The overall structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(A) A stereo view of the overall fold and domain organization showing the secondary structure elements within each module. Each element is labeled with the prefixes ‘H’ and ‘D’ to reflect their locations in the HPPK (blue) and DHPS (purple) domains, respectively. The N- and C-termini and the linker region (green) are labeled. Note that helix Dα8 in the canonical DHPS TIM-barrel is missing. (B) A surface representation of the view shown in (A) that highlights the position of the domain linker and the cleft within the DHPS module corresponding to the missing Dα8 TIM-barrel α-helix.</p

Interaction of the ^FtDHPS module with Compound 1.

<p>(A) Schematic comparison between the scaffolds of Compound 1 and DHP-PP. Compound 1 comprises a pterin-like core and is missing half of the B-ring as highlighted in orange. (B) Stereo view of Compound 1 (orange) bound within the pterin pocket of the TIM-barrel. Residues that make van der Waals and hydrogen-bond contacts are labeled and shown as pink sticks. The <i>F</i>o-<i>F</i>c simulated-annealing omit electron density for Compound 1 is shown as a blue mesh contoured at 3.5 σ.</p

Analytical ultracentrifugation of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.69 mg/ml in at 20°C and at a rotor speed of 60,000 rpm. (B) Absorbance scans at 280 nm at equilibrium are plotted <i>versus</i> the distance from the axis of rotation. The protein was centrifuged at 4°C for at least 24 h at each rotor speed of 15 k (red), 22 k (blue) and 27 k (black) rpm. The <i>solid lines</i> represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak K_D (2.7 mM). The loading protein concentration was 20 µM and the r.m.s. deviation for this fit was 0.0037 absorbance units.</p

Data Collection and Refinement Statistics.

<p>*Data were collected from a single crystal. Values in parentheses are for the highest-resolution shell.</p>a<p>R_free was calculated using 5% of the reflections.</p

Targeted modification to design new UC-112 analogs.

<p>Targeted modification to design new UC-112 analogs.</p

Heat map showing the GI₅₀ values (nM) for UC-112 and three analogs in the NCI-60 screening.

<p>High intensity (blue) cells indicate high activity and low intensity (red) cells indicate low activity. Average GI₅₀ values were calculated for each compound, separately.</p

Synthesis of compounds 6a-6g.

<p>Reagents and conditions: (a) R²CH₂OH, NaH, THF, reflux; (b) R²CH₂OH, 110 ^oC; (c) NH₄OH, H₂O, Et₂O, pH 8–10; (d) paraformaldehyde, pyrrolidine,EtOH, reflux.</p

Average GI₅₀ data for UC-112, compound 12c, 4c and 4g tested in NCI-60 anti-proliferative screening.

<p>Data is shown with mean ± SD as bar graph.</p

Design, Synthesis and Structure-Activity Relationship Studies of Novel Survivin Inhibitors with Potent Anti-Proliferative Properties

<div><p>The anti-apoptotic protein survivin is highly expressed in most human cancer cells, but has very low expression in normal differentiated cells. Thus survivin is considered as an attractive cancer drug target. Herein we report the design and synthesis of a series of novel survivin inhibitors based on the oxyquinoline scaffold from our recently identified hit compound UC-112. These new analogs were tested against a panel of cancer cell lines including one with multidrug-resistant phenotype. Eight of these new UC-112 analogs showed IC₅₀ values in the nanomole range in anti-proliferative assays. The best three compounds among them along with UC-112 were submitted for NCI-60 cancer cell line screening. The results indicated that structural modification from UC-112 to our best compound <b>4g</b> has improved activity by four folds (2.2 μM for UC-112 vs. 0.5 μM for <b>4g</b>, average GI₅₀ values over all cancer cell lines in the NCI-60 panel).Western blot analyses demonstrated the new compounds maintained high selectivity for survivin inhibition over other members in the inhibition of apoptosis protein family. When tested in an A375 human melanoma xenograft model, the most active compound <b>4g</b> effectively suppressed tumor growth and strongly induced cancer cell apoptosis in tumor tissues. This novel scaffold is promising for the development of selective survivin inhibitors as potential anticancer agents.</p></div