Preclinical Evaluation of the Novel Monoclonal Antibody H6-11 for Prostate Cancer Imaging

The biological properties of the novel monoclonal antibody (mAb) H6-11 and its potential utility for oncological imaging studies were evaluated using <i>in vitro</i> and <i>in vivo</i> assays. Immunoreactivity of H6-11 to the human prostate cancer PC-3 cell line and solid tumor xenografts was initially demonstrated using immunofluorescence staining; the specificity of H6-11 for prostate cancer was further evaluated using a commercial array of human prostate cancer and normal tissue samples (<i>n</i> = 49) in which H6-11 detected 95% of prostate adenocarcinomas. The <i>K</i>_d value of 61.7 ± 30 nM was determined using ¹²⁵I-labeled H6-11. Glycosylation analysis suggested the antigenic epitope of the glycan is an O-linked β-<i>N</i>-acetylglucoside (<i>O</i>-GlcNAc) group. Imaging studies of PC-3 tumor-bearing mice were performed using both optical imaging with NIR fluorescent dye-labeled H6-11 and microPET imaging with ⁸⁹Zr-labeled H6-11. These <i>in vivo</i> studies revealed that the labeled probes accumulated in PC-3 tumors 48–72 h postinjection, although significant retention in liver was also observed. By 120 h postinjection, the tumors were still evident, although the liver showed significant clearance. These studies suggest that the mAb H6-11 may be a useful tool to detect prostate cancer <i>in vitro</i> and <i>in vivo</i>

Synthesis of Fluorine-Containing Phosphodiesterase 10A (PDE10A) Inhibitors and the In Vivo Evaluation of F‑18 Labeled PDE10A PET Tracers in Rodent and Nonhuman Primate

A series of fluorine-containing PDE10A inhibitors were designed and synthesized to improve the metabolic stability of [¹¹C]­MP-10. Twenty of the 22 new analogues had high potency and selectivity for PDE10A: <b>18a</b>–<b>j</b>, <b>19d</b>–<b>j</b>, <b>20a</b>–<b>b</b>, and <b>21b</b> had IC₅₀ values <5 nM for PDE10A. Seven F-18 labeled compounds [¹⁸F]<b>18a</b>–<b>e</b>, [¹⁸F]<b>18g</b>, and [¹⁸F]<b>20a</b> were radiosynthesized by ¹⁸F-introduction onto the quinoline rather than the pyrazole moiety of the MP-10 pharmacophore and performed in vivo evaluation. Biodistribution studies in rats showed ∼2-fold higher activity in the PDE10A-enriched striatum than nontarget brain regions; this ratio increased from 5 to 30 min postinjection, particularly for [¹⁸F]<b>18a</b>–<b>d</b> and [¹⁸F]<b>20a</b>. MicroPET studies of [¹⁸F]<b>18d</b> and [¹⁸F]<b>20a</b> in nonhuman primates provided clear visualization of striatum with suitable equilibrium kinetics and favorable metabolic stability. These results suggest this strategy may identify a ¹⁸F-labeled PET tracer for quantifying the levels of PDE10A in patients with CNS disorders including Huntington’s disease and schizophrenia

Synthesis of Fluorine-Containing Phosphodiesterase 10A (PDE10A) Inhibitors and the In Vivo Evaluation of F‑18 Labeled PDE10A PET Tracers in Rodent and Nonhuman Primate

A series of fluorine-containing PDE10A inhibitors were designed and synthesized to improve the metabolic stability of [¹¹C]­MP-10. Twenty of the 22 new analogues had high potency and selectivity for PDE10A: <b>18a</b>–<b>j</b>, <b>19d</b>–<b>j</b>, <b>20a</b>–<b>b</b>, and <b>21b</b> had IC₅₀ values <5 nM for PDE10A. Seven F-18 labeled compounds [¹⁸F]<b>18a</b>–<b>e</b>, [¹⁸F]<b>18g</b>, and [¹⁸F]<b>20a</b> were radiosynthesized by ¹⁸F-introduction onto the quinoline rather than the pyrazole moiety of the MP-10 pharmacophore and performed in vivo evaluation. Biodistribution studies in rats showed ∼2-fold higher activity in the PDE10A-enriched striatum than nontarget brain regions; this ratio increased from 5 to 30 min postinjection, particularly for [¹⁸F]<b>18a</b>–<b>d</b> and [¹⁸F]<b>20a</b>. MicroPET studies of [¹⁸F]<b>18d</b> and [¹⁸F]<b>20a</b> in nonhuman primates provided clear visualization of striatum with suitable equilibrium kinetics and favorable metabolic stability. These results suggest this strategy may identify a ¹⁸F-labeled PET tracer for quantifying the levels of PDE10A in patients with CNS disorders including Huntington’s disease and schizophrenia

Design, Synthesis, and <i>In Vitro</i> and <i>In Vivo</i> Evaluation of an ¹⁸F‑Labeled Sphingosine 1‑Phosphate Receptor 1 (S1P₁) PET Tracer

Sphingosine 1-phosphate receptor 1 (S1P₁) plays a pivotal signaling role in inflammatory response; because S1P₁ modulation has been identified as a therapeutic target for various diseases, a PET tracer for S1P₁ would be a useful tool. Fourteen fluorine-containing analogues of S1P ligands were synthesized and their <i>in vitro</i> binding potency measured; four had high potency and selectivity for S1P₁ (S1P₁ IC₅₀ < 10 nM, >100-fold selectivity for S1P₁ over S1P₂ and S1P₃). The most potent ligand, <b>28c</b> (IC₅₀ = 2.63 nM for S1P₁) was ¹⁸F-labeled and evaluated in a mouse model of LPS-induced acute liver injury to determine its S1P₁-binding specificity. The results from biodistribution, autoradiography, and microPET imaging showed higher [¹⁸F]<b>28c</b> accumulation in the liver of LPS-treated mice than controls. Increased expression of S1P₁ in the LPS model was confirmed by immunohistochemical analysis (IHC). These data suggest that [¹⁸F]<b>28c</b> is a S1P₁ PET tracer with high potential for imaging S1P₁ <i>in vivo</i>

Structure Determination and Functional Analysis of a Chromate Reductase from <em>Gluconacetobacter hansenii</em>

<div><p>Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from <em>Gluconacetobacter hansenii</em> (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90–95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation.</p> </div

Data Collection and Structural Refinement Statistics of Gh-ChrR.

a<p>Rmerge = Σ|Ii − |/Σ, where Ii is the observed intensity and is the average intensity over symmetry equivalent measurements.</p>b<p>Rwork = Σ||Fobs| − |Fcalc||/Σ|Fobs|.</p>c<p>Rfree is the same as Rwork but for 5% of all reflections that were not used in crystallographic refinement (2114 reflections).</p

Structure proximal to bound FMN.

<p><b>A.</b> Electron density surrounding FMN and chloride ion (gray sphere) contoured at 1.0 σ. <b>B.</b> Schematic representation of hydrophobic contacts (arc with radiating spokes) and potential hydrogen bonds (dashed lines) between FMN and two monomeric units (chain A and C) of the Gh-ChrR tetramer. Atoms are color-coded: black = carbon, red = oxygen, blue = nitrogen. This image was produced using the program <i>LIGPLOT </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042432#pone.0042432-Wallace1" target="_blank">[62]</a>.</p

Putative Gh-ChrR NADH and substrate binding sites.

<p><b>A.</b> NADH was modeled into the Gh-ChrR structure by superimposing it with the NADH-containing structure of EmoB (PDB entry: 2VZJ, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042432#pone.0042432.s007" target="_blank">Figure S7</a>). The nicotinamide ring of NADH (primarily green stick model) is stacked on top of the isoalloxazine ring of FMN (primarily yellow stick model), and the adenosine part of NADH points to ribtyl group of FMN. The black arrow indicates the distance from C4N of NADH to the <i>si</i>-face of the FMN isoalloxazine ring. Residues N53, D54, E57, S100, R101 and F137 from chain A (cyan) and residues N85, P119, and T154 from chain C (gold) interact with NADH. <b>B.</b> The putative active site of Gh-ChrR shown with bound FMN (primarily yellow stick model) and a chloride ion (green sphere). The black arrow indicates the distance from the Cl⁻ to the <i>si</i>-face of the FMN isoalloxazine ring. Key residue R101 holding chloride ion in place is shown in a stick model. Critical residues for hydride transfer, N85 and Y86 from chain A (cyan) and S118 from chain C (gold) are shown in a stick model. The green dash lines indicate the distance (∼3 Å) between N of amide group of N85/Y86 and O4, and the distance (∼3 Å) between OG of hydroxyl group of S118 and O2.</p

Crystal structure of Gh-ChrR.

<p>Monomeric (<b>A</b>) and tetrameric (<b>B</b>) depictions of the 2.25 Å structure of Gh-ChrR showing the backbone fold, a space-filling model of bound FMN (elements color: red  =  oxygen, blue  =  nitrogen, gray  =  carbon) and bound chloride anion (green sphere). Secondary structural elements including the 3₁₀ helices (η) are numbered sequentially from the N-terminus. <b>C.</b> Electrostatic potentials at the solvent-accessible surface of Gh-ChrR. A stick model of the FMN molecule and the associated chloride ion (gray sphere) is highlighted. The electrostatic potential are drawn (Pymol) at a level of −71.817 <i>kT</i>/<i>e</i> (red) to +71.817 <i>kT</i>/<i>e</i> (blue), where <i>k</i> is the Boltzman’s constant, <i>T</i> is the absolute temperature, and <i>e</i> is the magnitude of the electron charge.</p

Catalytic Influence of Site-Directed Substitution of Putative Metal and Cofactor Ligands on NADH-Dependent Chromate Reduction Efficiency.

a<p>Values are based on triplicate measurements, where steady-state kinetic data for Gh-ChrR (5 µM) were measured at a constant NADH concentration (100 µM) and fit to the Michaelis-Menten equation.</p>b<p>Calculations based on a molecular mass of tetrameric Gh-ChrR, 80kDa, and four independent active sites.</p