Competitive receptor binding curves of selective isoquinolinone derivatives.

<p>Intact CHO cells expressing MT₁ or MT₂ were incubated with [³H]melatonin with or without different concentrations of selected tested compounds or unlabeled melatonin. Data represented mean ± SEM of at least 3 different trials performed in duplicates, and normalized to the maximal binding values (in the absence of tested compound). Estimation of maximal displacement and IC₅₀ and K_i were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113638#pone-0113638-t001" target="_blank">Table 1</a>.</p

OEOA was retained for a longer period in mouse blood plasma than OA.

<p>MS-MS product ion mass spectra of OEOA (A) and OA (B). (C) K562 cells were treated with OA or OEOA (1 µM) for 6 h. Culture media and cell lysates were collected at indicated time points for HPLC-MS/MS analysis. Data was represented as mean ± SEM in cell lysate compared to total exposure, n = 2 (* <i>p</i><0.05). (D) Concentrations of OA and OEOA in mouse plasma after intraperitoneal injection. Blood was collected from mice at different time points after single administration of OA and OEOA. Data was represented as mean ± SEM, n = 2 animals per time point (* <i>p</i><0.05).</p

OEOA attenuated phosphorylation of Rb protein in K562 and Jurket cells.

<p>(A) K562 and Jurket cells were treated with OEOA (0.1–10 µM) for 2 days, and the cell lysates were subjected to Western blot analysis for p-Rb and Rb. Actin served as an equal loading control. Histograms in (B) show the relative expression of p-Rb (normalized to actin) as compared to the vehicle-treated cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p

Blockade of ERK phosphorylation and Ca²⁺ mobilization by an isoquinolinone-based melatoninergic antagonist.

<p>(A) CHO cells expressing MT₁ or MT₂ were treated with the indicated concentrations of <b>7e</b> or luzindole in the absence or presence of a fixed concentration of melatonin (MLT) (for both MT₁ or MT₂) or <b>7b</b> (for MT₂ only). Other experimental details were as to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113638#pone-0113638-g005" target="_blank">Figure 5</a>. Data shown were representative blots of three separate trials. (B) CHO cells expressing MT₂ were treated with increasing concentrations (1 ρM – 1 µM) of melatonin in the absence or presence of 10 µM or 1 µM of <b>7e</b> or <b>7f</b>. Other experimental details were as to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113638#pone-0113638-g004" target="_blank">Figure 4</a>.</p

OEOA inhibited cell proliferation in leukemia cell lines.

<p>Different cell lines were treated with various concentrations of OEOA or OA for 2 days. Cell growth was measured by MTT assay: (A) K562, (B) HEL, (C) Jurket (D) HEKneo, and (E) HepG2, MCF<b>-</b>7 and HeLa cells. Data are mean ± SEM of three independent experiments (* <i>p</i><0.05).</p

OEOA did not induce cell death in K562 and HEL cells.

<p>K562 (A & C) and HEL (B & D) cells were treated with OEOA (1 µM) for 6 days. Cell viability was measured by trypan blue exclusion as described in Materials and Methods. Data are mean ± SEM of three independent experiments (* <i>p</i><0.05).</p

Synthesis of isoquinolinones 7a–g.

<p>a) MeNH₂, THF, r.t., ∼75%; b) DIC, <b>2</b>, CH₂Cl₂, r.t., 84–91%; c) Ac₂O, reflux, ∼100%; d) TsOH, toluene, heat, 71% for two steps; e) RX, K₂CO₃, DMF, r.t., ∼95%.</p

Synthesis and Functional Characterization of Substituted Isoquinolinones as MT₂-Selective Melatoninergic Ligands

<div><p>A series of substituted isoquinolinones were synthesized and their binding affinities and functional activities towards human melatonin MT₁ and MT₂ receptors were evaluated. Structure-activity relationship analysis revealed that substituted isoquinolinones bearing a 3-methoxybenzyloxyl group at C5, C6 or C7 position respectively (C5>C6>C7 in terms of their potency) conferred effective binding and selectivity toward the MT₂ receptor, with <b>15b</b> as the most potent compound. Most of the tested compounds were MT₂-selective agonists as revealed in receptor-mediated cAMP inhibition, intracellular Ca²⁺ mobilization and phosphorylation of extracellular signal-regulated protein kinases. Intriguingly, compounds <b>7e</b> and <b>7f</b> bearing a 4-methoxybenzyloxyl group or 4-methylbenzyloxyl at C6 behaved as weak MT₂-selective antagonists. These results suggest that substituted isoquinolinones represent a novel family of MT₂-selective melatonin ligands. The position of the substituted benzyloxyl group, and the substituents on the benzyl ring appeared to dictate the functional characteristics of these compounds.</p></div

Phosphorylation of ERK induced by isoquinolinone derivatives.

<p>CHO cells expressing MT₁ or MT₂ were serum-starved before treating with the indicated concentrations of melatonin or individual tested compounds. Resolved proteins were electrotransferred for immunodetection using phosphorylated ERK-specific antibody. Total amount of ERK was also detected similarly and no observable change of their expression levels has been found for all the treatments (not shown). Three individual trails yielded similar results as the representative blots shown in the figure.</p

OEOA induced G1 cell cycle arrest in K562 cells.

<p>(A) Cells were incubated with OEOA (1 or 10 µM) for 24 h. The distribution of cell cycle was examined by PI staining method. The table summarized the distribution of cells in OEOA-treated or control cells. Data represented mean ± SEM of three independent experiments (* <i>p</i><0.05). (B) K562 cells were cultured in the presence of OEOA (1 or 10 µM) for 2 days. Total proteins were collected for Western blot analysis to detect the expression of p27, Cdk4, Cdk6, Cyclin D1, Cyclin E and RAMP. Actin served as an equal loading control. Histograms on the right show the relative expression of various proteins (normalized to actin) as compared to the control cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p