Structure of MdOS.

<p>Structure of MdOS.</p

Predictive interaction between MdOS and VEGFR2 or EGFR by molecular docking.

<p>A, binding mode of MdOS and VEGFR2 kinase (PDB code: 1YWN). B, the detailed binding interactions between MdOS and VEGFR2 kinase. Hydrogen bonds are indicted by dashed lines. C, binding mode of MdOS and EGFR kinase (PDB code: 1XKK). D, the detailed binding interactions between MdOS and EGFR kinase. Hydrogen bonds are indicted by dashed lines.</p

MdOS directly targets intracellular tyrosine kinase.

<p>A, Cell entry and location of MdOS. SK-OV-3 cells were treated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003774#s4" target="_blank">materials and methods</a>, and photographed by Leica TCS confocal microscope. Bar, 10 µm. B, <i>Left</i>, starved SK-OV-3 cells were incubated with 100 µg/ml MdOS for 6 h, followed by stimulated with EGF directly or washed three times with serum-free medium then stimulated with EGF (+w). <i>Right</i>, SK-OV-3 cells were plated in six-well plates. 24 h after plating, medium was replaced with serum-free medium supplemented with indicated concentrations of MdOS for 6 h.</p

The inhibitory action of MdOS on angiogenesis.

<p>A, effect of MdOS on growth factor-stimulated proliferation of human microvascular endothelial cells (HMECs). HMECs proliferation was assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003774#s4" target="_blank">Materials and Methods</a>. B, effect of MdOS against HMEC tube formation on Matrigel. HMECs were plated on Matrigel in medium with various concentrations of MdOS for 8 h. <i>Left</i>, representative photographs (100×) of three independent experiments. <i>Right</i>, quantification of the inhibitory activity of MdOS on tube formation. C, effect of MdOS on microvessel outgrowth arising from rat aortic ring. Aortic rings were embedded in Matrigel in 96-well plates, then fed with medium containing various concentrations of MdOS for 6 days. <i>Left</i>, representative photographs (100×) of three independent experiments. <i>Right</i>, the area of microvessels was quantified and normalized to untreated controls. D, effect of MdOS on angiogenesis in a chorioallantoic membrane model. Glasscover-slip saturated with MdOS or normal saline was placed areas between preexisting vessels in the fertilized chicken eggs and incubated for 48 h. <i>Left</i>, representative photographs (100×) of three independent experiments. The Glasscover-slip was placed on the right side of the field. <i>Right</i>, the number of vessel branches was quantified and normalized to untreated controls. Data shown are mean±SD from three independent experiments.*, P<0.05; **, P<0.01, versus control.</p

Lineweaver Burke plot of ATP competition.

<p>VEGFR2 (A) and HER-2 (B) and EGFR (C) kinase assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003774#s4" target="_blank">Materials and Methods</a> in the presence of varying concentrations of ATP. Initial reaction velocity was expressed as the phosphorylation of poly(Glu, Tyr)_4∶1 substrate. All x, y data sets were multiplied by 100 for purposes of graphical presentation.</p

Inhibition of MdOS on cellular kinase phosphorylation and signal transduction.

<p>A–B, MdOS blocks HER-2 phosphorylation and its downstream signal transduction in SK-OV-3 cells (A) and NIH-3T3/neu cells (B). C–D, Inhibition of EGF-stimulated EGFR phosphorylation and signal transduction by MdOS in A431 (C) and MDA-MB-468 (D) cells. Serum-starved cells were incubated with indicated concentrations of MdOS for 6 h at 37°C, EGF (50 ng/ml) was added to the cultures during the last 15-min treatment. E–F, Inhibition of VEGF-stimulated VEGFR2 phosphorylation and signal transduction by MdOS. HMEC (E) and NIH-3T3/flk-1 (F) cells were starved, then incubated with indicated concentrations of MdOS for 6 h at 37°C, VEGF (50 ng/ml) was added to the cultures during the last 15-min treatment. Protein samples were separated by SDS-PAGE and probed using the indicated antibodies. Representative data are shown.</p

Interaction of VEGFR-2 (A) or HER-2 (B) or EGFR (C) or 6×his-tag (D) with MdOS by surface plasmon resonance analysis.

<p>VEGFR2 was injected at the concentrations of 2.56, 0.64, 0.32, 0.16 and 0.08 µM (from top to bottom); HER-2 was injected at the concentrations of 0.53, 0.26, 0.13, 0.07 and 0.03 µM (from top to bottom); EGFR was injected at the concentrations of 6, 3, 1.5, 0.75, 0.38, 0.19 and 0.09 µM (from top to bottom); 6×his-tag was injected at the concentrations of 100, 10, 1, 0.1, 0.01 µM. Sensorgram responses at equilibrium were plotted against each concentration of compounds and the equilibrium dissociation constant (K_D) of the binding system was calculated using BIAeval software 3.1.</p

Effects of MdOS on the activity of a panel of tyrosine kinases.

<p>Kinases activity was assayed by ELISA. Concentrations that cause 50% inhibition (IC₅₀) are shown as mean±SD of three to six separate experiments performed in duplicate. EGFR, epidermal growth factor receptor; VEGFR, vascular endothelial growth factor receptor; PDGFR, platelet derived growth factor receptor; FGFR, fibroblast growth factor receptor.</p

Discovery of a New Series of Naphthamides as Potent VEGFR‑2 Kinase Inhibitors

Inhibition of VEGFR-2 signaling pathway has already become one of the most promising approaches for the treatment of cancer. In this study, we describe the design, synthesis, and biological evaluation of a new series of naphthamides as potent inhibitors of VEGFR-2. Among these compounds, <b>14c</b> exhibited high VEGFR-2 inhibitory potency in both enzymatic and HUVEC cellular proliferation assays, with IC₅₀ values of 1.5 and 0.9 nM, respectively. Kinase selectivity profiling revealed that <b>14c</b> was a multitargeted inhibitor, and it also exhibited good potency against VEGFR-1, PDGFR-β, and RET. Furthermore, <b>14c</b> effectively blocked tube formation of HUVEC at nanomolar level. Overall, <b>14c</b> might be a promising candidate for the treatment of cancer

Discovery of 4,7-Diamino-5-(4-phenoxyphenyl)-6-methylene-pyrimido[5,4‑<i>b</i>]pyrrolizines as Novel Bruton’s Tyrosine Kinase Inhibitors

An alternative medicinal chemistry approach was conducted on Bruton’s tyrosine kinase (BTK) inhibitor <b>1</b> (ibrutinib) by merging the pyrazolo­[3,4-<i>d</i>]­pyrimidine component into a tricyclic skeleton. Two types of compounds were prepared, and their biochemical activities on BTK as well as stereochemistry effects were determined. Structural optimization focusing on the reactive binding group to BTK Cys481 and on the metabolic site guided by metabolic study were conducted. <b>7S</b> was identified as the most potent showing an IC₅₀ value of 0.4 nM against BTK and 16 nM against BTK-dependent TMD8 cells. Compared to <b>1</b>, <b>7S</b> was slightly more selective with strong inhibition on the B-cell receptor signaling pathway. In a TMD8 cell-derived animal xenograft model, <b>7S</b> showed a relative tumor volume of 5.3 at 15 mg/kg QD dosage that was more efficacious than <b>1</b> (RTV 6.6) at a higher dose of 25 mg/kg QD. All these results suggest <b>7S</b> as a new BTK inhibitor worthy of further profiling