Inhibition of tumor cell growth by Type I and Type II PTK6 inhibitors is independent of PTK6 expression levels in cells.

<p>(A) PTK6 expressions and activation (p-Y342 PTK6) in tumor cell lines and breast epithelial cells MCF10A cells analyzed by Western Blot. Engineered HEK293T cells overexpressing PTK6 WT was used as a positive control. (B) Detection of p-Y342 PTK6 in tumor cells MDA-MB-231 by confocal microscopy. Cells were treated with DMSO, 10 uM PTK6 inhibitor 21a, 10 uM PF-6698840 or 10 uM PTK6-negative control compound PF-6737007 for 2 hours. α-Tubulin (ubiquitously expressed in cells) and DAPI (restricted expression in nucleus) are shown in red and blue, respectively. p-PTK6 (green) was detected on the cell membrane of MDA-MB-231 cells. HEK293T cells do not have detectable PTK6 expression and were used as a negative control. (C) Cell growth inhibition by PTK6 inhibitors in PTK6-positive tumor cells and PTK6-negative HEK293T cells. Cells were treated with DMSO or compounds for 6 days in 2D or 7 days in 3D culture, and the cell growth inhibition was measured by Cell Titer-Glo on day 6 or 7.</p

Kinase selectivity of PTK6 inhibitors against more than 320 kinases.

<p>The % inhibition is calculated based on the biochemical kinase activity measured in the presence of 1uM compound relative to DMSO control. The color bar represents the level of inhibition: red, 100%; yellow, 50%; and green, 0% inhibition. Compounds 21a and 21c are Type I inhibitors that bind to the active form of PTK6; while PF-6683324 and PF-6698840 are Type II inhibitors that bind to the inactive form of PTK6. PF-6737007 is a structural analogue of PF-6698840 that shares similar kinase selectivity but does not inhibit PTK6 kinase activity. Kinases that are >80% inhibited by compounds at 1uM are marked in the table.</p

Cell growth inhibition by PTK6 inhibitors in PTK6-positive tumor cells and PTK6-negative HEK293T cells.

<p>Cell growth inhibition by PTK6 inhibitors in PTK6-positive tumor cells and PTK6-negative HEK293T cells.</p

Compound potency in biochemical kinase assay and p-PTK6 in-cell ELISA assay in engineered HEK293T cells overexpressing PTK6 WT.

<p>Compound potency in biochemical kinase assay and p-PTK6 in-cell ELISA assay in engineered HEK293T cells overexpressing PTK6 WT.</p

Inhibition of tumor cell growth by PTK6 inhibitors is independent of PTK6 kinase activity inhibition.

<p>(A) Tumor cell growth inhibition by PTK6 inhibitor PF-6698840 (circle) and PTK6 negative control compound PF-6737007 (square) in breast tumor cell lines MDA-MB-231 and MDA-MB-453. Similar potencies were observed in the tumor growth inhibition for both compounds despite their significant difference in the potency of inhibiting PTK6 kinase activity; (B) Dose dependent inhibition of p-PTK6 kinase activity (circle) and cell proliferation (square) by Type I inhibitor 21a and Type II inhibitor PF-6698840 in engineered MDA-MB-231 cells overexpressing PTK6 WT. The p-PTK6 kinase activity was assessed by In-Cell ELISA in which cellular levels of autophosphorylation of PTK6 at Y342 were measured by fluorescence signal (OdysseyCLx imager, LI-COR Bioscience) using anti-p-PTK6 antibody and fluorescent dye–labeled detection reagents. The cell proliferation was measured by Cell Titer-Glo over the course of 6 days. The data was normalized in reference to DMSO control and expressed as percentage of inhibition at various concentrations of inhibitor in the graph. The solid lines represent a non-linear regression curve fit to a dose-response equation defined in PRISM (Graphpad Inc.); (C) Western Blot analysis of p-PTK6 and total PTK6 in the engineered MDA-MB-231 cells in the presence and absence of PTK6 inhibitor. The level of p-PTK6 in engineered MDA-MB-231 cells overexpressing PTK6 WT was diminished by 1 hour treatment of PTK6 inhibitor 21a at 37°C (upper lane 4, treated with 21a vs. upper lane 3, treated with DMSO), while total PTK6 remained unchanged (lower lane 4 treated with 21a vs. lower lane 3 treated with DMSO). Lanes 1–2 represent parental MDA-MB-231 cells, in which p-PTK6 was not detected by western blot due to low level of expression.</p

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound <b>18</b>. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound <b>31.</b> Compound <b>31</b> displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound <b>18</b>. Inhibitor <b>31</b> also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound <b>18</b>. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound <b>31.</b> Compound <b>31</b> displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound <b>18</b>. Inhibitor <b>31</b> also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes

Correction to Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

Correction to Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitor