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

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

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

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

Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors

Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound <b>23</b>

Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors

Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound <b>23</b>

Discovery of Aryloxy Tetramethylcyclobutanes as Novel Androgen Receptor Antagonists

An aryloxy tetramethylcyclobutane was identified as a novel template for androgen receptor (AR) antagonists via cell-based high-throughput screening. Follow-up to the initial “hit” established <b>5</b> as a viable lead. Further optimization to achieve full AR antagonism led to the discovery of <b>26</b> and <b>30</b>, both of which demonstrated excellent in vivo tumor growth inhibition upon oral administration in a castration-resistant prostate cancer (CRPC) animal model

Discovery of Potent, Selective, and Peripherally Restricted Pan-Trk Kinase Inhibitors for the Treatment of Pain

Hormones of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), are known to activate the family of Tropomyosin receptor kinases (TrkA, TrkB, and TrkC). Moreover, inhibition of the TrkA kinase pathway in pain has been clinically validated by the NGF antibody tanezumab, leading to significant interest in the development of small molecule inhibitors of TrkA. Furthermore, Trk inhibitors having an acceptable safety profile will require minimal brain availability. Herein, we discuss the discovery of two potent, selective, peripherally restricted, efficacious, and well-tolerated series of pan-Trk inhibitors which successfully delivered three candidate quality compounds <b>10b</b>, <b>13b</b>, and <b>19</b>. All three compounds are predicted to possess low metabolic clearance in human that does not proceed via aldehyde oxidase-catalyzed reactions, thus addressing the potential clearance prediction liability associated with our current pan-Trk development candidate PF-06273340

Discovery of Potent, Selective, and Peripherally Restricted Pan-Trk Kinase Inhibitors for the Treatment of Pain

Hormones of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), are known to activate the family of Tropomyosin receptor kinases (TrkA, TrkB, and TrkC). Moreover, inhibition of the TrkA kinase pathway in pain has been clinically validated by the NGF antibody tanezumab, leading to significant interest in the development of small molecule inhibitors of TrkA. Furthermore, Trk inhibitors having an acceptable safety profile will require minimal brain availability. Herein, we discuss the discovery of two potent, selective, peripherally restricted, efficacious, and well-tolerated series of pan-Trk inhibitors which successfully delivered three candidate quality compounds <b>10b</b>, <b>13b</b>, and <b>19</b>. All three compounds are predicted to possess low metabolic clearance in human that does not proceed via aldehyde oxidase-catalyzed reactions, thus addressing the potential clearance prediction liability associated with our current pan-Trk development candidate PF-06273340