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The discovery of a highly potent and selective small molecule inhibitor 9 for in vitro target validation of MNK1/2 kinases is described. The aminopyrazine benzimidazole series was derived from an HTS hit and optimized by utilization of a docking model, conformation analysis, and binding pocket comparison against antitargets.

Two scaffolds based on 5,6-fused heterocyclic backbones were designed and synthesized as Raf kinase inhibitors. The scaffolds were assessed for in vitro pan-Raf inhibition, activity in cell proliferation and target modulation assays, and pharmacokinetic parameters.

A series of arylaminobenzimidazoles was designed and synthesized as Raf kinase inhibitors. Exploration of the structure-activity relationship resulted in compounds that are potent in vitro and show desirable in vivo properties.

Apo2L/TRAIL exhibits enhanced apoptotic activity in tumor xenograft models when used in combination with the topoisomerase 1 inhibitor CPT-11. To investigate the cellular mechanisms involved in this increased tumor-killing activity, a series of in vitro experiments were conducted using the human colon carcinoma cell line (HCT116). Apo2L/TRAIL induced a transient upregulation of DR5 mRNA, while CPT-11 increased both death and decoy receptor expression. Upregulation of decoy receptors by CPT-11 was partially inhibited by co-administration of Apo2L/TRAIL. CPT-11 treatment resulted in accumulation of cells at G(2)M-phase and correlated with a substantial increase in the protein levels of the cyclin-dependent kinase inhibitor p21. However, cells co-treated with CPT-11 and Apo2L/TRAIL, or pretreated with CPT-11 for up to 24 h followed by 2 h Apo2L/TRAIL, resulted in a caspase-dependent degradation of p21, reversal of G(2)-M phase arrest with a concomitant increase in apoptosis. The sequential treatment produced the greatest induction of DR5 and DR4, caspase-3-like cleavage/activation and p21 degradation, as well as increased apoptosis. These data indicate that the up-regulation of Apo2L/TRAIL ligand and its death receptors as well as cleavage of p21 protein in the Apo2L/TRAIL plus CPT-11 treatment contributes to the positive cooperation between these agents in enhancing tumor cell apoptosis.

The beta-catenin signal transduction pathway, which can be activated by secreted Wnt proteins, plays a key role in normal embryonic development and in malignant transformation of the mammary gland and colon. Here we demonstrate, for the first time, that Wnt and beta-catenin signaling also function in cells of the vasculature. RT-PCR analysis showed that primary endothelial and smooth muscle cell cultures, of both mouse and human origin, express members of the Wnt and Wnt receptor (Frizzled) gene families. Transfection of an expression vector for Wnt-1 into primary endothelial cells increased both the free pool of beta-catenin and the transcription from a Lef/tcf-dependent reporter gene construct. Expression of Wnt-1, but not Wnt-5a, also stimulated proliferation of primary endothelial cell cultures. These data show that Wnt and Frizzled proteins can regulate signal transduction, via beta-catenin, in endothelial cells. These findings suggest that Wnt signaling may feature in normal differentiation of the vasculature as well as in pathological settings where endothelial and smooth muscle proliferation is disturbed.

Wnt-1, a secreted glycoprotein, participates in development of the nervous system and contributes to mammary oncogenesis when overexpressed. We show that GSK3 activity is decreased in mouse mammary cells transformed by Wnt-1. These cells also exhibit a substantial Wnt-1 dependent increase in the uncomplexed population of beta-catenin. Wnt-1 signaling does not change the steady state level of either GSK3 alpha or GSK3 beta but instead leads to an increased association between GSK3 beta and beta-catenin. HGF/SF treatment of mouse mammary cells also leads to a transient decrease in GSK3 activity and a parallel, selective increase in the uncomplexed pool of beta-catenin. Both Wnt-1 and HGF/SF lead to nuclear accumulation of beta-catenin and activation of a LEF/Tcf responsive reporter gene. This study defines a pivotal signal transduction pathway, activated by both Wnt-1 and HGF/SF, leading to decreased GSK3 beta activity and consequently an increase in the free pool and nuclear accumulation of beta-catenin and changes in gene expression.