Discovery of novel small molecule activators of ß-catenin signaling.

Wnt/ß-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/ß-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a ß-galactosidase fragment complementation assay measuring ß-catenin nuclear entry revealed bona fide activators of ß-catenin signaling. The compounds stabilized cytoplasmic ß-catenin and activated ß-catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate ß-catenin signaling has yet to be determined, several key regulators of Wnt/ß-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced ß-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/ß-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/ß-catenin signaling.

Inhibition of Wnt/ß-catenin signaling by p38 MAP kinase inhibitors is explained by cross-reactivity with casein kinase Iδ/ɛ.

Wnt/ß-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a ß-galactosidase fragment complementation assay measuring ß-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated ß-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/ß-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated ß-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase Iδ and ɛ, which are known activators of Wnt/ß-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of ß-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/ß-catenin signaling.

Discovery and optimization of 1-(4-(pyridin-2-yl)benzyl)imidazolidine-2,4-dione derivatives as a novel class of selective cannabinoid CB2 receptor agonists.

Here, we report the identification and optimization of 1-(4-(pyridin-2-yl)benzyl)imidazolidine-2,4-dione derivatives as a novel chemotype with selective cannabinoid CB2 receptor agonist activity. 1 is a potent and selective cannabinoid CB2 receptor agonist (hCB2 pEC(50) = 8.6). The compound was found to be metabolically unstable, which resulted in low oral bioavailability in rat (F(po) = 4%) and possessed off-target activity at the hERG ion channel (pK(i) = 5.5). Systematic modification of physicochemical properties, such as lipophilicity and basicity, was used to optimize the pharmacokinetic profile and hERG affinity of this novel class of cannabinoid CB2 receptor agonists. This led to the identification of 44 as a potent, selective, and orally bioavailable cannabinoid CB2 receptor agonist (hCB2 pEC(50) = 8.0; hERG pK(i) < 4; F(po) = 100%), which was active in a rat spinal nerve ligation model of neuropathic pain.

Pharmacological characterization of receptor redistribution and beta-arrestin recruitment assays for the cannabinoid receptor 1.

Receptor redistribution and beta-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular beta-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution assay and 2 nonimaging-based beta-arrestin recruitment assays, Tango and PathHunter, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Galpha(i) coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via beta-arrestin recruitment and Galpha(i)-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed.