Identification of Akt pathway inhibitors using redistribution screening on the FLIPR and the IN Cell 3000 analyzer.

The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization.

Carbamoylcholine homologs: synthesis and pharmacology at nicotinic acetylcholine receptors.

In a recent study, racemic 3-(N,N-dimethylamino)butyl-N,N-dimethylcarbamate (1) was shown to be a potent agonist at neuronal nicotinic acetylcholine receptors with a high selectivity for nicotinic over muscarinic acetylcholine receptors [Mol. Pharmacol. 64 (2003) 865-875]. Here we present the synthesis and pharmacological characterization of a series of analogs of, where the methyl group at C-3 has been replaced by different alkyl substituents. Ring systems have been incorporated into the carbon backbone of some of the molecules, or the amino group has been build into ring systems. Furthermore, the (+)- and (-)-enantiomers of have been separated, and X-ray crystallography has revealed that (-)-1 possesses (S)-configuration. The compounds have been characterized pharmacologically at recombinant nicotinic receptor subtypes. The structure-activity relationship study has provided valuable insight into the mode of interactions of and its analogs with neuronal nicotinic acetylcholine receptors.

Carbamoylcholine homologs: novel and potent agonists at neuronal nicotinic acetylcholine receptors.

The classic muscarinic acetylcholine receptor (mAChR) agonist carbamoylcholine (carbachol) does not seem to be the most obvious lead for the development of selective ligands at nicotinic acetylcholine receptors (nAChRs). In the past, however, N-methylations of carbachol have provided N-methylcarbamoylcholine and N,N-dimethylcarbamoylcholine (DMCC), which predominantly display nicotinic activity. In this study, 12 homologous analogs of DMCC and its corresponding tertiary amine, N,N-dimethylcarbamoyl-N,N-dimethylaminoethanol, were synthesized and their binding affinities to native mAChR and nAChR sites estimated. One of the compounds in the series, 3-N,N-dimethylaminobutyl-N,N-dimethylcarbamate (7), displayed low nanomolar binding affinity to nAChRs and a 400-fold selectivity for nAChRs over mAChRs. Hence, a new series of compounds was synthesized in which alkyl and aryl groups and different ring systems were introduced in the carbamate moiety of 7. In a [3H]epibatidine binding assay, the Ki values of 7 and its analogs at rat alpha2beta2, alpha4beta2, alpha2beta4, alpha3beta4, and alpha4beta4 nAChRs, stably expressed in mammalian cell lines, ranged from low nanomolar to midmicromolar concentrations, whereas all of the compounds displayed weak binding to an alpha7/5-HT3 chimera and to native mAChRs. Compound 7 and its analogs were determined to be agonists at the alpha3beta4 nAChR subtype. This series includes the most potent and selective nicotinic agonists structurally derived from ACh to date. Furthermore, the compounds are tertiary amines, implying some advantages in terms of bioavailability pertinent to future in vivo pharmacological studies. Finally, observations made in the study hold promising perspectives for future development of ligands selective for specific nAChR subtypes.