Interaction of alpha2-Adrenergic Receptors With G-Proteins

A wide variety of hormones and neurotransmitters have been shown to produce intracellular signals mediated by guanine-nucleotide binding proteins (G-proteins). These have been shown to form a link between receptors on the extracellular face of the cell membrane, and their intracellular second-message-generating effectors, such as phospholipase C and adenylyl cyclase. An increasing number of different heterotrimeric G-proteins have been isolated, with varying degrees (50-95%) of sequence homology. This similarity of sequence has raised questions of specificity of interaction of G-proteins: do individual G-proteins interact only with specific receptors and effectors, or are they more promiscuous in their interaction? This question has been addressed by examining the specificity of coupling of G-proteins to alpha2-adrenergic receptors in two model systems

Discovery and Characterization of 2-Anilino-4- (Thiazol-5-yl)Pyrimidine Transcriptional CDK Inhibitors as Anticancer Agents

The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription, i.e., CDK7 and especially CDK9. The screening cascade employs cellular phenotypic assays based on mitotic index and nuclear p53 protein accumulation. This permitted us to classify compounds into transcriptional, cell cycle, and mitotic inhibitor groups. We describe the characterization of the transcriptional inhibitor class in terms of kinase inhibition profile, cellular mode of action, and selectivity for transformed cells. A structural selectivity rationale was used to optimize potency and biopharmaceutical properties and led to the development of a transcriptional inhibitor, 3,4-dimethyl-5-[2-(4-piperazin-1-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one, with anticancer activity in animal models

<i>In vitro</i> and <i>in vivo</i> pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine and CYC202

In vitro and in vivo pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin- dependent kinase inhibitors olomoucine, bohemine and CYC202 Purpose: To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin- dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model. Experimental Design: The in vitro activity of the agents was determined in a human tumor panel using the sulforhodamine B assay. The concentration and time dependence was established in HCT116 cells. Molecular biomarkers, including RB phosphorylation and cyclin expression, were assessed by Western blotting. Pharmacokinetic properties were characterized in mice following analysis by liquid chromatography-tandem mass spectrometry. Based on these studies, a dosing regimen was developed for CYC202 that allowed therapeutic exposures in the HCT116 tumor xenograft. Results: The antitumor potency of the agents in vitro was in the order olomoucine (IC50, 56 mu mol/L) < bohemine (IC50, 27 mu mol/L) < CYC202 (IC50,15 mu mol/L), corresponding to their activities as cyclin-dependent kinase inhibitors. Antitumor activity increased with exposure time up to 16 hours. The agents caused inhibition of RB and RNA polymerase 11 phosphorylation and depletion of cyclins. They exhibited relatively rapid clearance following administration to mice. CYC202 displayed the slowest clearance from plasma and the highest tumor uptake, with oral bioavailability of 86%. Oral dosing of CYC202 gave active concentrations in the tumor, modulation of pharmacodynamic markers, and inhibition of tumor growth. Conclusions: CYC202 showed therapeutic activity on human cancer cell lines in vitro and on xenografts. Pharmacodynamic markers are altered in vitro and in vivo, consistent with the inhibition of cyclin-dependent kinases. Such markers may be potentially useful in the clinical development of CYC202 and other cyclin-dependent kinase inhibitors