Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to the pathogenesis of restenosis. Thus, drugs interfering with cell cycle progression in VSMC are promising candidates for an antirestenotic therapy. In this study, we pharmacologically characterize, a novel derivative of the cyclin-dependent kinase (CDK) inhibitor roscovitine (ROSC), in PDGF-BB-activated VSMC. Cell proliferation was quantified measuring DNA synthesis via 5-bromo-2Ј-deoxyuridine incorporation. Analysis of cell cycle distribution was done by flow cytometry using propidium iodide-stained nuclei. Key regulators of the cell cycle and relevant signaling pathways were dissected by Western blot analyses. In addition, in vitro kinase assays and in silico studies regarding the pharmacokinetic profile of both compounds were performed. LGR1406 shows a stronger (IC 50 ϭ 3.0 M) antiproliferative activity than ROSC (IC 50 ϭ 16.9 M), halting VSMCs in G 0 /G 1 phase of the cell cycle, whereas ROSC does not arrest but rather delays cell cycle progression. Neither of the compounds interferes with early PDGF-BB-induced signaling pathways (p38, extracellular signal-regulated kinase 1/2, c-Jun NH 2 -terminal kinase, Akt, signal transducer and activator of transcription 3), and both inhibit CDKs, with LGR1406 exerting a slightly higher potency against CDK1/2 and 4 than ROSC. Expression of cyclins A and E as well as hyperphosphorylation of the pocket proteins retinoblastoma protein and p107 are negatively affected by both compounds, although to a different extent. In silico calculations predicted a much higher metabolic stability for LGR1406 compared with ROSC. Altogether, ROSC derivatives, such as LGR1406 seem to be promising compounds for further development in antirestenotic therapy.Developing strategies against restenosis, the renarrowing of an artery after angioplastic interventions, remains an important goal of vascular biology and pharmacological research. Currently, drug-eluting stents have become the treatment of choice for patients undergoing percutaneous coronary revascularization (Kukreja et al., 2008). Current marketed first-generation drug eluting stents use drugs such as rapamycin and paclitaxel targeting mammalian target of rapamycin and tubulin, respectively (Windecker and Jü ni, 2008). Unfortunately, some concerns have been raised recently due to a potential increased risk of late-stent thrombosis (Steffel et al., 2008). Therefore, the identification of new drug candidates interfering with vascular smooth muscle cell (VSMC) proliferation using mechanisms other than rapamycin and paclitaxel is an important pharmacological topic.Roscovitine (ROSC) has been characterized as a selective inhibitor of cyclin dependent kinases (CDK) 1, 2, and 5 in enzyme-based assays (Meijer et al., 1997), inhibiting the proliferation of various cell types ranging from numerous cancer cell lines to keratinocytes and fibroblasts. The ob-

ABSTRACT:N-Dealkylation is a commonly observed metabolic reaction for drugs containing secondary and tertiary amines. On searching the literature, it is obvious that this reaction is far less common among cytochrome P450 2D6 catalyzed reactions compared with other cytochromes P450. The CYP2D6 pharmacophore and characteristic features in the active site cavity suggest a favored substrate orientation that prevents N-dealkylation from occurring. In this study, the literature was searched for N-dealkylated and non-Ndealkylated CYP2D6 substrates. The hypothesis that was suggested and confirmed demonstrated that N-dealkylation occurs by this enzyme when the preferred site of metabolism is blocked toward other oxidative metabolic pathways. An interesting observation was also that addition of stable groups at preferred sites of metabolism generally improved the metabolic stability but also resulted in retained or increased inhibition of the enzyme. In addition, the effect of pH on N-and O-dealkylation of dextromethorphan was shown to be consistent with the hypothesis that an ionized amino function favored substrate dockings resulting in O-dealkylation.Cytochrome P450 2D6 (CYP2D6) is a polymorphic member of the cytochrome P450 superfamily important for the metabolism of a variety of xenobiotics (Guengerich, 2003). In drug discovery research it is of great interest to identify whether a compound is a substrate or not, and considerable effort has been put into the development of models to identify substrates and/or inhibitors of CYP2D6. Several pharmacophore models have been published identifying common features in CYP2D6 substrates and inhibitors (Koymans et al., 1992;de Groot et al., 1997de Groot et al., , 1999Lewis et al., 1997). The pharmacophore comprises basic nitrogen atom 5-7 or 10 Å from the site of oxidation, a flat hydrophobic region near the site of oxidation and a negative molecular electrostatic potential coplanar with this hydrophobic region (see examples in Fig. 1). The intramolecular distances of 5 and 7 Å were combined by Koymans et al. (1992) in a model assuming a carboxylate group as an anchor point in the protein with one of the oxygen atoms interacting with the "5-Å substrates" and the other with the "7-Å substrates." Since several substrates with approximately 10 Å between the nitrogen atom and the site of oxidation were also known, refined models were constructed including this intramolecular distance (Lewis et al., 1997). Even though the early pharmacophore models are rather crude, they have been successful in predicting the influence of CYP2D6 in the metabolism of xenobiotics as well as predicting possible sites of metabolism [e.g., 75% predictability (de Groot et al., 1999)].Structural information of CYP2D6 from homology models (de Groot et al., 1996;De Rienzo et al., 2000) and a crystal structure of ligand-free CYP2D6 (Rowland et al., 2006) revealed two acidic amino acids, Glu216 and Asp301, and two phenylalanine residues, Phe120 and Phe483, in the active site cavity. This was compatible with t...

NF enhances endothelial NO release most likely by promoting specific dephosphorylation of eNOS-Thr495 via PP1 in vitro and may be a promising compound to enhance endothelial function in vivo.

No abstract

No abstract