A novel roscovitine derivative potently induces G1-phase arrest in platelet-derived growth factor-BB-activated vascular smooth muscle cells.

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 N-5-(2-aminocyclohexyl)-N-7-benzyl-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine-5,7-di-amine (LGR1406), 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-2'-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) = 3.0 muM) antiproliferative activity than ROSC (IC(50) = 16.9 muM), 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.

The molecular basis of CYP2D6-mediated N-dealkylation: balance between metabolic clearance routes and enzyme inhibition.

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-N-dealkylated 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.

A Maillard reaction product enhances eNOS activity in human endothelial cells.

Nitric oxide (NO) produced by the endothelial nitric oxide synthase (eNOS) is an important signaling molecule in the cardiovascular system. Although dietary factors can modulate eNOS activity, putative effects of processed food are barely investigated. We aimed to examine whether the model Maillard reaction product 3-hydroxy-2-methyl-1-propyl-4(1H)-pyridone (HMPP), formed from maltol or starch and propylamine, affects the eNOS system. Incubation of EA.hy926 endothelial cells with 30-300 microM HMPP for 18 h enhanced endothelial NO release measured with the fluorescent probe diaminofluorescein-2 and eNOS activity determined by the [14C]L-arginine-[14C]L-citrulline conversion assay. HMPP increased NO production also in two different types of primary human endothelial cells. Protein levels of eNOS and inducible NO synthase remained unaltered by HMPP. HMPP inhibited eNOS activity within the first 2-4 h, whereas it potently increased eNOS activity after 12-24 h. Levels of eNOS phosphorylation, expression of heat-shock protein 90, caveolin-1 and various antioxidant enzymes were not affected. Intracellular reactive oxygen species remained unchanged by HMPP. This is the first study to demonstrate positive effects of a Maillard reaction product on eNOS activity and endothelial NO production, which is considered favourable for cardiovascular protection.

Norfuraneol dephosphorylates eNOS at threonine 495 and enhances eNOS activity in human endothelial cells.

AIM: Pentoses are widely abundant in organic food. Thermal treatment of pentoses leads to the formation of norfuraneol (NF). The aim of this study was to show whether NF, which is taken up regularly, for example with cooked food, affects the human endothelial nitric oxide synthase (eNOS) system. METHODS AND RESULTS: The study was performed using cultured human umbilical vein endothelial cells (HUVEC), HUVEC-derived EA.hy926 cells, and bovine aortic endothelial cells. Nitric oxide (NO) release and eNOS activity were measured using diaminofluorescein-2 and [14C]L-arginine/[14C]L-citrulline conversion. Levels of (phospho-)eNOS were detected by western blotting. Reactive oxygen species (ROS) production was assessed using 2',7'-dichlorodihydrofluorescein diacetate. Pharmacokinetic parameters of NF were calculated by VolSurf software. NF dose dependently increased eNOS activity and NO release (30-300 microM), but did not affect total eNOS protein or cellular ROS levels. The increase in eNOS activity coincided with specific dephosphorylation of eNOS-Thr495, known to enhance eNOS activity. Inhibition of protein phosphatase 1 (PP1) by calyculin A, tautomycetin, or siRNA against PP1 reversed NF-induced eNOS-Thr495 dephosphorylation. Phosphorylation at eNOS-Ser1177 was not significantly altered by NF. Inhibition of protein kinase C with bisindolylmaleimide I (GFX) or calphostin C mimicked the effect of NF. In contrast to GFX, however, NF had no effect on phorbol-12-myristate-13-acetate-induced endothelial ROS formation. In silico, NF is stable towards CYP3A4 metabolism, shows low protein binding, and high tissue distribution. CONCLUSION: 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.