Abstract
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 (IC50 = 3.0 μM) antiproliferative activity than ROSC (IC50 = 16.9 μM), halting VSMCs in G0/G1 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 NH2-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.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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This work was supported by the European Union within the sixth framework program project “ProKinase Research” [Grant LSHB-CT-2004-503467]; the Czech Grant Agency [Grant 8301/08/1649]; and the Czech Ministry of Education [Grant MSM 6198959216].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.060327
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ABBREVIATIONS:
- VSMC
- vascular smooth muscle cell
- Ang II
- angiotensin II
- BrdU
- 5-bromo-2′-deoxyuridine
- CDK
- cyclin-dependent kinase
- DMSO
- dimethyl sulfoxide
- ERK
- extracellular signal-regulated kinase
- HSA
- human serum albumin
- ISS
- in-stent stenosis
- MAPK
- mitogen-activated protein kinase
- PBS
- phosphate-buffered saline
- PDGF
- platelet-derived growth factor
- Rb
- retinoblastoma protein
- ROSC
- roscovitine
- LGR1406
- N-5-(2-aminocyclohexyl)-N-7-benzyl-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine-5,7-di-amine
- PI
- propidium iodide
- VD
- volume distribution
- ANOVA
- analysis of variance.
- Received August 14, 2009.
- Accepted November 2, 2009.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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