CVT-313, a Specific and Potent Inhibitor of CDK2 That Prevents Neointimal Proliferation

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0. 5 μm in vitro. Inhibition was competitive with respect to ATP (K i = 95 nm), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8. 5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1. 25 to 20 μm. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation. The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0. 5 μm in vitro. Inhibition was competitive with respect to ATP (K i = 95 nm), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8. 5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1. 25 to 20 μm. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation. Cell cycle progression in mammalian cells is regulated by a family of cyclin-dependent protein kinases (CDKs), 1The abbreviations used are: CDK, cyclin-dependent kinase; Rb, retinoblastoma gene product; FACS, fluorescence-activated cell sorter. that include CDK1, CDK2, CDK3, CDK4, and CDK6 (1Pines J. Semin. Cancer Biol. 1994; 5: 305-313PubMed Google Scholar). CDK2 is a serine/threonine kinase whose activity is essential for the G1 to S transition during cell division. A number of proteins have been shown to be substrates for CDK2 phosphorylation, and among them are the retinoblastoma gene product (Rb) and other related pocket proteins, members of the E2F transcription factor family, cyclin E, and members of the CDK inhibitory proteins. It is also postulated that CDK2 phosphorylates and regulates proteins involved in DNA replication (2Morgan D. O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2935) Google Scholar, 3Sherr C. J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4977) Google Scholar). Two lines of evidence suggest that CDK2 activity is essential for cell proliferation; microinjection of antibodies directed against CDK2 blocks the progression of human diploid fibroblasts into S phase (4Tsai L. H. Lees E. Faha B. Harlow E. Riabowol K. Oncogene. 1993; 8: 1593-1602PubMed Google Scholar, 5Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1128) Google Scholar), and overexpression of a dominant negative mutant of CDK2 in human osteosarcoma cells has a similar effect (6van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (976) Google Scholar). The crucial role of CDK2 in controlling cell cycle progression suggests that CDK2 is an attractive target for treatment of aberrant cell proliferation. Smooth muscle cell proliferation is largely responsible for restenosis following angioplasty (7Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar). A recent study has shown that CDK2 is activated very early after endothelial denudation in the rat carotid artery model of restenosis (8Wei G. L. Kransinski K. Kearney M. Isner J. M. Walsh K. André V. Circ. Res. 1997; 80: 418-426Crossref PubMed Scopus (111) Google Scholar) ; moreover, antisense oligonucleotides directed against CDK2 were shown to be effective in reducing neointima formation in this model (9Morishita R. Gibbons G. H. Ellison K. E. Nakajima M. von der Leyen H. Zhang L. Kaneda Y. Ogihara T. Dzau V. J. J. Clin. Invest. 1994; 93: 1458-1464Crossref PubMed Scopus (267) Google Scholar, 10Abe J. Zhou W. Taguchi J. Takuwa N. Miki K. Okazaki H. Hurokawa K. Kumada M. Takuwa Y. Biocham. Biophys. Res. Commun. 1994; 198: 16-24Crossref PubMed Scopus (103) Google Scholar). Arguably, the restenosis model can be used as a “proof of principle” for developing CDK2 inhibitors as drug candidates for the treatment of diseases related to aberrant cell proliferation. Olomoucine is a purine analog of ATP and is a specific inhibitor of CDK1 and CDK2 (11Vesely J. Havlicek L. Strand M. Blow J. J. Donella-Deana A. Pinna L. Letham D. S. Kato J. Detivaud L. Leclerc S. Meijer L. Eur. J. Biochem. 1994; 224: 771-786Crossref PubMed Scopus (613) Google Scholar). Its potency, however, is relatively low. Using the crystal structure of CDK2 (12De Bondt H. L. Rosenblatt J. Jancarick J. Jones H. D. Morgan D. O. Kim S. Nature. 1993; 363: 595-602Crossref PubMed Scopus (834) Google Scholar) and computer-aided drug design, a combinatorial library strategy (13Norman T. C. Gray N. S. Koh J. T. Schultz P. G. J. Am. Chem. Soc. 1996; 118: 7430-7431Crossref Scopus (140) Google Scholar, 14Nugiel D. A. Cornelius L. A. M. Corbett J. W. J. Org. Chem. 1997; 62: 201-203Crossref PubMed Scopus (93) Google Scholar) generated a large number of purine analogs. While the synthesis and structure activity relations of these compounds will be described elsewhere, 2S. R. Schow, R. T. Lum, D. Shiffman, R. Mackman, and M. W. Wick, manuscript in preparation. the present communication describes the biological effects of CVT-313, a representative compound from this purine-based library. All chemicals were purchased from Sigma and all tissue culture media and reagents were purchased from Life Technologies, Inc. , except where indicated. Baculovirus homogenization buffer was 10 mm HEPES (pH 7. 4), 10 mm NaCl, 5 mm EDTA, 1 mmdithiothreitol, 1 mm Pefabloc (Boehringer Mannheim), 1 μg/ml leupeptin, and 5 μg/ml aprotonin. Activation buffer was 10 mm HEPES (pH 7. 4), 10 mm MgCl2, and 1 mm ATP. Lysis buffer was 50 mm HEPES (pH 7. 4), 250 mm NaCl, 0. 1% Triton X-100, 50 mmNaF, 80 mm β-glycerophosphate, 0. 1 mmNa3VO4, 1 mm Pefabloc, 1 μg/ml leupeptin, 5 μg/ml aprotonin, and 1 mm iodoacetamide. Baculovirus constructs expressing human cyclin AΔ171, CDK2, cyclin H, and CDK7 were a kind gift from David Morgan (University of California, San Francisco). Recombinant proteins were expressed in Sf9 insect cells. Cells were homogenized using a Dounce homogenizer in baculovirus homogenization buffer. CDK2-cyclin A complexes were activated by mixing with a 0. 1 volume of CDK7/cyclin H extracts in activation buffer and incubating at 25 °C for 1 h. The CDK2-cyclin A complex was purified as described previously (15Fisher R. P. Morgan D. O. Cell. 1994; 78: 713-724Abstract Full Text PDF PubMed Scopus (559) Google Scholar). Human cyclin E was amplified from RNA made from MRC-5 cells by reverse transcription-polymerase chain reaction using the following primers: 5′-CGCGGATCCATGAAGGAGGACGGCGGCGC-3′ and 5′-TGCTCTAGATCACGCCATTTCCGGCCCGC-3′. The cDNA was cloned into pFASTBAC HTb (Life Technologies, Inc. ), generating a histidine tag at the amino-terminal end of cyclin E. Recombinant cyclin E was expressed in Sf9 cells, mixed with protein extracts of Sf9 cells expressing CDK2, and activated as described above. The CDK2-cyclin E complex was purified by nickel resin chromatography utilizing the histidine tag of cyclin E, as recommended by the vendor (Life Technologies, Inc. ). Human CDK4 cDNA was amplified from RNA made from MRC-5 cells by reverse transcription-polymerase chain reaction using the following primers: 5′-CGCGGATCCATGGCTACCTCTCGATATGAGCC-3′ and 5′-CCCAAGCTTTCACTCCGGATTACCTTCATCC-3′. The cDNA was cloned into pFASTBAC1 (Life Technologies, Inc. ). Human cyclin D1 cDNA (a kind gift from Steven Reed, Scripps Institute) was cloned into a pFASTBAC HTb vector, generating a histidine tag at the amino-terminal end of cyclin D1. CDK4/cyclin D/CDK7/cyclin H were co-expressed in Sf9 cells, and the activated CDK4-cyclin D complex was isolated by nickel resin chromatography. Human CDK1/cyclin B was purchased from New England Biolabs. Rat brain protein kinase C was purchased from Pierce. Bovine protein kinase A was purchased from Boehringer Mannheim. Murine recombinant mitogen-activated protein kinase was purchased from Upstate Biotechnology. CDK2/cyclin A, CDK2/cyclin E, and CDK1/cyclin B were incubated with 1 μg of histone H1 (Life Technologies, Inc. ) or 1 μg of glutathioneS-transferase-Rb (Santa Cruz Biotechnology) as indicated, in 10 mm MgCl2 with 50 μm ATP and 0. 3 μCi of γ-32PATP (3000Ci/mmol, NEN Life Science Products) in a total volume of 20 μl. Reactions were carried out for 25 min at 30 °C. Reactions were stopped by the addition of 2 μl of 0. 5 m EDTA. Samples were blotted onto Whatman P81 phosphocellulose paper and washed three times with 150 mmphosphoric acid. Filters were blotted dry, mixed with scintillation fluid, and quantitated by liquid scintillation spectrometry (Beckman LS 6500). All other in vitro kinase assays were performed by the same method, except that the protein kinase C assay contained 0. 2 μg of phosphotidyl-l-serine as an activator of proein kinase C. CDK4/cyclin D1 assay used 1 μg of glutathioneS-transferase-Rb (Santa Cruz Biotechnology) as the substrate in 10 mm Hepes (pH 7. 4), 10 mmMgCl2, with 10 μm ATP and 1 μCi of γ-32PATP (3000Ci/mmol). The protein kinase A assay used 50 μm Kemptide (Life Technologies, Inc. ) as the substrate in 10 mm MgCl2, 1 μg/μl bovine serum albumin, 12. 5 mm Tris (pH 7. 5) with 100 μm ATP. The mitogen-activated protein kinase assay contained 10 μg of myelin basic protein (Life Technologies, Inc. ) as the substrate in 18. 75 mm MgCl2 with 125 μm ATP. All cell lines were purchased from ATCC and grown as recommended except for neonatal rat vascular smooth muscle cells that were a kind gift from Mark Majesky (Baylor College of Medicine). These cells were grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum. Proliferation assays were carried out using the nonradioactive CellTiter 96 kit (Promega) after 48-h exposure. For FACS analysis of DNA content, cells were trypsinized, fixed in 70% ice-cold ethanol, and treated with 0. 1 mg/ml RNase A and 40 μg/ml propidium iodide for 1 h at 37 °C. Samples were sent to Cytometry Associates (San Diego) for analysis. Proteins were extracted from cells from two 9. 6-cm2 wells using 0. 2 ml of lysis buffer. Equal volumes (40 μl) of cleared cell lysate were separated on a 6% polyacrylamide gels, blotted onto nitrocellulose membrane, and probed with Rb antibodies (Santa Cruz Biotechnology). Blots were developed using the BM chemiluminescent system (Boehringer Mannheim). Pairs of male Sprague-Dawley rats (Charles River) aged 2–3 months (400–500 g) were housed under a normal 12-h light/dark cycle in standard plastic “shoe box” cages with standard laboratory chow and water available ad libitum. Prior to surgery, the animals were anesthetized with 1. 3 ml/kg ketamine/xylazine mixture (58% ketamine hydrochloride, 100 mg/ml; 42% xylazine hydrochloride, 20 mg/ml) injected intraperitoneally. After preparing the ventral cervical area for aseptic surgery, the bifurcation of the left common carotid artery was exposed, and a small vascular clamp was applied to the internal carotid artery. A deflated, saline-filled 2 F Fogarty catheter (Baxter) was inserted via an arteriotomy in the external carotid artery. The catheter was advanced proximally into the common carotid artery up to the aortic arch. To denude and injure the artery, the balloon was inflated with 20 μl of saline and then gently pulled back to the bifurcation. The procedure was repeated three times. Immediately after removing the balloon catheter, a delivery cannula (PE10 polyethylene tubing) was inserted, and 65 units of heparin sodium salt (Sigma, H-4898) in 1 ml of saline were injected. Following the heparin injection, a second vascular clamp was used to bisect the common carotid and allow for the infusion of drug or control solutions to one half of the injured artery. A loose silk ligature was used to mark the point where the clamp bisected the artery. This suture mark was later used to determine the treated and untreated sections of the artery when the animal was sacrificed. Before infusing control or drug solutions, the artery was backflushed with 1 ml of sterile saline. 100 μl of control or drug solution were flushed through the artery to wash out the saline and fill the lumen with the solution under study. A 3-0 Prolene ligature was placed around the external carotid to hold the cannula in place and create a closed system. Approximately 1 atm of steady pressure was applied to the delivery syringe over a 15-min incubation period. After the 15-min incubation period, the cannula and vascular clamp bisecting the common carotid artery were removed, to reestablish blood flow and flush residual solution out of the artery, before a 3-0 silk suture was applied to permanently ligate the external carotid artery. Finally, the vascular clamp was removed from the internal carotid artery, the neck wound was closed, and the animals were allowed to recover. Throughout the 14-day recovery period, a clinically relevant dose of aspirin (162. 5 mg/L) was added to the animals' water to prevent thrombosis. Fourteen days after the balloon catheter injury to the carotid artery, the animals were anesthetized, as described above. Buffered formalin (10%) was perfused at physiological pressure through the aortic arch. The left carotid artery was removed (from the carotid bifurcation to the aortic arch) and bisected at the loose silk ligature that demarked the treated and untreated boundary. The treated and untreated sections were each cut into three equal sections and mounted in cryomolds. The arteries were sectioned in a cryotome into 10-μm slices. Samples were taken randomly at 100-μm intervals and mounted on slides for staining and analysis. Fifteen samples from both treated and untreated segments were used, for a total of 30 samples from each animal. The slides were stained with hematoxylin and eosin and analyzed with the 4× objective of a light microscope (Olympus) and a digitizing tablet (JS-2, Jandel Scientific). Sigmascan software (Jandel Scientific) was used to determine the neointimal area of the digitized image. CVT-313 (2-bis- (hydroxyethyl) amino-6- (4-methoxybenzyl-amino) -9-isopropyl-purine, Fig. 1), was by the at and of synthesis will be described A large library of compounds were and tested for to CDK2 activity. CVT-313 was identified as a potent inhibitor of CDK2/cyclin A IC50 = 0. 5 It is potent at CDK2/cyclin E, using histone or recombinant Rb as a The of Rb by the CDK2-cyclin A complex was to inhibition by CVT-313 = It is this is Rb is a substrate of CDK2/cyclin E of CDK2/cyclin A, or we used a of cyclin A in assay system. CVT-313 was tested for effect on two related cyclin-dependent kinases. For inhibition of CDK1 a higher concentration was required = and for inhibition of CDK4 a 430-fold higher concentration was required = To the of inhibition of CVT-313, three other serine/threonine ATP kinases were also The concentration of CVT-313 required for half inhibition of mitogen-activated protein kinase and protein kinase A was at a higher than that required to CDK2, protein kinase C was inhibited at all at μm of the inhibition of CDK2 activity by CVT-313 to be The of inhibition of CDK2 by CVT-313 were shown in that the inhibition was competitive with respect to ATP and the i was 95 CDK2 activity. activity assays were carried out as described under and the standard of each The IC50 were from these as CDK2/cyclin A, 0. 5 CDK1/cyclin CDK4/cyclin mitogen-activated protein kinase protein kinase A analysis of CVT-313 CDK2/cyclin A activity was using histone as ATP were and 50 μm. CVT-313 were 0. 1 and 0. 2 μm A was from the of Using normal and cell the effects of CVT-313 on cell proliferation was and The IC50 for growth inhibition from 1. 25 to 20 μm. To the growth inhibition by CVT-313 was cell MRC-5 cells diploid were exposed to MRC-5 cells, a large number of MRC-5 cells that had been by serum for h contained DNA A After h of serum a relatively large of the cells into S with DNA 2 and 5 CVT-313 was added to cells h after serum the DNA of of the cells was 2 or with very cells than S phase D under similar culture the concentration of CVT-313 was to FACS analysis cells with 2 cells with and very cells in S These suggest that cells arrest at the G1/S and at a higher concentration of CVT-313, at the concentration of CVT-313, of the cells are arrested at the G1/S boundary. These observations are with the role of CDK1 and CDK2 in controlling and G1/S and with at CVT-313 CDK2 a higher concentration of CVT-313 is to CDK1 activity. were also in CVT-313 be used to cells at the G1/S boundary. MRC-5 cells were treated with CVT-313 for h and then analyzed by Fig. 5 F that of these cells had a 2 DNA content, with a G1/S After of CVT-313 from the growth medium and cells the cell that the inhibition of cell proliferation was 50 for added to cells in tissue normal vascular smooth muscle neonatal aortic smooth muscle was using CellTiter 96 system as described under and IC50 were from the of the inhibition Rat vascular smooth muscle cells. in a analysis of MRC-5 analysis of propidium iodide MRC-5 cells. cells after serum serum by serum serum by serum at which CVT-313 at 12. 5 μm was added for h. E, same CVT-313 concentration was cells exposed to CVT-313 at μm for h. same as h after drug has been H, same h after drug has been Cell was using CellTiter 96 system as described under and IC50 were from the of the inhibition of the in of CDK2 is to CVT-313 inhibited Rb hyperphosphorylation in MRC-5 cells that had been by serum analysis was used to determine the of Rb after serum and h after with a hyperphosphorylation of Rb be FACS analysis of cells that were in that these cells contained 2 DNA with Rb hyperphosphorylation to into S phase Cell. 1995; Full Text PDF Scopus Google Scholar). CVT-313 was added at or h after serum Rb hyperphosphorylation was The effect on Rb hyperphosphorylation was when CVT-313 was added h after of Rb by CDK2 had have also Rb hyperphosphorylation when CVT-313 was added at the of serum These be the of CDK4 with that CVT-313 CDK4 activity in vitro. To the in of CVT-313, we the injured rat carotid artery model of restenosis. In this is to neointima formation by smooth muscle cell and proliferation (7Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar). of drug in this animal model has two the to endothelial denudation from animal to control a higher with treated this by one half of the injured and used the other half as an internal untreated to the of the the that can when a sections from each carotid are analyzed randomly tissue each of the Using this we that exposure of the denuded carotid artery to the salt of CVT-313 in saline solution for min under neointima formation by 80% in each animal treated with CVT-313 is at 70% inhibition of the neointimal in treated animal. Two of CVT-313 and were reducing neointimal area by the dose tested in neointimal sections from rat carotid arteries days after endothelial denudation the of CVT-313 in restenosis in the rat carotid These “proof of principle” the of CDK2 as an target and that CVT-313 is an candidate of evaluation in other animal models of of and normal rat A, normal rat same as A, rat carotid days after endothelial same E, same as C and treated with CVT-313 as described in the same as E, Kim for the crystal structure of CDK2, David Morgan for on CDK2 and N. for