A Novel Class of Cyclin-dependent Kinase Inhibitors Identified by Molecular Docking Act through a Unique Mechanism
2009; Elsevier BV; Volume: 284; Issue: 43 Linguagem: Inglês
10.1074/jbc.m109.055251
ISSN1083-351X
AutoresPatrick Corsino, Nicole A. Horenstein, David A. Ostrov, Thomas C. Rowe, Mary E. Law, Amanda Barrett, George Aslanidi, W. Douglas Cress, Brian K. Law,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe cyclin-dependent kinase (Cdk) family is emerging as an important therapeutic target in the treatment of cancer. Cdks 1, 2, 4, and 6 are the key members that regulate the cell cycle, as opposed to Cdks that control processes such as transcription (Cdk7 and Cdk9). For this reason, Cdks 1, 2, 4, and 6 have been the subject of extensive cell cycle-related research, and consequently many inhibitors have been developed to target these proteins. However, the compounds that comprise the current list of Cdk inhibitors are largely ATP-competitive. Here we report the identification of a novel structural site on Cdk2, which is well conserved between the cell cycle Cdks. Small molecules identified by a high throughput in silico screen of this pocket exhibit cytostatic effects and act by reducing the apparent protein levels of cell cycle Cdks. Drug-induced cell cycle arrest is associated with decreased Rb phosphorylation and decreased expression of E2F-dependent genes. Multiple lines of evidence indicate that the primary mechanism of action of these compounds is the direct induction of Cdk1, Cdk2, and Cdk4 protein aggregation. The cyclin-dependent kinase (Cdk) family is emerging as an important therapeutic target in the treatment of cancer. Cdks 1, 2, 4, and 6 are the key members that regulate the cell cycle, as opposed to Cdks that control processes such as transcription (Cdk7 and Cdk9). For this reason, Cdks 1, 2, 4, and 6 have been the subject of extensive cell cycle-related research, and consequently many inhibitors have been developed to target these proteins. However, the compounds that comprise the current list of Cdk inhibitors are largely ATP-competitive. Here we report the identification of a novel structural site on Cdk2, which is well conserved between the cell cycle Cdks. Small molecules identified by a high throughput in silico screen of this pocket exhibit cytostatic effects and act by reducing the apparent protein levels of cell cycle Cdks. Drug-induced cell cycle arrest is associated with decreased Rb phosphorylation and decreased expression of E2F-dependent genes. Multiple lines of evidence indicate that the primary mechanism of action of these compounds is the direct induction of Cdk1, Cdk2, and Cdk4 protein aggregation. Uncontrolled cell proliferation is one of the defining features of cancer. Cdks 3The abbreviations used are: CdkCyclin-dependent kinaseGFPgreen fluorescent proteinNSCNomenclature Standards CommitteeDTPDevelopmental Therapeutics ProgramRbretinoblastomaPBSphosphate-buffered salineErk1/2extracellular signal-regulated kinase 1 and 2. are serine/threonine protein kinases that play key roles in controlling cell cycle progression (1Deshpande A. Sicinski P. Hinds P.W. Oncogene. 2005; 24: 2909-2915Crossref PubMed Scopus (356) Google Scholar, 2Sánchez I. Dynlacht B.D. Semin. Cell Dev. Biol. 2005; 16: 311-321Crossref PubMed Scopus (228) Google Scholar). The concerted activities of Cdks result in chromatin condensation, nuclear envelope breakdown, and the up-regulation of genes involved in nucleotide synthesis and DNA replication, among other events. Cdks have long been considered ideal targets for anti-cancer drugs, owing to their importance in the cell cycle. As a result, many Cdk inhibitors have been developed, some of which have progressed to clinical trials. Roscovitine (Selicilib) and flavopiridol (Alvocidib) are examples of Cdk inhibitors that have passed Phase I clinical trials (3Benson C. White J. De Bono J. O'Donnell A. Raynaud F. Cruickshank C. McGrath H. Walton M. Workman P. Kaye S. Cassidy J. Gianella-Borradori A. Judson I. Twelves C. Br. J. Cancer. 2007; 96: 29-37Crossref PubMed Scopus (225) Google Scholar, 4Christian B.A. Grever M.R. Byrd J.C. Lin T.S. Curr. Opin. Oncol. 2007; 19: 573-578Crossref PubMed Scopus (49) Google Scholar) and have entered Phase II clinical trials (6Schwartz G.K. Ilson D. Saltz L. O'Reilly E. Tong W. Maslak P. Werner J. Perkins P. Stoltz M. Kelsen D. J. Clin. Oncol. 2001; 19: 1985-1992Crossref PubMed Scopus (201) Google Scholar, 7Shapiro G.I. Supko J.G. Patterson A. Lynch C. Lucca J. Zacarola P.F. Muzikansky A. Wright J.J. Lynch Jr., T.J. Rollins B.J. Clin. Cancer Res. 2001; 7: 1590-1599PubMed Google Scholar). 4C. Belani (2006) Efficacy Study of Oral Seliciclib to Treat Non-Small Cell Lung Cancer, ClinicalTrials.gov, identifier: NCT00372073. However, these drugs, as well as most other Cdk inhibitors, are ATP-competitive. The disadvantage of using a therapeutic strategy involving ATP competition is that all kinases possess an ATP-binding site, leading to the potential for reduced target specificity. Recently, advances have been made in identifying Cdk inhibitors that act through novel mechanisms. One example of this effort is the identification of a series of cyclic peptides designed to mimic the structure of the Cdk inhibitor p27. These compounds bind to the substrate recognition site of Cdk complexes and inhibit their kinase activity in vitro (8Andrews M.J. McInnes C. Kontopidis G. Innes L. Cowan A. Plater A. Fischer P.M. Org. Biomol. Chem. 2004; 2: 2735-2741Crossref PubMed Scopus (58) Google Scholar). Another example is the use of a small, 39-amino acid peptide that inhibits the kinase activity of Cdk2 by mimicking the inhibitory effects of the pRb2/p130 spacer domain (9Bagella L. Sun A. Tonini T. Abbadessa G. Cottone G. Paggi M.G. De Luca A. Claudio P.P. Giordano A. Oncogene. 2007; 26: 1829-1839Crossref PubMed Scopus (36) Google Scholar). These approaches are promising, but rely on peptide-based inhibitors that have inherent disadvantages for use as therapeutic agents. Cyclin-dependent kinase green fluorescent protein Nomenclature Standards Committee Developmental Therapeutics Program retinoblastoma phosphate-buffered saline extracellular signal-regulated kinase 1 and 2. The use of knockout mice has recently generated much information about the role of Cdks with respect to cell cycle regulation. For instance, mice lacking Cdk2, Cdk4, or Cdk6 are all viable (10Berthet C. Aleem E. Coppola V. Tessarollo L. Kaldis P. Curr. Biol. 2003; 13: 1775-1785Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 11Malumbres M. Hunt S.L. Sotillo R. Martín J. Odajima J. Martín A. Dubus P. Ortega S. Barbacid M. Adv. Exp. Med. Biol. 2003; 532: 1-11Crossref PubMed Scopus (34) Google Scholar, 12Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (613) Google Scholar). Furthermore, cells lacking both Cdk4 and Cdk6 proliferate almost normally (13Malumbres M. Sotillo R. Santamaría D. Galán J. Cerezo A. Ortega S. Dubus P. Barbacid M. Cell. 2004; 118: 493-504Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). More recently it has been discovered that mouse embryonic fibroblast cells are able to cycle in the absence of Cdk2, Cdk4, and Cdk6, needing only Cdk1 to complete cell division (14Santamaría D. Barrière C. Cerqueira A. Hunt S. Tardy C. Newton K. Cáceres J.F. Dubus P. Malumbres M. Barbacid M. Nature. 2007; 448: 811-815Crossref PubMed Scopus (749) Google Scholar). In light of the fact that Cdks are able to functionally replace one another, highly selective Cdk inhibitors that target only one type of cell cycle Cdk may not be as effective as anti-tumor agents that inhibit Cdk1, Cdk2, Cdk4, and Cdk6. An inhibitor that acts on multiple cell cycle Cdks would therefore have a greater probability of inhibiting tumor cell growth by ensuring that the cell cycle is arrested. Here we report the identification of a novel structural pocket present on Cdk2 that is conserved on Cdks 1, 4, and 6. Using a high throughput in silico screening procedure we have identified compounds that decrease the function of Cdks in cells through binding to this site. The two protein crystal structures used for identification and in silico screening of the structural site were the Cyclin A-Cdk2 and p27kip1-Cyclin A-Cdk2 complexes (RCSB Protein Data Bank codes: 1FIN (15Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massagué J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar) and 1JSU (16Russo A.A. Jeffrey P.D. Patten A.K. Massagué J. Pavletich N.P. Nature. 1996; 382: 325-331Crossref PubMed Scopus (801) Google Scholar), respectively). A molecular surface of 1JSU was prepared using the MSROLL program, which was then used as input for the sphere-generating program SPHGEN. A cluster of spheres that was shown to be within the pocket of interest was then selected and edited manually to leave a cluster of 21 spheres. The SHOWBOX program was used to construct a three-dimensional rectangle, 4 Å in every direction from the sphere cluster. The program CHIMERA was used to convert the PDB file of 1JSU into the appropriate mol2 format. The box file that was generated was then used as input for the GRID program, which calculates information concerning the steric and electrostatic environment within the box of the 1JSU mol2 file. DOCK was used to screen the entire NCI/Developmental Therapeutics Program (DTP) database of small molecules (which consisted of ∼140,000 small molecules at the time of docking) within the 1JSU grid, with the selected spheres as theoretical binding sites. CHIMERA was subsequently used to rank the small molecule output based on predicted energy scores composed of electrostatic interactions and van der Waals' forces. The top 40 compounds were obtained from the NCI/DTP for cell culture testing. All of the programs listed for this procedure were part of the DOCK5.0 suite developed at University of California, San Francisco (17Moustakas D.T. Lang P.T. Pegg S. Pettersen E. Kuntz I.D. Brooijmans N. Rizzo R.C. J. Comput. Aided Mol. Des. 2006; 20: 601-619Crossref PubMed Scopus (358) Google Scholar). Protein structure visualization and image generation were performed using PyMOL software (DeLano Scientific, Palo Alto, CA). All experiments involving mammalian cell culture were performed using Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (35-011-CV, Mediatech, Inc., Manassas, VA). BT549 and HCT116 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). QBI-293A kidney cells were obtained from Quantum (Montreal, Canada). Chemical reagents and solvents were purchased from Sigma-Aldrich and Acros (Morris Plains, NJ). Both synthesized compounds displayed spectroscopic data consistent with the proposed structures. NSC43067 (1-(5-methylthiophen-2-yl)-3-phenylpropenone): solid NaOH (1.78 g, 44.5 mm) was dissolved in 50 ml of water/ethanol (2:1, v/v) with cooling in an ice bath. Sequential addition of 5-methyl-2-acetyl thiophene (5.00 g, 35.6 mm) and benzaldehyde (3.77 g, 35.6 mm) to the cooled solution of NaOH was followed by rapid stirring on ice for 2 h. The mixture was stored overnight at 4 °C, resulting in the formation of an oily solid. The solid was removed by vacuum filtration, and the filtrate was then concentrated in vacuo. The residue was dissolved in hot absolute ethanol and left to cool to provide 4.04 g (50% yield) of NSC43067 as flaky yellow crystals; melting point, 93–95 °C. NSC63002 (2-(4-methoxy-phenyl)-3-pyridin-2-yl-acrylonitrile): The compound was prepared in 26% yield as described in a previous study (18Magarian E.O. Nobles W.L. J. Pharm. Sci. 1969; 58: 1166-1167Abstract Full Text PDF PubMed Scopus (1) Google Scholar) (melting point, 69–70 °C (literature: 69.5–70.5). For [3H]thymidine incorporation assays, cells were plated in 24-well plates at a density of 30,000 cells per well and allowed to incubate overnight at 37 °C. Following treatment periods, cells were incubated for a further 2 h at 37 °C with 40 μl per well [3H]thymidine (0.1 mCi per ml diluted in PBS, NET027005MC, PerkinElmer Life Sciences). Cells were then fixed with 10% trichloroacetic acid for 15 min, followed by a further two washes with 10% trichloroacetic acid. The DNA was then dissolved with 300 μl of 0.2 n sodium hydroxide. For each well, 100 μl was counted in a Beckman Coulter LS6500 multipurpose scintillation counter (Beckman Coulter, Fullerton, CA). For cell viability assays, drug activities were determined using a high-throughput CellTiter-Blue assay. Cells (1200–6000; 24-μl volumes) were plated in each well of 384-well plates and incubated overnight at 37 °C, 5% CO2. The next day, the drugs were diluted in media, and 6 μl of these dilutions was added to appropriate wells using an automated pipetting station. Four replicate wells were used for each drug concentration and an additional four control wells received a diluent control without drug. Drug dilutions generally consisted of 1.5-fold dilutions from a maximum final concentration of 100 μm. The cells were incubated with the drug for 72–120 h. At this time, 5 μl of CellTiter-Blue reagent (Promega, Madison, WI) was added to each well. Cell viability was assessed by the ability of the remaining viable cells to bioreduce resazurin to resorufin. Resazurin is dark blue in color and has little intrinsic fluorescence until it is reduced to resorufin (579 nm Ex/584 nm Em). The change in fluorescence was measured with a Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). The fluorescence data were transferred to a spreadsheet program to calculate the percent viability relative to the four replicate cell wells that did not receive drug. IC50 values were determined using a sigmoidal equilibrium model regression using XLfit version 4.3.2 (ID Business Solutions Ltd.). The IC50 value was defined as the concentration of drug required for a 50% reduction in growth/viability. For flow cytometry analysis, cells were plated at a density of 300,000 cells per plate in P100 dishes. Following treatment, cells were removed from the plate by trypsin digestion and suspended in a solution containing 3.4 mm sodium citrate, 75 μm propidium iodide, 0.1% Triton X-100, and 5 μg/ml RNase A. Samples were analyzed on a BD Biosciences FACSort flow cytometer. Cells were lysed with extraction buffer (0.1% Triton X-100, 20 mm HEPES, pH 7.6, 1 mm EDTA, 1 mm EGTA, 0.1% β-mercaptoethanol, 5% glycerol, 10 nm microcystin, 1 mm sodium orthovanadate, and 40 mm sodium pyrophosphate) followed by sonication. Extracts were cleared by centrifugation at 16,000 × g for 20 min. The cleared supernatant was then analyzed for total protein concentration with Bradford protein assay dye reagent (500-0006, Bio-Rad), and all extracts were normalized to the lowest protein concentration. The extracts were boiled with one-third volume of 4× SDS sample buffer (60 mm Tris, pH 6.7, 24 mm EDTA, 200 mm SDS, 40% glycerol, 300 μm bromphenol blue, 0.4% β-mercaptoethanol) for 5–10 min. Samples were resolved on SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were immunoblotted using antibodies specific for either actin (sc-1616-P, Santa Cruz Biotechnology, Santa Cruz, CA), Cdk1 (sc-54, Santa Cruz Biotechnology), Cdk2 (sc-163, Santa Cruz Biotechnology), Cdk4 (sc-601, Santa Cruz Biotechnology), E2F1 (sc-193, Santa Cruz Biotechnology), Cyclin A (sc-596, Santa Cruz Biotechnology), Cyclin D1 (MS-210-PO, NeoMarkers, Freemont, CA), Cyclin E (sc-198, Santa Cruz Biotechnology), B-myb (sc-725, Santa Cruz Biotechnology), p-Rb (#9309, Cell Signaling, Danvers, MA), p-Rb-780 (#9307, Cell Signaling), p-Rb-807/811 (#9308, Cell Signaling), Erk1/2 (sc-93, Santa Cruz Biotechnology), p38 (sc-535, Santa Cruz Biotechnology), Hsp70 (sc-94, Santa Cruz Biotechnology), Hsp90 (sc-13119, Santa Cruz Biotechnology), Hsc70 (SPA-815, Stressgen, Victoria BC, Canada), p21 (sc-6246, Santa Cruz Biotechnology, OP64 and OP76, Calbiochem, San Diego, CA), p27 (554069, BD Biosciences, San Jose, CA), or Caspase 3 (sc-7148, Santa Cruz Biotechnology). Cyclin E-Cdk2 complexes (#7524, Cell Signaling) were diluted to a concentration of 12 μg/ml and preincubated with either the vehicle or NCI compounds for 24 h at room temperature. The kinase/treatment mixture was then mixed with the substrate solution for a final concentration of 6 μg/ml Cyclin-Cdk, 25 μg/ml GST-Rb (sc-4112, Santa Cruz Biotechnology), 18 μCi/ml [γ-32P]ATP (BLU002A250UC, PerkinElmer Life Sciences), 0.1 μm unlabeled ATP, 50 mm HEPES, pH 7.5, 10 mm magnesium chloride, 2.5 mm EGTA, 1 mm dithiothreitol, 0.1 mm sodium fluoride, and 0.1 mm sodium orthovanadate. Reactions were allowed to incubate for 1 h at 37 °C, followed by quenching with one-third volume 4× SDS sample buffer and boiling for 5 min. Samples were resolved on SDS-PAGE gels, fixed, and stained with Coomassie dye. Radioactive bands were quantified using a Beckman Coulter LS6500 multipurpose scintillation counter. Green Fluorescent Protein (GFP) fused to Cdk4 was stably expressed in 293A cells. GFP cDNA was amplified from the pAcGFP-tubulin plasmid (Clontech) using the following PCR primers: 5′-TTTTGGATCCGATATCCCACCATGGTGAGCAAGGGCGCCGAG-3′ and 5′-TTTTGGATCCCTTGTACAGCTCATCCATGCC-3′. Following amplification, the GFP PCR product was purified by chloroform/phenol extraction and ethanol precipitation. The PCR product was subsequently digested with BamHI. The previously described pcDNA3 plasmid encoding Cdk4-His6 contains a BamHI site 5′ to Cdk4-His6 (19Chytil A. Waltner-Law M. West R. Friedman D. Aakre M. Barker D. Law B. J. Biol. Chem. 2004; 279: 47688-47698Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). This plasmid was also digested with BamHI, followed by treatment with calf intestinal alkaline phosphatase for 1 h at 37 °C. Vector and insert DNA were ligated for 18 h at room temperature, creating a construct that contains cDNA encoding GFP in-frame with cDNA encoding Cdk4-His6. The orientation of the insert was confirmed by EcoRV digestion. Ten micrograms of the GFP-Cdk4/pcDNA3 construct was transfected into 293A cells using Lipofectamine (18324-020, Invitrogen). Cells that stably retained the plasmid were first selected by treating the cells with medium containing 500 μg/ml G418 Sulfate (61–234-RG, Cellgro), followed by one round of cell sorting for GFP-positive cells using a FACSAria cell sorter (BD Biosciences) and the Diva program (version 6.1). Clonal lines were then established for experimental analysis. Mink lung epithelial cells (Mv1Lu) stably expressing a Cyclin D1-Cdk2 fusion protein or E2F1 were described previously (19Chytil A. Waltner-Law M. West R. Friedman D. Aakre M. Barker D. Law B. J. Biol. Chem. 2004; 279: 47688-47698Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (92) Google Scholar). Cell extracts were prepared as described in the previous section. Samples were centrifuged at either 16,000 × g for 20 min or 150,000 × g for 1 h. In both cases, the supernatants were removed and normalized for protein concentration. The pellets were extracted with 300 μl of extraction buffer containing 1.0% Triton X-100, 20 mm HEPES, pH 7.6, 1 mm EDTA, 1 mm EGTA, 0.1% Triton X-100, 0.1% β-mercaptoethanol, 5% glycerol, 10 nm microcystin, 1 mm sodium orthovanadate, and 40 mm sodium pyrophosphate, and 300 μl of 2× SDS sample buffer. The extracted pellets were normalized using the same dilution factors as for the supernatant fraction, sonicated vigorously, and boiled for 5–10 min. Cells for immunofluorescence studies were plated onto glass coverslips in 6-well plates. Cells were treated for 24 h with either vehicle control or the indicated NSC compounds. After a further 24-h incubation, the cells were fixed with 1% paraformaldehyde in PBS for 20 min, followed by a 10-min incubation with the quench solution (50 mm ammonium chloride plus 0.5% Triton X-100 in PBS) in the case of Cdk4 visualization. Alternatively, cells were fixed with paraformaldehyde followed by incubation with ice-cold 100% methanol for Hsp70 and γ-tubulin visualization. In either case, the coverslips were subsequently incubated with antibody buffer (5% goat serum plus 0.5% Triton X-100 in PBS) in a humidified chamber for 1 h. Primary antibody staining was performed using an antibody for Cdk4 (sc-601, Santa Cruz Biotechnology), Hsp70 (sc-24, Santa Cruz Biotechnology) or γ-tubulin (sc-17787, Santa Cruz Biotechnology) at a 1:100 dilution in antibody buffer, or no primary antibody as a control for nonspecific staining, for 2 h. Following primary antibody incubation, the coverslips were washed four times with PBS and incubated with a goat α-Rabbit Fluor 488 (A11008, Invitrogen, Molecular Probes, Carlsbad, CA) or a goat α-Mouse Cy3 secondary antibody (81-6515, Zymed Laboratories Inc., Carlsbad, CA) for 1 h at a 1:200 dilution in antibody buffer. Following four additional washes with PBS, coverslips were mounted onto slides with Vectashield plus 4′,6-diamidino-2-phenylindole (H-1200, Vector Laboratories, Burlingame, CA) to visualize nuclei. Slides were viewed on a Leica TCS SP2 AOBS spectral confocal microscope. Images were collected and processed using Leica Confocal Software (Leica Microsystems, Wetzlar, Germany) Version 2.61, Build 1537. GFP-Cdk4 293A cells were plated, treated, and processed in the same manner as for immunofluorescence. However, instead of antibody incubation, fixed and quenched cells were mounted directly onto slides with Vectashield plus 4′,6-diamidino-2-phenylindole. GFP-Cdk4 cells used for time-lapse microscopy were plated at a density of 450,000 cells per plate in fluorodish plates (FD35–100, World Precision Instruments, Sarasota, FL). Cells were visualized on a Leica TCS SP2 AOBS spectral confocal microscope. We examined the differences between several crystal structures of Cdk2 to identify a novel inhibitor binding site on Cdks. A side-by-side comparison of the structures of the catalytically active Cyclin A-Cdk2 complex and the catalytically inactive p27-Cyclin A-Cdk2 complex revealed the formation of a structural pocket, present only in the inhibited, p27-bound form of Cyclin A-Cdk2 (Fig. 1A). We hypothesized that a small molecule could bind to this pocket and stabilize the Cyclin A-Cdk2 complex in an "open" conformation which would mimic the p27-bound form of Cyclin A-Cdk2, and would thereby inactivate the enzyme catalytically. A sequence alignment of Cdks 1, 2, 4, and 6 revealed that the residues that comprise the pocket are relatively well conserved between these Cdks, indicating that this pocket is likely to be present in the other cell cycle Cdks (Fig. 1B). The pocket is in close proximity to the ATP binding site (two of the residues that make up the pocket are part of the GXGXXG motif). However, the bulk of the pocket is distinct from the ATP-binding region. We performed a high-throughput in silico molecular docking screen on the p27-Cyclin A-Cdk2 crystal structure using the University of California, San Francisco program suite DOCK5.0. Approximately 140,000 small molecules from the NCI/DTP data base were docked into the pocket, designated by the appropriate spheres and scoring grid, and ranked according to their predicted binding energies. The top 40 compounds were ordered from the NCI/DTP and tested in cell culture. Fig. 1C shows a selection of the most active compounds as determined by assays described in later sections, with their predicted energy scores. [3H]Thymidine incorporation assays were performed to examine the influence of the compounds on cell proliferation. Initially, all 40 compounds ordered from the NCI/DTP were tested in cell culture at a concentration of 100 μm to determine if any cytostatic effect could be measured (data not shown). The most promising compounds were subsequently used in more detailed dose-response [3H]thymidine incorporation assays, as well as in further mechanistic studies. The BT549 human breast cancer cell line and the HCT116 human colon cancer cell line exhibited decreased cell proliferation as measured by [3H]thymidine incorporation after treatment with compounds NSC43067, NSC43042, and NSC63002 for 24 h (Fig. 2A). Cell cycle analyses of each of these two cell lines were performed by flow cytometry of propidium iodide stained cells after treatment with the same compounds for 24 h. In both cell lines, and for both compounds, an arrest at the G0/G1 phase of the cell cycle is observed at lower concentrations, but an accumulation of cells in the G2/M cell cycle phase is apparent at higher concentrations (Fig. 2, B and C). Importantly, the profiles from these experiments reveal a lack of a sub-G1 population, indicating that apoptosis is not a significant contributing factor to the observed decrease in cell proliferation. This assay confirms that the decrease in DNA synthesis as determined by [3H]thymidine incorporation is associated with cell cycle arrest. IC50 values were also determined for compounds NSC63002 and NSC117024 by Cell Titer-Blue assay in several other breast and lung cancer cell lines (supplemental Fig. S1). Total levels of several Cdks were analyzed by immunoblot to determine whether the compounds affect Cdk abundance. BT549 cells were treated with compounds NSC43067, NSC269621, and NSC63002 at 100 μm and 200 μm for 24 or 48 h. All three compounds significantly reduced the levels of soluble Cdk1, Cdk2, and Cdk4, especially at the highest concentrations and longest time points. The levels of several Cyclins were also affected, but to a lesser extent. This result was not due to a universal effect on all cellular proteins, because the levels of the protein phosphatase PP2A and the structural protein actin did not change appreciably (Fig. 3A). Additionally, no cleaved Caspase 3 bands were observed, indicating that apoptotic cell death did not play a major role in the effects of the NSC compounds on the cells, consistent with the flow cytometry results in Fig. 2. E2F-1 is a transcription factor that plays a crucial role in mediating Cdk-initiated cell cycle progression. Early cell cycle (G1 to S phase) Cdk activity leads to the phosphorylation of the E2F inhibitor Rb, resulting in its release from E2F family proteins and increased E2F-dependent transcription (for review see Ref. 21Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar). Activation of E2F-dependent transcription is thought to be one of the primary cell cycle-related functions of the G1/S Cdks. Therefore, E2F-1 overexpression would be expected to result in the partial reversion of the Cdk-reducing effects of the compounds. To examine this possibility, we used a mink lung epithelial cell line (Mv1Lu) engineered to overexpress E2F-1 (Mv1Lu-E2F1–11) (20Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (92) Google Scholar). Treatment of the parental Mv1Lu cells with 200 μm NSC63002 resulted in an almost complete reduction of phosphorylation of Rb at serine 780, as well as a decrease in the levels of several E2F-1 dependent gene products including Cyclin A, B-Myb, and E2F-1 itself (Fig. 3B). Levels of Cdk1 and Cdk4 were also found to decrease. E2F1 overexpression resulted in a diminished response to compound NSC63002 compared with the effect observed in the parental cells. A decrease in E2F dependent gene products, as well as serine 780 phosphorylation of Rb, was observed, albeit the inhibition was weaker than that observed in the parental cells. The effect of NSC63002 on Rb phosphorylation and E2F-depdendent transcription was also dampened in an Mv1Lu cell line that expresses a constitutively active CyclinD1-Cdk2 fusion protein (19Chytil A. Waltner-Law M. West R. Friedman D. Aakre M. Barker D. Law B. J. Biol. Chem. 2004; 279: 47688-47698Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Although the levels of the CyclinD1-Cdk2 fusion protein decreased upon drug treatment, Cyclin D1 alone did not. This would indicate that the effects of the compounds are Cdk-dependent. In all of the cell lines, there was no observable change in Erk1/2, and p38, two kinases closely related to the Cdks, or in the loading control, actin. Finally, there was no change in levels of the heat shock proteins Hsc70 or Hsp70, indicating that the compound did not simply induce a general stress response in the cells. In vitro kinase assays were performed using purified CyclinE-Cdk2 complexes to determine whether the compounds directly inhibit Cdk activity. After a 24-h preincubation with the NSC compounds, Cdk-dependent phosphorylation of Rb was dramatically reduced as compared with the vehicle-treated samples (Fig. 3C). Together, these data indicate that the NSC compounds decrease Cdk abundance in cultured cells and directly inhibit Cdk activity in in vitro kinase assays. A decrease in Cdks 1, 2, and 4 was observed as early as 4 h after treatment (Fig. 4A). Time-course experiments were performed to ensure that Cdk down-regulation by the compounds occurred in parallel with cell cycle arrest. BT549 cells treated with 200 μm NSC43042 or NSC63002 were incubated for 4, 8, 12, or 24 h before measuring cell proliferation by [3H]thymidine incorporation (Fig. 4B). The cytostatic effects of both compounds were observed as early as 4 h after treatment, in accord with the apparent decrease in Cdk levels. Taken together, these data suggest that the cytostatic effects of the compounds are mediated through a decrease in soluble Cdk levels. Initially, we examined the possibility that the compounds increase protein degradation of the Cdks through proteasomal degradation. Co-treatment of cells with proteasome inhibitors, such as lactacystin or 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone, did not affect compound-induced Cdk ablation, indicating that the effect was not induced by proteasomal degradation of the Cdks (data not shown). Consistent with this result, transfection
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