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Structure-based design of apotent purine-based cyclin-dependent kinase inhibitorThomas G. Davies1, Johanne Bentley2, Christine E. Arris2,F. Thomas Boyle3, Nicola J. Curtin2, Jane A. Endicott1,Ashleigh E. Gibson2, Bernard T. Golding2, Roger J. Griffin2, Ian R. Hardcastle2, Philip Jewsbury3,Louise N. Johnson1, Veronique Mesguiche2, David R. Newell2, Martin E.M. Noble1, Julie A. Tucker1,Lan Wang2 and Hayley J. Whitfield2,4

1Laboratory of Molecular Biophysics and Department of Biochemistry,University of Oxford, Oxford, OX1 3QU, UK. 2Cancer Research Unit andDepartment of Chemistry, University of Newcastle, Newcastle upon Tyne,NE2 4HH, UK. 3AstraZeneca Pharmaceuticals, Alderley Park, Cheshire,SK10 4TG, UK. 4This work is dedicated to the memory of Hayley J. Whitfield.

Published online: 16 September 2002, doi:10.1038/nsb842

Aberrant control of cyclin-dependent kinases (CDKs) is a central feature of the molecular pathology of cancer. Iterativestructure-based design was used to optimize the ATP-competitive inhibition of CDK1 and CDK2 by O6-cyclohexyl-methylguanines, resulting in O6-cyclohexylmethyl-2-(4′-sulfamoylanilino)purine. The new inhibitor is 1,000-foldmore potent than the parent compound (Ki values for CDK1 = 9 nM and CDK2 = 6 nM versus 5,000 nM and12,000 nM, respectively, for O6-cyclohexylmethylguanine).The increased potency arises primarily from the formation oftwo additional hydrogen bonds between the inhibitor andAsp 86 of CDK2, which facilitate optimum hydrophobicpacking of the anilino group with the specificity surface ofCDK2. Cellular studies with O6-cyclohexylmethyl-2-(4′-sulfamoylanilino) purine demonstrated inhibition of MCF-7cell growth and target protein phosphorylation, consistentwith CDK1 and CDK2 inhibition. The work represents thefirst successful iterative synthesis of a potent CDK inhibitorbased on the structure of fully activated CDK2–cyclin A.Furthermore, the potency of O6-cyclohexylmethyl-2-(4′-sulfamoylanilino)purine was both predicted andfully rationalized on the basis of protein–ligandinteractions.

The cyclin-dependent kinases (CDKs) are increasinglyrecognized as important targets for therapeutic interven-tion in various proliferative disease states, including cancer1. Inhibitors of CDK have entered clinical trials to treat cancer2,3. We recently reported the identificationof a newly discovered ATP-competitive purine-based

inhibitor, O6-cyclohexylmethylguanine (NU2058, Fig. 1a), whichhas a Ki of 5 µM for CDK1 and 12 µM against CDK2 but essen-tially no activity against CDK4 (ref. 4). An important feature ofNU2058 is that it has a cellular pharmacological profile that is dis-tinct from that of the benchmark inhibitors olomoucine andflavopiridol4. Furthermore, the crystal structure of NU2058 incomplex with monomeric CDK2 revealed protein–ligand inter-actions that were distinct from ATP and the 6-aminopurineinhibitors olomoucine5, roscovitine6 and purvalanol7. Com-parison of the binding modes of NU2058, the 6-aminopurineCDK inhibitors and the natural product indirubin8 suggest thatsubstitution at the N2 position of NU2058 would be tolerated andmight lead to inhibitors with increased potency.

Here we demonstrate that structure-based development anditerative biological evaluation can be used to rapidly optimizeCDK1 and CDK2 inhibition and identify inhibitors withnanomolar potency. Notably, this work is the first successful useof the fully active CDK2–cyclin A complex in a prospective drugdesign program.

NU2058 in complex with phosphoCDK2–cyclin AAs a starting point for the optimization of NU2058, we deter-mined the structure of the NU2058 inhibitor bound to the fullyactive Thr 160-phosphorylated CDK2–cyclin A spacing complex(T160pCDK2–cyclinA) (Fig. 1b). Structural rearrangementsassociated with CDK2 activation suggest that the use of the fullyactive complex is important for detailed atom-targeted, structure-based design9.

NU2058 in complex with T160pCDK2–cyclinA, as withmonomeric CDK2, forms a triplet of hydrogen bonds betweenits purine ring and the hinge region of CDK2 (residues 81–84)(Fig. 1c). In addition, several van der Waals and hydrophobiccontacts are made between the purine ring and the top and bot-tom of the ATP-binding cleft. An edge–face aromatic–aromaticcontact is formed between the purine ring and Phe 80. The O6-cyclohexylmethyl substituent is accommodated in the bind-ing site for the ribose moiety of ATP and forms highly comple-mentary packing and hydrophobic interactions with an apolarpocket in the Gly-rich loop (residues 9–19).

Structure-activity relationships for the O6-position haveshown that a cyclic hydrophobic substituent such as cyclohexyl-methyl seems to be optimal10, and we have maintained this

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Fig. 1 Inhibitor structures and binding of NU2058 toT160pCDK2–cyclinA. a, Chemical structures of NU2058, NU6094,NU6086 and NU6102. b, T160pCDK2–cyclinA–NU2058 structure.T160pCDK2–cyclinA is drawn in ribbon representation. CDK2 ispurple and cyclin A is gold. NU2058 bound in the CDK2 activesite cleft is shown as a surface representation in green. c, CDK2–NU2058 hydrogen bond interactions. Schematic repre-sentation of the conserved hydrogen bonds between backboneatoms of CDK2 residues Leu 83 and Glu 81, located in the hingeregion, and NU2058. Hydrogen bonds are drawn as dotted lines.Arrows pointing towards Phe 80 and Lys 89 indicate the orienta-tion of the inhibitor within the active site.

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group in subsequent compounds. To increase the potency ofNU2058, we considered addition and elaboration of functionalgroups at the N2 position of the purine. Assuming a similarbinding mode to NU2058, any groups added to N2 would pro-ject out of the ATP-binding cleft toward the solvent and contactthe ‘specificity surface’ of CDK2 (ref. 11), with consequences forboth potency and selectivity.

Studies with NU2058 derivativesSeveral compounds that pack aromatic moieties with the speci-ficity surface of CDK2 have been developed, such as the pur-valanols7 and the indirubins8,12. Taking this into account, we

initially synthesized 2-anilino-6-cyclohexylmethoxy-purine (NU6094; Fig. 1a), which contains an anilinogroup at the purine C2 position of NU2058. This com-pound has an IC50 of 1.00 ± 0.03 µM for CDK2 and issimilarly active against CDK1 (Table 1), representingover a 10-fold increase in affinity for CDK2 relative tothe parent compound NU2058.

To confirm the binding mode of NU6094 and ratio-nalize the observed increase in affinity, the structure ofNU6094 in complex with T160pCDK2–cyclinA wasdetermined (Fig. 2a). Within the ATP-binding cleft,NU6094 adopts the same binding mode as NU2058. Theanilino group projects out of the adenine site though alargely hydrophobic tunnel constituted by the side chainsof Phe 82 and Ile 10 (Fig. 2b), and packs against thekinase surface, forming a π–π stacking interaction withthe peptide backbone between Asn 85 and Asp 86. Theincrease in potency associated with this additional group

likely arises from desolvation of the hydrophobic plane of theanilino group on binding. Assuming the measured IC50 valuesare proportional to the Kd for the CDK2–inhibitor interaction,the relative free energy difference between NU2058 and NU6094(∆∆G°2058→6094) is ∼ –7 kJ mol–1. The upper limit for the free energy associated with desolvation of hydrophobic groups hasbeen estimated13 to be –136 J mol–1 Å–2. The binding of NU6094buries ∼ 50 Å2 of apolar surface, leading to a calculated free ener-gy change of –6.8 kJ mol–1, which is in good agreement with theexperimentally measured free energy difference.

The planes of the purine and anilino rings are tilted relative toeach other with an interplanar angle of 50°. A search of the

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Table 1 Inhibition of CDK activity and human MCF-7 breast carcinomacell growth by O6-cyclohexylmethylguanine CDK inhibitors

Inhibitor CDK inhibition (µM)1 Cell growth inhibition2

CDK1–cyclin B1 CDK2–cyclin A3 CDK4–cyclin D1 GI50 (µM)3

NU20584 5 ± 15 12 ± 3 >100 32 ± 7NU6094 1.6 ± 0.1 1.00 ± 0.03 16 ± 5 11 ± 2NU6086 2.2 ± 0.4 2.3 ± 0.3 >100 26 ± 1NU6102 0.009 ± 0.001 0.006 ± 0.0005 1.6 ± 1.0 8 ± 1

1Enzyme activities were assayed as described (see Methods), and values in italicsare Ki determinations and in normal text are IC50 results.2Growth inhibition was determined over 48 h exposure.3GI50 is the mean concentration required to inhibit cell growth by 50%.4Inhibitor structures are shown in Fig. 1a.5Data shown are the mean of at least three independent experiments ± standarddeviation.

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Fig. 2 Binding of NU6094, NU6086 and NU6102 to T160pCDK2–cyclinA. a, T160pCDK2–cyclinA–NU6094 structure. Selected CDK2residues are drawn in ball-and-stick representation, with carbonatoms colored green (inhibitor) and yellow (CDK2). The final 2Fo – Fc

electron density contoured at 0.24 e– Å–3 for NU6094 is included.Hydrogen bonds in all panels except (b) are denoted by dashedlines. b, NU6094 bound to the CDK2 active site. NU6094 is depictedas yellow CPK spheres. The CDK2 molecular surface is colored byatom type: carbon, oxygen and nitrogen atoms are green, red andblue, respectively. The figure illustrates the complementarity of theNU6094 anilino ring to the shape of the hydrophobic tunnel lead-ing to the specificity surface. c, T160pCDK2–cyclinA–NU6086 struc-ture. NU6086 and selected CDK2 residues are rendered in ball-andstick-representation, with carbon atoms colored as in (a). Both con-formers of the anilino ring (denoted I and II) are included. The final2Fo – Fc electron density for NU6086 is contoured at 0.24 e– Å–3.d, T160pCDK2–cyclinA–NU6102 structure. NU6102 and selectedCDK2 residues are rendered in ball-and-stick representation, withcarbon atoms colored as in (a). The final 2Fo – Fc electron density forNU6102 is contoured at 0.24 e– Å–3. e, NU6102 bound to the CDK2active site. The CDK2 molecular surface is rendered in transparentgray so that interactions between the NU6102 sulfonamide groupand the backbone nitrogen and side chain oxygen of Asp 86 are visible. Hydrogen bonds are depicted by dotted lines. NU6102 isrendered in ball and stick, with carbon atoms colored green.

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Cambridge Structural Database (CSD) for small molecule crystalstructures14 that contain two aromatic rings coupled by a singlenitrogen showed the modal angle to be ∼ 48°, close to thatobserved for NU6094 bound to CDK2. The binding of NU6094 toCDK2 may, therefore, have the additional advantage that the con-formation of the complex will be relatively strain-free and entrop-ically favored because of pre-organization of the free ligand.

The observation for the purvalanols that CDK2 has a preferencefor chlorine atoms on the specificity surface7 led to the synthesisand testing of 6-cyclohexylmethoxy-2-(3′-chloroanilino)purine(NU6086, Fig. 1a). However, the affinity of NU6086 for bothCDK2 and CDK1 (Table 1) was not improved over NU6094. Thecrystal structure of NU6086 in complex with T160pCDK2–cyclinA (Fig. 2c) revealed a mode of inhibitor binding identical toNU6094. Initial difference density for the chloro-substituted ani-lino group indicated the presence of two partially occupied con-formations. In conformer I, the meta chloro atom forms van derWaals contacts with Phe 82 and His 84, whereas in conformer II itforms a hydrogen bond with the protonated form of Asp 86 (rCl–O = 3.0 Å). The packing and interplanar anilino-purine angleis different in each case, with the interaction between Asp 86 andthe inhibitor causing a 13° rotation of the anilino ring for con-former II relative to conformer I and NU6094.

The development of NU6102The NU6094 and NU6086 results demonstrate that increasedaffinity could be achieved through the addition of an anilinogroup at C2 of NU2058. To further increase the potency of suchcompounds, a sulfonamide group was introduced at the anilinopara position of NU6094 in an attempt to form a hydrogen bondto Lys 89.

O6-cyclohexylmethoxy-2-(4′-sulfamoylanilino)purine (NU6102;Fig. 1a) was synthesized and found to be a highly potent CDKinhibitor, with Ki values of 9 ± 1 nM and 6 ± 0.5 nM for CDK1and 2, respectively (Table 1). The crystal structure ofT160pCDK2–cyclinA in complex with NU6102 reveals the inter-actions formed and provides an explanation for its tight binding.NU6102 adopts an identical binding mode to the other N2-sub-stituted purines discussed above. The O6-cyclohexyl groupsuperimposes closely with both NU6086 and NU6094 in theinhibitor-bound complexes, and the usual triplet of hydrogenbonds are formed between the purine core and the backbone ofresidues Leu 83 and Glu 81 (Fig. 2d). Although the anilino grouppacks closely with the specificity surface, the sulfonamide doesnot form the designed hydrogen bond with Lys 89. Instead, theNH2 group of the sulfonamide donates a hydrogen bond to a sidechain oxygen of Asp 86 (rNH2–O = 2.9 Å), and one sulfonamideoxygen accepts a hydrogen bond from the backbone nitrogen ofAsp 86 (rNH–O = 3.1 Å) (Fig. 2e). The formation of these inter-actions with good geometry leads to rotation of the anilinogroup by 13°, as observed for conformer II of NU6086. Whenthis occurs, the packing between the surface of the protein andthe plane of the anilino ring is enhanced and stabilized. With

NU6102 in essentially its lowest energy conformation, the sul-fonamide group is positioned to form protein–ligand hydrogenbonds with good geometry. Furthermore, this ‘fine-tunes’ theorientation of the anilino group for optimum packing with thekinase, as well as for stabilizing hydrophobic contacts. The resultis a 1,000-fold increase in affinity relative to NU2058 and main-tenance of selectivity for CDK1 and CDK2 over CDK4. To con-firm the selectivity of NU6102, the compound wasindependently tested as an inhibitor of 28 kinases, includingCDK2–cyclin A in the same assay system, at an ATP concentra-tion of 100 µM. The only kinases to be inhibited by NU6102under these conditions with IC50 values <1 µM were CDK2(30 nM), Rho-dependent protein kinase II (ROCKII at 600 nM),phospholipid dependent kinase 1 (PDK1 at 800 nM) and dual-specificity tyrosine phosphorylated and regulated kinase 1A(DYRK1A at 900 nM). Thus, NU6102 has at least 10-fold selec-tivity for the target kinases CDK1 and 2.

Cellular effects of CDK inhibitorsAll compounds inhibited growth of MCF-7 cells in a concentration-dependent manner, with NU6102 being the mostpotent (Table 1). The activities of NU2058 and NU6102 onG1/S-associated CDKs were further examined by investigatingtheir ability to inhibit phosphorylation of downstream CDK tar-get proteins, the retinoblastoma protein (pRb) and CDK1. pRbcontains 16 consensus sites for CDK phosphorylation that havebeen characterized as either specific for CDK4–cyclin D1,CDK2–cyclin E or CDK2–cyclin A, or able to be phosphorylatedby combinations of these kinases15. Both NU2058 and NU6102induced a concentration-dependent decrease in pRb hyperphos-phorylation (Fig. 3a). Furthermore, phosphorylation of pRb atThr 821 (Fig. 3b), a site that is specific for CDK2-mediated phos-phorylation events, was markedly reduced after treatment with

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Fig. 3 Inhibition of CDK2- and CDK1-specific phosphorylation events byNU2058 and NU6102. MCF-7 cells were serum starved for 24 h and thenreleased into medium containing 10% (v/v) serum and NU2058 orNU6102 at appropriate concentrations for 24 h. Cell lysates were ana-lyzed by western blotting for a, retinoblastoma phosphorylation (higher(hyperphosphorylation) and lower (hypophosphorylation) molecularweight pRb species indicated by arrows); b, CDK2-specific pRb phospho-rylation at Thr 821; and c, actin. Cycling MCF-7 cells were treated with0.5 µg ml–1 nocodazole for 24 h in the presence of NU2058 or NU6102,and cell lysates were analyzed for d, CDK1-phosphorylated nucleolin ande, actin. Blots are representative of three independent experiments.

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NU2058 and NU6102 (50% inhibition at 31 ± 7 and 5 ± 2 µM,respectively). Phosphorylation of the CDK1 target protein was measured using the TG3 antibody, which binds toCDK1–cyclin B phosphorylated nucleolin16 (Fig. 3d). Cellsarrested in M-phase by treatment with 0.5 µg ml–1 nocodazolefor 24 h were found to contain high levels of phosphorylatednucleolin, and the presence of NU2058 or NU6102 inhibitednucleolin phosphorylation (50% inhibition at 39 ± 7 and 3 ± 1 µM, respectively) in a concentration-dependent manner.

DiscussionThis paper describes the development of a CDK inhibitor withnanomolar potency using structural information initiallyderived from a complex of the inhibitor NU2058 with fully acti-vated CDK2–cyclin A. An important feature of NU2058 was thatit binds to monomeric CDK2 in a conformation distinct fromATP and other adenine-based CDK inhibitors4. Furthermore,NU2058 has a pattern of activity against a panel of tumor celllines that is distinct from that of the known CDK inhibitorsflavopiridol and olomoucine4.

In the current study, molecules were designed to probe inter-actions with the ‘specificity surface’ on CDK2. Structural analy-ses had indicated that an aromatic ring at the N2 position ofNU2058 would improve inhibitory activity against CDK1 andCDK2. This was found to be the case, with the resulting com-

pound maintaining specificity for the inhibition ofCDK1/2 over CDK4. Additions to the anilino ring atthe meta position, including chlorine (NU6086),bromine, fluorine or methoxy (data not shown), didnot improve or reduced CDK-inhibitory activity andtumor cell growth inhibition. In contrast, substitutionsat the para position, exemplified by addition of a sulfonamide group, substantially improved activityagainst CDK1 and CDK2. Furthermore, excellentspecificity over CDK4 (∼ 1,000-fold) and 27 otherkinases was maintained, and inhibition of tumor cellgrowth was enhanced. The structure of a complex ofNU6102 with T160pCDK2–cyclinA revealed that thetriplet of hydrogen bonds observed in theNU2058–T160pCDK2–cyclinA structure was main-tained, and additional hydrogen bonds involvingAsp 86 were formed. These interactions facilitate opti-mal hydrophobic packing of the anilino group with thespecificity surface of CDK2. Consistent with inhibitionof CDK1 and CDK2, both CDK2-specific pRb phos-phorylation and CDK1-specific nucleolin phosphoryl-ation was inhibited by NU6102. Further modificationof the NU6102 structure is necessary to improve thepharmacological properties of these compounds. Inparticular, despite in vitro Ki values <10 nM againstCDK1 and CDK2, micromolar concentrations arerequired to inhibit cell growth and CDK activity inwhole cells (Table 1; Fig. 3); poor cellular penetrationmay explain this discrepancy.

In summary, this paper describes the prospectiveapplication of structure-based drug design to the devel-opment of inhibitors of CDK2, where a fully activeCDK–cyclin complex has been used. The resultingcompound, NU6102, is selective and one of the mostactive CDK2 inhibitors described so far. The potency ofthe compound has been predicted and is fully rational-ized on the basis of protein–ligand interactions.

MethodsChemistry. The synthesis of the CDK inhibitors used in these stud-ies has been described (NU2058)4 or will be described elsewhere(NU6094, NU6086 and NU6102).

Expression and purification T160pCDK2–cyclinA, and crystal-lization of T160pCDK2–cyclinA–inhibitor complexes. Thr 160-phosphorylated CDK2–cyclin A was purified as described17. Crystalswere obtained by the hanging drop method, using protein at a con-centration of 10 mg ml–1, 5% (v/v) dimethylsulfoxide (DMSO) and ∼ 5 mM inhibitor. The reservoir solution contained 0.8 M KCl and1.2 M (NH4)2SO4 in 40 mM HEPES, pH 7.0, and 5 mM dithiothreitol(DTT). The protein was incubated with inhibitor on ice for 1 h andspun through a 0.22 µM filter before setting up crystallization trials.

X-ray crystallography data collection and processing. Beforedata collection, crystals were briefly immersed in cryo-protectant,which consisted of mother liquor and 24% (v/v) glycerol (NU2058and NU6094 structures) or 8 M sodium formate (NU6086 andNU6102 structures), before flash-freezing using an OxfordCryostream at 100 K. Data were collected using a rotating anodesource and Mar image plate (NU6094) or a MarCCD at ID14-EH1 atthe ESRF (NU2058, NU6086 and NU6102). Data processing was car-ried out using MOSFLM18 and SCALA19.

Structure solution and refinement. The structures were solvedby molecular replacement (MR) using MOLREP18. A high-resolution,well-refined structure of the T160pCDK2–cyclinA complex with

748 nature structural biology • volume 9 number 10 • october 2002

Table 2 X-ray data collection and refinement statistics for the four structures

Data collectionNU2058 NU6094 NU6086 NU6102

Space group C2221 P212121 P212121 P212121

Cell dimensions (Å)a 124.03 73.99 74.11 74.23b 193.83 134.73 134.95 135.35c 157.49 148.12 148.33 148.46

Maximal resolution (Å) 2.1 2.5 2.0 2.0Observations 245,195 137,122 251,715 21,174Unique reflections 110,338 66,509 101,035 101,670Completeness (%) 92.5 94.1 93.9 94.5Rmerge

1 0.083 0.079 0.075 0.079Mean I / σ (I) 5.5 8.2 6.7 5.2Highest resolution bin (Å) 2.15–2.10 2.59–2.50 2.06–2.00 2.04–2.00Completeness (%) 82.7 80.8 75.5 61.2Rmerge

1 0.406 0.378 0.522 0.289Mean I / σ (I) 1.7 1.9 1.4 2.5

RefinementProtein atoms 8,942 8,942 8,942 8,942Other atoms

Inhibitor 37 48 59 56Water 410 535 723 703

Resolution range (Å) 20–2.1 20–2.5 20–2.0 20–2.0Rconv

2 (%) 22.2 23.8 23.5 23.6Rfree

3 (%) 25.8 33.2 29.4 28.6Mean B-factor (Å2)

Protein 44.6 56.6 46.1 39.9Ligand 43.0 52.6 39.1 41.2Solvent 50.5 61.5 50.5 45.1

1Rmerge = ΣhΣj |Ih,j – I-h| / ΣhΣj|Ih,j|, where Ih,j is the jth observation of reflection h.2Rconv = Σh||Fo| – |Fc|| / Σh|Fo|, where Fo and Fc are the observed and calculated structurefactor amplitudes respectively for the reflection h.3Rfree is equivalent to Rconv for a 5% subset of reflections not used in the refinement.

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another inhibitor (J.A.E, L.N.J & M.E.M.N, unpub. data) was used asthe starting model for MR, and a clear solution was obtained withtwo CDK2–cyclin A heterodimers in the asymmetric unit. This modelwas subjected to rigid-body refinement followed by individualatomic refinement using REFMAC20. Strict noncrystallographicrestraints were applied initially and gradually relaxed during refine-ment. Inhibitor models were built using SYBYL (Tripos), and modelbuilding and fitting were carried out in O21. Water molecules wereadded using ARP22. Data collection, processing and refinement sta-tistics are given in Table 2.

Kinase assays. Inhibition of CDK1–cyclin B1 and CDK2–cyclin Awas assayed as described4. Inhibition of CDK4–cyclin D1 was deter-mined in a similar assay using an assay buffer composed of 50 mMHEPES, 10 mM MnCl2, 1 mM DTT, 100 µM sodium fluoride, 100 µMsodium vanadate, 10 mM sodium glycerophosphate, 5 µg ml–1

aprotinin, 2.5 µg ml–1 leupeptin, 100 µM phenylmethylsulfonylflouride (PMSF) and a recombinant retinoblastoma peptide(encoding residues 792–928) as substrate. CDK4–cyclin D1 (giftfrom AstraZeneca Pharmaceuticals) was prepared as a GST-taggedcomplex from baculoviral-infected insect cell lysate. The final ATPconcentration in each assay was 12.5 µM. The IC50 concentration foreach inhibitor is the concentration required to inhibit enzymeactivity by 50% under the assay conditions used. The activity ofNU6102 against 28 kinases, including CDK2–cyclin A, was studied asdescribed23 using an ATP concentration of 100 µM (data obtainedin collaboration with P. Cohen, University of Dundee, UK).

MCF-7 cell growth inhibition. The effects of compounds on thegrowth of MCF-7 human breast carcinoma cells was investigated.Cells were plated at a density of 1 × 103 per well in 96-well platescontaining RPMI medium (supplemented with 10% (v/v) fetal calfserum) (Gibco), allowed to grow for 72 h and then treated with arange of inhibitor concentrations (1–100 µm) for 48 h. Cell growth,relative to control cells treated with 1% (v/v) DMSO, was measuredusing the cell proliferation assay kit (Boehringer).

Western blotting. Cells were lysed (20% (v/v) glycerol, 4% (w/v)SDS and 100 mM Tris-HCl, pH 6.8) and heated to 85 °C for 5 min.Following addition of equal volume of sample buffer (0.001% (w/v)bromophenol blue and 100 mM DTT), 20 µg protein was resolved on3–8% polyacrylamide Tris-acetate (pRb) and 4–20% (nucleolin)NUPAGE gels (Invitrogen) and blotted onto Hybond ECL nitro-cellulose membrane (Pharmacia) using NUPAGE transfer buffer.Blots were blocked in TTBS (20 mM Tris, 140 mM NaCl and 0.1 %(v/v) Tween 20, pH 7.6) containing 5 % (w/v) dried milk for 1 h andincubated overnight in primary antibody (either retinoblastoma-specific (1:250, sc-50 Santa Cruz), retinoblastoma-phosphospecific(1:1000, pT821 Biosource International) or nucleolin-specific (1:500,TG3 a generous gift from P. Davies, Albert Einstein College ofMedicine, New York)). Labeled proteins were detected usingSupersignal West Dura extended duration ECL substrate (Pierce).

Coordinates. Coordinates and structure factors for the four struc-tures (NU2058, NU6094, NU6086 and NU6102) have been depositedwith the Protein Data Bank (accession codes 1H1P, 1H1Q, 1H1R and1H1S, respectively).

AcknowledgmentsWe would like to thank T. Hunt for his gift of the human CDK2 and cyclin A3cDNAs and C. Man for the Saccharomyces cerevisiae CIV1 sequence. We thankN. Hanlon, N. Brown and D. Barford for the development of the CDK2-CIV1 co-expression strategy. We also thank the beamline staff at ESRF, Grenoble,France, who provided excellent facilities and advice during data collection. We wish to acknowledge the use of the EPSRC’s Database Service at Daresbury.At the LMB, the authors would like to thank I. Taylor, E. Garman, A. Lawrie, R. Bryan, Y. Huang, K. Measures and S. Lee, and L. Meijer at CNRS StationBiologique, Roscoff, for advice on the nucleolin western blotting procedure. This research was supported by grants from Cancer Research UK, The RoyalSociety UK, Medical Research Council UK, BBSRC and AstraZeneca PLC UK.

Competing interests statementThe authors declare that they have no competing financial interests.

Correpsondence should be addressed to D.R.N. email: herbie.newell@ncl.ac.uk

Received 25 April, 2002; accepted 5 August, 2002.

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