Báo cáo khoa học: Alternative binding modes of an inhibitor to two different kinases doc

Alternative binding modes of an inhibitor to two different kinases Erika De Moliner*, Nick R. Brown and Louise N. Johnson Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7- tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC 50 1.6 l M )(Sarnoet al. (2001) FEBS Letts. 496, 44–48). Three other kinases [cyclin- dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3b] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2– cyclin A at 2.2 A ˚ resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals con- tacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases. Keywords: protein kinase inhibitors; cyclin-dependent protein kinase 2; CK2; tetrabromobenzotriazole. Protein kinases catalyze critical post-translational phos- phorylations of proteins in almost all intracellular signaling pathways. Protein kinases have become popular targets for inhibitors and there have been dramatic successes in a few cases in the design of specific inhibitors that have found effective clinical application [1] or as tools for probing signaling pathways [2,3]. Almost all available protein kinase inhibitors target the ATP substrate binding site. This buried nonpolar cavity, which contains specific hydrogen-bonding groups, provides a ready recognition site for the design of a whole variety of different compounds [4]. Many of these compounds are now undergoing clinical trials [5]. The key recognition features of the ATP binding site are conserved in all the 518 putative protein kinases in the human genome [6]. Nevertheless small differences in the constellation of residues adjacent to the site have allowed, through chemical screening and structure-based methods, high affinity com- pounds to be developed that are selective for just a few kinases [7]. The selectivity of kinase inhibitors is a key feature for their success either in the clinic or in the laboratory. Several inhibitors are less specific than originally envisaged and may target other kinases with similar affinities [8]. Thus it is useful to be able to predict the likely interactions of an inhibitor designed to target one particular kinase with other kinases. It is generally assumed that the mode of binding of an inhibitor to one kinase is likely to be similar to the mode of binding to other kinases. For example, staurosporine, a nonselective kinase inhibitor with nanomolar K i for many protein kinases, has been shown to bind in almost identical modes to four different kinases [cyclin-dependent protein kinase 2 (CDK2) [9], cyclic AMP-dependent protein kinase (PKA) [10], C-terminal Src kinase (CSK) [11] and leukocyte specific kinase (LCK) [12]]. Critical interactions that involve two specific hydrogen bonds and extensive non-polar interactions are very similar in each of the kinase structures solved [13]. Results reported in this manuscript indicate that similar binding of a compound to different kinases cannot always be assumed. 4,5,6,7-Tetrabromobenzotriazole (TBB) belongs to a class of compounds related to the commercially available 1-(b- D -ribofuranosyl)-5,6-dichlorobenzimidazole (DRB). TBB, like DRB, was found to inhibit kinases, but exhibited a greater specificity than DRB. Investigation of the inhi- bitory power of TBB with a panel of 33 protein kinases showed highest potency for CK2 (originally called casein Correspondence to L. N. Johnson, Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Rex Richards Building, Oxford OX1 3QU, UK. Fax: 01865 285353, Tel.: 01865 275365, E-mail: louise@biop.ox.ac.uk Abbreviations: CDK2, cyclin-dependent protein kinase 2; CK2, casein kinase 2; GSK, glycogen synthase kinase; pCDK2–cyclin A, phosphoThr160-CDK2–cyclin A complex; PhK, phosphorylase kinase; PKA, cyclic AMP-dependent protein kinase; TBB, 4,5,6,7- tetrabromobenzotriazole. Note: a web site is available at http://www.biop.ox.ac.uk *On leave of absence from: Department of Organic Chemistry and CNR Biopolymer Research Center, University of Padova, Via Marzolo 1, 35131 Padova, Italy. (Received 26 April 2003, revised 29 May 2003, accepted 2 June 2003) Eur. J. Biochem. 270, 3174–3181 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03697.x kinase 2) (human CK2: IC 50 ¼ 1.6 l M at 100 l M ATP) [14]. TBB also inhibited three other kinases with less potency: CDK2 (IC 50 ¼ 15.6 l M ), phosphorylase kinase (IC 50 ¼ 8.7 l M ) and glycogen synthase kinase 3b (GSK3b) (IC 50 ¼ 11.2 l M ). All other kinases tested had IC 50 values 50-fold greater than that for CK2. CK2 is very pleiotropic with >300 different substrates known [15,16]. Several of these proteins are implicated in cellular functions such as signal transduction, gene expression and control of the circadian rhythm [17]. The kinase is constitutively active and appears to lack the strict regulation that is a significant feature of most other kinases. CK2 has a possible role in oncogenic events and exploitation by viruses [16] and hence is a target for drug design. The structure of CK2 in complex with TBB has defined the important interactions made by this compound at the ATP binding site [18]. Cyclin-dependent kinase 2 (CDK2) plays a key role in the regulation of the eukaryotic cell cycle. Through phosphory- lation of selected target proteins, CDK2 in association with cyclin E promotes the transition between G1 and S phase, and in association with cyclin A promotes progression through and exit from S phase. For full activity, CDK2 requires phosphorylation of a threonine residue in the activation segment of the kinase (Thr160) in addition to association with a cyclin molecule. The structural basis of CDK2 activation mechanisms [19], substrate specificity [20,21] and small molecule inhibitor recognition [22,23] are well understood. CDK2 belongs to the same CMGC family [6] of protein kinases as CK2 and has 33% identity in amino acid sequence. The interaction site on the kinase domain of CDK2 with cyclin A is mimicked in CK2a by interaction of the N-terminal region with the CK2 core structure [24]. We have determined the structure of the phospho-CDK2–cyclin A complex with TBB at 2.2-A ˚ resolution. It is found that although TBB binds at the ATP recognition site of CDK2, it adopts a different orientation and makes different interactions from those made with CK2. The differences arise from the rather broad specificity of the bromine atoms but they also involve contacts to the nitrogens of TBB. The results indicate that similar binding modes of an inhibitor to different kinases cannot be assumed. Materials and methods Crystal preparation and data collection Human pCDK2–cyclin A, the fully active form of CDK2 in which Thr160 is phosphorylated, was prepared as previ- ously described [20]. TBB was gift from D. Shugar (University of Warsaw, Poland). TBB was dissolved in dimethylsulfoxide to obtain a 100-m M stock solution and was co-crystallized with pCDK2–cyclin A using the sitting drop vapor diffusion method. A solution containing 1 lL of pCDK2–cyclin A (in 10 m M Hepes pH 7.4, 150 m M NaCl, 2 m M EDTA, 0.01% azide, 0.01% MTG) at a concentration of 10 mgÆmL )1 was preincubated with 0.5 m M TBB and mixed with an equal volume of precipitant solution containing 1.25 M (NH 4 ) 2 SO 4 ,0.85 M KCl, 100 m M Hepes, pH 7. Crystals grew in 1 week at a temperature of 277 K. Before mounting, crystals were soaked for <1 s in 8 M sodium formate cryoprotectant solution. Crystallographic data for the pCDK2–cyclin A–TBB complex were collected on beam line ID14 EH1 at ESRF, Grenoble to 2.22 A ˚ resolution with wavelength 0.934 A ˚ and temperature 100 K, The space group of the crystals is P2 1 2 1 2 1 , with two molecules in the asymmetric unit and cell parameters a ¼ 73.54 A ˚ ,b ¼ 133.95 A ˚ ,c ¼ 148.42 A ˚ .The Matthews coefficient for two molecules of pCDK2–cyclin A per asymmetric unit is V M is 3.00 A ˚ 3 ÆDa )1 1 giving a solventcontentof57.4%. Structure determination and refinement Data were processed with MOSFLM [25], SCALA and other programs in the CCP4 suite [26]. The structure was solved by molecular replacement with MOLREP [27,28] using as a starting model the coordinates of a 2.3-A ˚ resolution pCDK2–cyclin A–inhibitor complex from which the inhi- bitor and water molecules had been removed [7]. After rigid body refinement, SIGMA - A weighted [29] |2Fo-Fc| and |Fo-Fc| maps were calculated and showed clear electron density for the inhibitor TBB in the ATP binding site pocket. The ligand was added and the structure was refined using alternating cycles of maximum likelihood refinement (CNS suite [30]) and manual rebuilding [ QUANTA Ó (Version 98.1111) and O [31]]. Waters were added in the last cycles of refinement. The results are summarized in Table 1. The crystals contain two pCDK2–cyclin A molecules per asymmetric unit with the molecules of the A (pCDK2) and B (cyclin A) chains better ordered than those of the C (pCDK2) and D (cyclin A) chains as judged from the temperature factors (Table 1). There are no significant structural differences between the two complexes. The two complexes superimpose with a root mean squared difference in Ca coordinates of 0.7 A ˚ with the greatest differences occurring in the flexible loop regions. The loop residues Table 1. Summary of data collection and refinement statistics for the pCDK2–cyclin A–TBB complex. Numbers in brackets refer to the highest resolution range. Data collection Maximum resolution (A ˚ ) 2.22 (2.30) Independent reflections 72014 (6225) Multiplicity 2.8 (2.2) I/r? 5 10.9 (3.2) R merge 0.050 (0.232) Completeness (%) 98.5 (98.5) Refinement Reflections used in refinement 67955 Protein atoms (+TBB) 8938 (+26 TBB) Solvent molecules 242 R/R free 21.9/25.7 Root mean square on bonds distances (A ˚ ) 0.008 Root mean square on bond angles (°) 1.50 Mean protein B factors (A ˚ 2 ) Chain A 29.3, chain B 30.1, chain C 48.3, chain D 43.1 Mean TBB B factors (A ˚ 2 ) Chain E 63.6, chain F 77.9 Ó FEBS 2003 Dual binding modes of a protein kinase inhibitor (Eur. J. Biochem. 270) 3175 220–240 of the C molecule of pCDK2 has poorly defined electron density, because of lattice contacts with the C-terminal region of the A molecule of pCDK2. We use chain A (pCDK2) for reference in describing the structures. Checks on the stereochemistry of the pCDK2–cyclin A complex with PROCHECK [32] indicated that 96% of residues were in the allowed or additionally allowed regions of the Ramachandran plot. The pCDK2 residue Val164 in both molecules is outside the allowed region. This residue has well-defined electron density and its unusual conformation is stabilized by a contact to an arginine residue, Arg169. The two TBB molecules bind in similar manner to each of the CDK2 subunits making equivalent contacts. Coordinates Coordinates have been deposited in the Protein Data Bank (1P5E). Results Interactions between TBB and pCDK2–cyclin A Phospho-CDK2–cyclin A (pCDK2–cyclin A) exhibits a typical kinase fold comprised of an N-terminal and a C-terminal lobe. The N-terminal lobe is composed of 5 antiparallel b-strands and one a helix (the C helix). The glycine rich loop is located in the loop between strands b1 and b2 and is flexible. The C-terminal lobe is mainly a helical and is connected to the N-terminal domain by the hinge region. The C-terminal lobe includes the activation segment, the stretch of chain that runs between the conserved DFG and APE motifs in protein kinases and which carries the phosphorylated threonine residue, pThr160. The ATP substrate-binding site is located between the two lobes. The adenine moiety of ATP makes two crucial hydrogen bonds to main chain atoms of the hinge region. The N1 atom of the adenine hydrogen bonds to the main chain nitrogen of Leu83 while the N6 group hydrogen bonds to the main chain oxygen of residue Glu81. The crystal structure of pCDK2–cyclin A in complex with TBB solved at 2.2 A ˚ resolution was refined to final crystallographic R and R free values of 21.9 and 25.7%, respectively (Table 1). TBB (Fig. 1A) binds in the region of the ATP binding site (Fig. 1B). The overall structure of the pCDK2–cyclin A–TBB complex is similar to the pCDK2– cyclin A–ATP structure [33] with no major rearrangements of structural elements. The root mean squared difference in Ca positions measured with O [31] is 0.66 A ˚ for the full- length CDK2 and 0.96 A ˚ for the N-terminal lobes. There are small shifts in the side chains of residues Val18, Val64 and Phe80 in order to accommodate TBB. The side chains of Lys89 and Gln131 assume different conformations in the TBB bound pCDK2–cyclin A complex, although only Lys89 makes contact to TBB through a water molecule. These external residues participate in inhibitor binding for certain high affinity CDK2 inhibitors [7] and conforma- tional changes are also seen in these inhibitor complexes. The glycine-rich loop maintains almost the same confor- mation in the ATP and TBB complexes, despite the flexibility in this region. Interactions between TBB and CDK2 include both polar and non-polar interactions (Fig. 2A and Table 2). Two of the bromine atoms, Br5 and Br6, interact with electroneg- ative atoms of the protein backbone in the hinge region, namely the carbonyl oxygen atoms of Glu81 and Leu83, respectively. Further contributions to binding are made by polar contacts between Br7 through water to the NZ atom of Lys89, following the conformational change of this side chain, and between Br4 through water to the main chain nitrogen of Asp145. The nitrogen N3 of the triazole ring hydrogen bonds through water to the NZ of Lys33. Two of these hydrogen-bonding residues (Asp145 and Lys33) are conserved in eukaryotic protein kinases and are important Fig. 1. TBB binding to pCDK2–cyclin A. (A) Schematic representation of the structure of pCDK2 (yellow) and cyclin A (magenta) in complex with TBB (carbon atoms, green; nitrogen atoms, blue; and bromine atoms, cyan). TBB binds at the ATP binding site in the region between theN-andC-terminallobesandmakescontactswithresiduesinthe hinge region. (B) Details of TBB fit to the final SIGMAA weighted 2Fo-Fc electron density map. The map is contoured at levels corres- ponding to 1 r (blue contours) and 4 r (red contours). The position of ATP is shown superimposed (carbon atoms: black). These figures and those in Fig. 2 were prepared with AESOP (M.E.M.Noble,unpub- lished work). 3176 E. De Moliner et al. (Eur. J. Biochem. 270) Ó FEBS 2003 for ATP recognition. TBB binding to pCDK2–cyclin A exploits many van der Waals contacts with hydrophobic side chains from residues of the ATP binding site that include Ile10, Val18, Ala31, Val64, Phe80, Phe82, Leu83, and Leu134 (Fig. 2a and Table 2). TBB fits neatly into this hydrophobic pocket. Each bromine atom makes five to 10 van der Waals contacts. Br4 contacts the p electrons from the aromatic ring of Phe80. Comparison of pCDK2–cyclin A–TBB complex with CK2–TBB complex Phospho-CDK2 and CK2 exhibit similar protein kinase folds. Superposition of the catalytic domains of the human pCDK2–cyclin A–TBB complex and the Zea mays CK2– TBB complex [18] gives an rms difference in Ca positions of 1.4 A ˚ for the full length kinase domains and 1.6 A ˚ for the N-terminal lobes. Neither of the two kinases exhibits major changes in protein structure on TBB binding. However the inhibitor TBB occupies significantly different orientations at the ATP binding sites in the two complexes (Fig. 2A,B). The planes of the aromatic rings are parallel but the ring of TBB in the CDK2 structure is 0.7 A ˚ higher (i.e. towards the N-terminal lobe) than in the CK2 structure. Within the plane of TBB, there is a relative rotation of about 30° and a shift of about 1.4 A ˚ (Fig. 2C). CDK2’s TBB bromine atoms are located deeper in the ATP binding site compared with CK2. In CK2 two of TBB bromine atoms (Br6 and Br7) protrude towards the exit of the cavity. In CK2, TBB makes van der Waals contacts to residues Val45 (Ile10), Arg47 (Glu12), Val53 (Val18), Ile66 (Ala31), Lys68 (Lys33), Val95 (Val64), Val116 (Leu83), His160 (Gln131), Met163 (Leu134), Ile174 (Ala144) and Asp175 (Asp145), where the corresponding residues in CDK2 are given in brackets (Fig. 2B and Table 2). Despite the shift towards the exit of the cavity, the nonpolar contacts from CK2 to TBB are slightly more numerous (73 contacts) than those of CDK2 to TBB (52 contacts). The closer fit of TBB to the ATP site of CK2 is also demonstrated by the molecular surfaces that are buried. In CDK2, 257 A ˚ 2 on CDK2 and 187 A ˚ 2 of TBB Fig. 2. Details of the interactions of TBB with pCDK2 and CK2. Polar contacts to the bro- mine atoms and hydrogen bonds from nitro- gen atoms are shown as black dashed lines. (A) Stereo diagram of TBB bound to pCDK2 (pCDK2 carbon atoms are yellow, TBB car- bon atoms are green, TBB bromine atoms are cyan). Ala144 is shown for reference although it does not make any van der Waals inter- actions with TBB. (B) Stereo diagram of TBB bound to CK2 (CK2 carbon atoms of residues in contact with TBB are orange, CK2 hinge region carbon atoms, which do not contact TBB, are white, TBB carbon atoms are dark green, TBB bromine atoms are magenta). (C) Superposition of TBB bound to pCDK2 (carbon atoms, green; bromine atoms, cyan) and TBB bound to CK2 (carbon atoms, dark green; bromine atoms, magenta). There is a shiftofabout2.5A ˚ and a rotation of about 30° between the two TBB molecules. Ó FEBS 2003 Dual binding modes of a protein kinase inhibitor (Eur. J. Biochem. 270) 3177 become buried on forming the complex and give a total molecular surface buried of 444 A ˚ 2 .InCK2,277A ˚ 2 on CK2 and 199 A ˚ 2 of TBB become buried on forming the complex and give a total of 476 A ˚ 2 . TBB is about 96% buried in CDK2 but is 100% buried in CK2. In the CK2–TBB complex, each bromine atom makes between four and seven van der Waals contacts and two bromine atoms also make polar contacts. One bromine atom, Br7, contacts the NE of Arg47 (distance 3.0 A ˚ ). A second bromine, Br4, contacts the main chain nitrogen of Asp175 (Asp145) though a water molecule, a similar interactiontothatmadebyTBBBr4inCDK2.The nitrogen N2 of the triazole ring hydrogen bonds to Lys68 and the nitrogen N3 to a water molecule, which in turn hydrogen bonds to a second water but the distances are just too large to make contact to the NZ of Lys68 (Lys33 in CDK2). Sequence and structural differences between CDK2 and Ck2 result in differences in the residues located immediately above and beneath the plane of the inhibitor in the two TBB complexes. Above the plane, Val53 of CK2 is in a lower position with respect to the equivalent residue Val18 of CDK2. The more bulky residue Ile66 in CK2 replaces Ala31 in CDK2. Below the plane, the more bulky residue Ile174 in CK2 replaces Ala144 in CDK2 (Fig. 2A,B). The result of these sequence changes is that the space available for inhibitor binding in CDK2 is greater than in CK2. This allows TBB in CDK2 to penetrate deeper into the cavity and to establish the interactions with the hinge region. The substitutions of Ala31 and Ala144 in CDK2 by Ile66 and Ile174 in CK2, respectively, appear to be the major changes that encourage TBB to different positions in CDK2 and CK2. If TBB is placed in its position as in CK2 in CDK2 there are no bad contacts but many fewer van der Waals contacts. So it seems that the CK2 position of TBB is not excluded in CDK2 but that the considerably fewer contacts (because of CDKs wider site) results in absence of binding at this site and preference for the experimentally observed site in CDK2 which is more buried and where more contacts are made. If TBB is placed in its position as in CDK2 in CK2, there are a few short contacts ( 3A ˚ ) between two nitrogens of the triazole and Val53 side chain and no hydrogen bonds, but otherwise the contacts are acceptable. Again it seems that the major incentive for the alternative mode of binding is in finding more effective interactions rather than exclusion from the other site. Comparison of ATP binding site between CDK2 and CK2 CK2 is unusual in that it can utilize both ATP and GTP. Comparison of the nucleotide binding sites in CK2 [34] shows that the region that follows the hinge is largely responsible for allowing utilization of ATP and GTP in CK2. In CK2, the region from the hinge to the end of the aD helix has fewer residues (13 residues from 117 to 128) compared with CDK2 (16 residues from 85 to 99). This opens up a pocket that can be accommodated by the guanine base on binding GTP to CK2. However these differences at the nucleotide-binding site do not affect the TBB binding site. Comparison ATP binding modes in CDK2 and in CK2 show that the binding is very similar. There is a small shift between the two adenine moieties Table 2. Interactions between TBB and pCDK2 and CK2. TBB atom Polar contacts (<3.4 A ˚ ) Van der Waals contacts (<4.4 A ˚ ) pCDK2 contacts CK2 contacts pCDK2 contacts CK2 contacts Residue atom Distance (A ˚ ) Residue atom Distance (A ˚ ) TBB atom Residue (number of contacts) Residue (number of contacts) Br4 Wat2 OH2 3.0 Wat1 OH2 3.4 Br4 Val64(1), Phe80(7) Ile66(3), Val95(1), Ile174(2) Br5 Glu81 O 3.0 Br5 Ala31(1), Phe80(1), Glu81(2), Phe82(1), Leu83(4), Leu134(1) Ile66(3), Val116(1) Br6 Leu83 O 3.0 Br6 Ile10(1), Phe82(2), Leu83(5), Leu134(1) Val45(2), His160(2), Met163(1) Br7 Wat43 OH2 Ile10 O 3.1 3.2 Arg47 NE 3.0 Br7 Ile10(5) Gly46(1), Arg47(6), Val53(2), His160(2) N2 Lys68 NZ 3.3 N1 Val18(3) Val53(1), Lys68(3), Asp175(3) N3 Wat241 OH2 2.5 Wat1 OH2 2.7 N2 Val18(2) Val53(1), Lys68(3), Asp175(6) N3 Val18(1) Lys68(3), Asp175(3) C2 Val18(3) Val53(3), Ile174(1) C3 Val18(2) Val53(2), Ile174(1) C4 Leu134(1) Val53(1), Ile66(1), Ile174(2) C5 Ala31(1), Leu134(1) Val53(1), Ile66(1), Ile174(1) C6 Ile10(1), Leu134(1), Ala31(1) Val53(3), Ile174(1) C7 Ile10(1), Val18(1), Leu134(1) Val53(3), His160(1), Ile174(1) 6 Total 52 73 3178 E. De Moliner et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (about 1 A ˚ ) which tracks the similar displacement of the two hinge regions but the contacts between the adenines and the hinge regions are the same in both CDK2 and CK2. Thus although there are structural and sequence differences at the ATP binding sites between CDK2 and CK2, these differences do not lead to significantly different binding modes of ATP in these kinases. Discussion The conservative character of the ATP binding site has been considered a drawback in the design of selective kinase drugs. The results presented here show that the conserved ATP binding pockets of two different kinases can bind the same inhibitor in different ways by exploiting different features despite structure homology. These observations could exacerbate the problem in the design of selective kinase inhibitors. Comparison of TBB binding to pCDK2 and to Zea mays CK2 shows positional differences of up to 2.5 A ˚ and a difference in rotation of 30°. As a consequence the interactions of TBB with pCDK2 and CK2 are different. In CDK2 TBB binds deeper and the bromine atoms contact the carbonyl oxygens of main chain atoms in the hinge region between the N- and C-terminal lobes. In CK2 the inhibitor is displaced towards the exit of the cavity. In CDK2 TBB makes more polar contacts but fewer nonpolar contacts than in binding to CK2. TBB is selective for CK2. Sarno et al. [14] measured an IC 50 of 1.6 l M for TBB inhibition of human CK2. The IC 50 of TBB for Zea mays CK2 has not been reported but comparison of human and Zea mays CK2 structures [35] shows that in the vicinity of the TBB and ATP binding sites all residues are identical. The rms difference in Ca atoms for the N-terminal lobes is 0.5 A ˚ . It is reasonable to assume that the IC 50 for TBB inhibition of Mays CK2 is similar to that of human. The IC 50 value for TBB inhibition of CDK2 is 15.6 l M , indicating a 10-fold lower affinity, assuming that the kinetic constant can be equated approximately with the binding constant. This is not a large difference in affinity. It corresponds to a difference in free energy of only 5.5 kJÆmol )1 , but in terms of drug design such a difference could be significant in allowing moderation of doses to target one kinase and not other kinases. We note that the shape of CK2 ATP binding cavity is smaller than that of CDK2, largely because of substitutions of two alanine residues for isoleucine (Ala31 and Ala144 in CDK2 are replaced by Ile66 and Ile174 in CK2) and that its shape, as measured by buried molecular surfaces, is just slightly more complementary to TBB in CK2 than in CDK2 (a total of molecular surface area of 476 A ˚ 2 is buried on binding TBB to CK2 compared with a total of 444 A ˚ 2 on binding to pCDK2). When tested against a panel of 33 kinases [14], TBB exhibited inhibitory properties against only two other kinases, PhK and GSK, with IC 50 values of 8.7 and 11.2 l M , respectively. Although the structures of both these kinases are known, the unexpected result with CDK2, which shows that TBB can bind in different modes to different kinases, indicates that we should be cautious in attempts to predict or rationalize the inhibitory properties against these kinases. Other kinases such as CK1, Chk1, PKA, PDK1, PKCa,PKBa, CDK1–cyclin B showed negligible inhibition at 10 l M TBB. It is interesting that on binding to CDK2, TBB is able to exploit polar interactions between two bromines and the main chain electronegative carbonyl oxygens in the hinge region (residues Glu81 and Val83). A search through the IsoStar database [36] shows that polar interactions of aromatic Br atoms are relatively uncommon. However, it has been observed [37] that carbon-bonded halogens (with the exception of fluorine) can make contacts with electro- negative atoms such as oxygen, nitrogen and sulfur and that the contact distance can be smaller than the sum of van der Waals radii in the direction of the bond connecting the C atom and the halogen. This has been explained in terms of an anisotropic electron distribution of the halogen atoms, which results in a decreased repulsive wall and an increase in the electrostatic attraction in the direction of the carbon– halogen bonds. This sort of interaction is weaker than a hydrogen bond, being in the range of 8 kJÆmol )1 .Inastudy of the binding of bromophenols to transthyretin [38] it was observed that one bromine atom in the pentabromophenol complex contacted only waters in a hydrogen bonding network while the other bromines made largely nonpolar contacts. The planar nature of the pentabromophenol structure meant that only two of the five bromine atoms could occupy the halogen sites that are recognized by the natural ligand, thyroxine. The other bromine atoms of pentabromophenol occupied different sites than those occupied by the iodines of thyroxine. A dual binding mode was observed with pentabromophenol in which the bromine contacting the polar groups remained constant but there was a 90° rotation of the aromatic ring that placed the other four bromines at different sites. It appears that while some bromine recognition sites have sufficient features to direct specificity, other sites that employ mostly hydrophobic contacts have weak specificity. Thus bromine can be accommodated in several different pockets and this appears to be the situation that accounts for the differential binding of TBB to pCDK2 and CK2. CDK2 is a frequent target for specific inhibition. Over 30 CDK2–inhibitor complexes have been elucidated by struc- tural studies [23] and many more structures are unpublished. Among the first compounds studied were substituted purines. Compounds such as olomoucine [39], roscovitine [40], purvalanol [41] and H717 [42] bound at the ATP binding site with the purine ring overlapping the site occupied by the adenine of ATP but with the purine in a quite different orientation, a result that could be rationalized by the structures. The bulky substituents on these com- pounds occupied different pockets as demanded by their geometry and accounted for differences in potency. A further compound, isopentenyladenine [43], adopted yet a third different orientation for its purine (i.e. different from the ATP and olomoucine-like binding modes) but one which is similar to a series of guanine substituted com- pounds [7]. Also reviewed in [22,23], these results show that different but related compounds bind to the same enzyme, namely CDK2, in different binding modes depending on the substituents. In our current work we have addressed the complementary problem, namely does the same compound bind to related enzymes in a similar binding mode? There are many instances where a high affinity ligand (e.g. staurosporine) has been observed to bind in a similar mode to different kinases. Indeed the observation of the Ó FEBS 2003 Dual binding modes of a protein kinase inhibitor (Eur. J. Biochem. 270) 3179 crystal structure of the radio-sensitizing drug UCN-01 (7-hydroxystaurosporine) bound to pCDK2–cyclin A [44] allowed a prediction of how UCN-01 might bind with higher affinity to Chk1 kinase, the likely natural target for anticancer action. The prediction was borne out by structural studies with Chk1 [45]. On the other hand the analysis of the FAD-containing proteins [46] has revealed that no single ÔpharmacophoreÕ exists for binding FAD, although most exhibit a conserved site for pyrophosphate recognition. In a further extreme example, the compound flavopiridol, which is in phase II trials as an anticancer drug, binds quite differently to CDK2 [47] and to glycogen phosphorylase [48]. While it should not surprise us that a ligand might bind differently to nonhomologous enzymes, the present work indicates that different binding modes can be encountered in the binding of a ligand to homologous enzymes. Thus extrapolation of results from one system to another system might be misleading. This seems more likely to arise when the IC 50 valuesaremorethan1l M ,asinthe present example, than in instances such as in the stauro- sporine recognition by protein kinases where IC 50 values are less than 100 n M . 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