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

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 co

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 (IC501.6 lM) (Sarno et 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 Kifor 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)

kinase 2) (human CK2: IC50¼ 1.6 lM at 100 lM ATP)

[14] TBB also inhibited three other kinases with less

potency: CDK2 (IC50¼ 15.6 lM), phosphorylase kinase

(IC50¼ 8.7 lM) and glycogen synthase kinase 3b (GSK3b)

(IC50¼ 11.2 lM) All other kinases tested had IC50values

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-mM 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 mMHepes pH 7.4, 150 mMNaCl, 2 mMEDTA, 0.01%

azide, 0.01% MTG) at a concentration of 10 mgÆmL)1

was preincubated with 0.5mM TBB and mixed with an

equal volume of precipitant solution containing 1.25M

(NH4)2SO4, 0.85M KCl, 100 mM 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 P212121, with two molecules in the asymmetric unit and cell parameters a¼ 73.54 A˚, b ¼ 133.95A˚, c ¼ 148.42 A˚ The Matthews coefficient for two molecules of pCDK2–cyclin

A per asymmetric unit is VM is 3.00 A˚3ÆDa)1

solvent content of 5 7.4%

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 withMOLREP [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-Aweighted [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
(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)

I/r?

Refinement Reflections used in refinement 67955 Protein atoms (+TBB) 8938 (+26 TBB)

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

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 withPROCHECK[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 Rfree 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, Br5and 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 (Asp145and 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 the N- and C-terminal lobes and makes contacts with residues in the 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).

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˚2on CDK2 and 187 A˚2of 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

shift of about 2.5 A˚ and a rotation of about

30 between the two TBB molecules.

become buried on forming the complex and give a total molecular surface buried of 444 A˚2 In CK2, 277 A˚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 interaction to that made by TBB Br4 in CDK2 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 ( 3 A˚) 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 85to 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

˚ )

˚ )

(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

2.5A˚ 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

IC50of 1.6 lMfor TBB inhibition of human CK2 The IC50

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 0.5A˚ It is reasonable to assume that

the IC50for TBB inhibition of Mays CK2 is similar to that

of human The IC50value for TBB inhibition of CDK2 is

15.6 lM, 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˚2is 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 IC50 values of 8.7 and

11.2 lM, 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 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 In a study

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

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 pharmacophore 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 IC50values are more than 1 lM, as in the

present example, than in instances such as in the

stauro-sporine recognition by protein kinases where IC50 values

are less than 100 nM

Acknowledgements

We are grateful to staff at the ESRF, Grenoble, France, for help during

measurements at the diffraction beam-line, to T G Davies for

providing the coordinates of the CDK2 complex before publication and

to David Shugar for providing TBB We thank the Medical Research

Council for financial support.

References

1 Druker, B.J (2002) Perspectives on the development of a

mole-cularly targeted agent Cancer Cell 1, 31–36.

2 Lee, J.C., Laydon, J.T., McDonnell, P.C., Gallagher, T.F.,

Kumar, S., Green, D., McNulty, D., Blumenthal, M.J., Heys,

J.R., Landvatter, S.W et al.

2 (1994) A protein kinase involved in

the regulation of inflammatory cytokine biosynthesis Nature 372,

739–746.

3 Cuanda, A & Alessi, D.R (2000) Use of kinase inhibitors to

detect signalling pathways Methods Mol.Biol.99, 161–175.

4 Garcia-Echeverria, C., Traxler, P & Evans, D.B (2000) ATP

site-directed competitive and irreversible inhibitors of protein kinases.

Med.Res.Rev.20, 28–57.

5 Cohen, P (2002) Protein kinases – the major drug targets of the

twenty-first century? Nat.Rev.Drug Disc.1, 309–315.

6 Manning, G., Whyte, D.B., Martinez, R., Hunter, T &

Sudarsanan, S (2002) The protein kinase complement of the

hu-man genome Science 298, 1912–1934.

7 Davies, T.G., Bentley, J., Arris, C.E., Boyle, F.T., Curtin, N.J.,

Endicott, J.A., Gibson, A.E., Golding, B.T., Griffin, R.J.,

Hard-castle, I.R., Jewsbury, P., Johnson, L.N., Mesguiche, V., Newell,

D.R., Noble, M.E.M., Tucker, J.A., Wang, L & Whitfield, H.J.

(2002) Structure-based design of a potent purine-based cyclin

dependent kinase inhibitor Nat.Struct.Biology 9, 745–749.

8 Davies, S.P., Reddy, H., Caivano, M & Cohen, P (2000)

Speci-ficity and mechanism of action of some commonly used protein

kinase inhibitors Biochem.J.351, 95–105.

9 Lawrie, A.M., Noble, M.E.M., Tunnah, P.R., Brown, N.R.,

Johnson, L.N & Endicott, J.A (1997) Protein kinase inhibition by

staurosporine: details of the molecular interaction determined by

X-ray crystallographic analysis of a CDK2-staurosporine

com-plex Nat.Struct.Biol.4, 796–801.

10 Prade, L., Engh, R.A., Girod, A., Kinzel, V., Huber, R & Boss-meyer, D (1997) Staurosporine-induced conformational changes

of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential Structure 5, 1627–1637.

11 Lamers, M.B.A.C., Antson, A.A., Hiubbard, R.E., Scott, R.K & Williams, D.H (1999) Structure of the protein tyrosine kinase domain of C-terminal Src kinase (Csk) in complex with stauro-sporine J.Mol.Biol.285, 713–725.

12 Zhu, X., Kim, J.L., Newcomb, J.R., Rose, P.E., Sover, D.R., Toledo, L.M., Zhao, H & Morgenstern, K.A (1999) Structural analysis of the lymphocyte specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors Structure

7, 651–661.

13 Engh, R.A & Bossmeyer, D (2002) Structural aspects of protein kinase control – role of conformational flexibility Pharmacol Ther 93, 99–111.

14 Sarno, S., Reddy, H., Meggio, F., Ruzzene, M., Davies, S.P., Donella-Deana, A., Shugar, D & Pinna, L.A (2001) Selectivity

of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor

of protein kinase CK2 (casein kinase 2) FEBS Lett 496, 44–48.

15 Meggio, F & Pinna, L.A (2003) One-thousand-and-one sub-strates of protein kinase CK2? FASEB J 17, 349–368.

16 Pinna, L.A & Meggio, F (1997) Protein kinase CK2 (casein kinase 2) and its implication in cell division and proliferation Prog.Cell.Cycle Res.3, 77–97.

17 Lin, J.-M., Kilman, V.L., Keegan, K., Paddock, B., Emery-Le, M., Rosbach, M & Allade, R (2002) A role for casein kinase 2a

in the Drosophila circadian clock Nature 420, 816–820.

18 Battistutta, R., De Moliner, E., Sarno, S., Zanotti, G & Pinna, L.A (2001) Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzo-triazole Protein Sci 10, 2200–2206.

19 Pavletich, N.P (1999) Mechanisms of cyclin dependent regula-tions: structures of cdks, their cyclin activators, and cip and INK4 inhibitors J.Mol.Biol.287, 821–828.

20 Brown, N.R., Noble, M.E.M., Endicott, J.A & Johnson, L.N (1999) The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases Nat.Cell Biol.

1, 438–443.

21 Lowe, E.D., Tews, I., Cheng, K.-Y., Brown, N.R., Gul, S., Noble, M.E.M., Gamblin, S.J & Johnson, L.N (2002) Specificity determinants of recruitment peptides bound to phospho-CDK2/ cyclin A Biochemistry 41, 15625–15634.

22 Fischer, L., Endicott, J.A & Meijer, L (2002) Cyclin-dependent kinase inhibitors Progress in Cell Cycle Research (Meijer, L., Jezequel, A & Roberge, M., eds), pp 235–248 Station Biologique

de Roscoff Life in Progress Editions, Roscoff.

3

23 Knockaert, M., Greengard, P & Meijer, L (2002) Pharmacolo-gical inhibitors of cyclin-dependent kinases, Trends Pharmacol Sci 23, 417–425.

24 Niefind, K., Guerra, B., Pinna, L.A., Issinger, O.-G & Schom-burg, D (1998) Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 A˚ resolution EMBO J 17, 2451–2462.

25 Leslie, A.G.W (1999) Integration of macromolecular diffraction data Acta Cryst D10, 1696–1702.

26 CCP4 (1994) The CCP4 (Collaborative Computational Project Number 4) suite: programmes for protein crystallography Acta Cryst.D.D50, 760–763.

27 Vagin, A & Teplyakov, A (1997) MOLREP: an automated program for molecular replacement J.Appl.Cryst.30, 1022–1025.

28 Vagin, A & Teplyakov, A (2000) An approach to multi-copy search in molecular replacement Acta Cryst D 56, 1622–1624.

29 Read, R.J (1986) Improved coefficients for map calculation using partial structures with errors Acta Crystallogr A42, 140–149.

30 Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros,

P., Grosse Kuntsleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M.,

Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T & Warren,

G.L (1998) Crystallography and NMR system: a new software

suite for macromolecular structure determination Acta Cryst.D.

54, 905–921.

31 Jones, T.A., Zou, J.Y., Cowan, S.W & Kjeldgaard, M (1991)

Improved method for building models in electron density maps

and the location of errors in these models Acta Crystallogr A47,

110–119.

32 Laskowski, R.A., MacArthur, M.W., Moss, D.S & Thornton,

J.M (1993) PROCHECK: a programme to check the

stereochemical quality of protein structures J.Appl.Cryst.26,

283–291.

33 Russo, A., Jeffrey, P.D & Pavletich, N.P (1996) Structural basis

of cyclin dependent kinase activation by phosphorylation Nat.

Struct.Biol.3, 696–700.

34 Niefind, K., Putter, M., Guerra, B., Issinger, O.G & Schomburg,

D (1999) GTP plus water mimic ATP in the active site of protein

kinase CK2 Nat.Struct.Biol.6, 1100–1103.

35 Niefind, K., Guerra, B., Ermakowa, I & Issinger, O.G (2001) The

crystal structure of human CK2: insights into basic properties of

the CK2 holoenzyme EMBO J 20, 5320–5331.

36 Bruno, U., Cole, J.C., Lommerse, J.P.M., Rowland, R.S.,

Taylor, R & Verdonk, M.K (1997) IsoStar: a library of

information about non–bonded interactions J.Computer-Aided

Mol.Design 11, 525–537.

37 Lommerse, J.P.M., Stone, A.J., Taylor, R & Allen, F.H (1996)

The nature and geometry of intermolecular interactions between

halogens and oxygen or nitrogen J.Am.Chem.Soc.118,

3108–3116.

38 Ghosh, M., Meerts, I.A.T.M., Cook, A., Bergman, A., Brouwer,

A & Johnson, L.N (2000) Structure of human transthyretin

complexed with bromophenols: a new mode of binding Acta

Crystallogr D56, 1085–1095.

39 Schulze-Gahmen, U., Brandsen, J., Jones, H.D., Morgan, D.O.,

Meijer, L., Vesely, J & Kim, S.H (1995) Multiple modes of ligand

recognition: crystal structures of cyclin-dependent protein kinase 2

in complex with ATP and two inhibitors, olomoucine and iso-pentenyladenine Proteins 22, 378–391.

40 De Azevedo, W.F., Leclerc, S., Meijer, L., Havlicek, L., Strnad, M & Kim, S.H (1997) Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine Eur.J.Biochem.243, 518–526.

41 Gray, N.S., Wodicka, L., Thunnissen, A.M., Norman, T.C., Kwon, S., Espinoza, F.H., Morgan, D.O., Barnes, G., LeClerc, S., Meijer, L., Kim, S.H., Lockhart, D.J & Schultz, P.G (1998) Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors Science 281, 533–538.

42 Dreyer, M.K., Borcherding, D.R., Dumont, J.A., Peet, N.P., Tsay, J.T., Wright, P.S., Bitonti, A.J., Shen, J & Kim, S.H (2001) Crystal structure of human cyclin-dependent kinase 2 in complex with the adenine-derived inhibitor H717 J.Med.Chem.44, 524–530.

43 Schulze-Gahmen, U., de Bondt, H.L & Kim, S.H (1996) High resolution crystal structures of human cyclin depedent protein kinase 2 with and without ATP; bound waters and natural ligands

as guides for inhibitor design J.Med.Chem.39, 4540–4546.

44 Johnson, L.N., De Moliner, E., Brown, N.R., Song, H., Barford, D., Endicott, J.A & Noble, M.E.M (2002) Structural studies with inhibitors of the cell cycle regulatory kinase cyclin-dependent protein kinase 2 Pharmacol.Ther.93, 1–12.

45 Zhao, B., Bower, M., McDevitt, P.J., Zhao, H., David, S.T., Johanson, K.O., Green, S.M., Concha, N.O & Zhou, B.-B.S (2002) Structural basis for Chk1 inhibition by UCN-o1 J.Biol Chem 277, 46609–46615.

46 Dym, O & Eisenberg, D (2001) Sequence strucrture analysis of FAD-containing proteins Protein Sci 10, 1712–1728.

47 De Azevedo, W.F., Nueller-Dickermann, H.-J., Schulze-Gahmen, U., Worland, P.J., Sausville, E.A & Kim, S.-H (1996) Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase Proc.Natl Acad.Sci.USA 93, 2735–2740.

48 Oikonomakos, N.G., Schnier, J.B., Zographos, S.E., Skamnaki, V.T., Tsitsanou, K.E & Johnson, L.N (2000) Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site J.Biol.Chem.275, 34566–34573.