flavopiridol inhibits glycogen phosphorylase by binding at ... · substrate affinity). allosteric...

Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site

Nikos G. Oikonomakos‡||, Joachim B. Schnier§, Spyros E. Zographos‡, Vicky T.

Skamnaki‡, Katerina E. Tsitsanou‡, and Louise N. Johnson¶

From the ‡Institute of Biological Research and Biotechnology, The National Hellenic

Research Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece, §Department of

Biological Chemistry, Tupper Hall, University of California, One Shields Avenue, Davis, CA

95616, USA, and the ¶Laboratory of Molecular Biophysics, University of Oxford, South

Parks Road, Oxford OX1 3QU, UK.

||To whom correspondence should be addressed:

Name: Nikos G. Oikonomakos

Tel: +301-7273761

Fax: +301-7273758

E-mail: [email protected]

Running title: Flavopiridol binding to glycogen phosphorylase

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 2, 2000 as Manuscript M004485200 by guest on Septem

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Summary

Flavopiridol (L86-8275), [(-)-cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-

hydroxy-1-methyl)-piperidinyl]-4H-benzopyran-4-one], a potential antitumour drug,

currently in phase II trials, has been shown to be an inhibitor of muscle glycogen

phosphorylase (GP) and to cause glycogen accumulation in A549 NSCLC cells [Kaiser, A.,

Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E.M. and Schnier, J.B., submitted]. Kinetic

experiments reported here show that flavopiridol inhibits GPb with an IC50= 15.5 µM. The

inhibition is synergistic with glucose resulting in a reduction of IC50 for flavopiridol to 2.3

µM and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition,

we determined the structures of GPb complexed with flavopiridol, GPb complexed with

caffeine, and GPa complexed with both glucose and flavopiridol at 1.76 Å, 2.30 Å, and 2.23

Å resolution, and refined to crystallographic R values of 0.216 (Rfree =0.247), 0.189 (Rfree

=0.219), and 0.195 (Rfree =0.252), respectively. The structures provide a rational for

flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the

allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine

binds. Flavopiridol intercalates between the two aromatic rings of Phe285 and Tyr613. Both

flavopiridol and glucose promote the less active T-state through localisation of the closed

position of the 280s loop which blocks access to the catalytic site, thereby explaining their

synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that

of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the

inhibitor can be used to provide specific and potent binding to two different enzymes.

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Introduction

Flavopiridol (L86-8275, Scheme I), a flavonoid, has been shown to be a potent,

competitive inhibitor (with respect to ATP) of cyclin-dependent kinases (CDKs1) with IC50

values between 0.2 and 0.4 µM (1) and to have antiproliferative and cytotoxic activity on

certain tumour cell lines in vitro and in vivo (2-5). The compound is currently in phase II

trials, the first CDK inhibitor to be tested in clinical trials (5-6). X-ray crystallographic

analysis has provided evidence that des-chloroflavopiridol (L86-8276) binds to the ATP

binding site of CDK2 (7-8), and also a structural basis for the development of novel CDK

modulators, as therapeutic agents for cancer therapy (9-11).

Recently, it was found that flavopiridol significantly inhibited both rabbit muscle

glycogen phosphorylase b (GPb) (IC50=1 µM ) and glycogen phosphorylase a (GPa)

(IC50=2.5 µM), but AMP activated GPa was poorly inhibited by flavopiridol2. Furthermore,

flavopiridol treatment of A549 non-small cell lung carcinoma (NSCLC) cells resulted in an

increase in glycogen accumulation. These findings raise the possibility that this antitumour

drug may also interfere with glucose homeostasis in addition to the effects on the cell cycle

through CDK inhibition.

GP (E.C. 2.4.1.1), a key-enzyme in the regulation of glycogen metabolism, catalyses

the degradative phosphorolysis of glycogen to glucose 1-phosphate (glucose-1-P). In

muscle, glucose-1-P is utilised via glycolysis to generate metabolic energy, but in the liver it

is mostly converted to glucose, which is the output for the benefit of other tissues (12). The

enzyme exists in two interconvertible forms: the dephosphorylated form, GPb (low activity,


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low substrate affinity), and the Ser14-phosphorylated form, GPa (high activity, high

substrate affinity). Allosteric activators, such as AMP or inhibitors such as ATP, glucose-6-

P, glucose and caffeine can alter the equilibrium between a less active T state and a more

active R state or vice versa. The structures of T and R state GP have been characterised (13-

15).

Because of its central role in glycogen metabolism, GP has been exploited as a target

for structure-assisted design of compounds that might prevent unwanted glycogenolysis

under high glucose conditions that may be relevant to the control of diabetes (16-24). GP

contains at least 6 potential regulatory sites: the Ser14-phosphate recognition site, the

allosteric site that binds AMP, IMP, ATP and glucose-6-P, the catalytic site that binds the

substrates glycogen and glucose-1-P, and also glucose and glucose analogues, the inhibitor

site, which binds caffeine and related compounds, the glycogen storage site, and a new

allosteric inhibitor site situated at the dimer interface which binds the potential antidiabetic

drug CP320626 (24) (Fig. 1).

In order to provide a stereochemical explanation for flavopiridol inhibition of

glycogen phosphorylase, we have cocrystallised GPb with flavopiridol, and GPa with both

glucose and flavopiridol and determined the structures of the complexes by x-ray

crystallographic methods at 1.76 Å and 2.23 Å resolution, respectively. The structural results

show that flavopiridol binds at the inhibitor site, the site where caffeine binds, located 10 Å

from the catalytic site. In order to compare the flavopiridol binding site with the caffeine

binding site we determined the structure of GPb complexed with caffeine at 2.30 Å

resolution. The detailed interactions of flavopiridol with the protein provide a structural


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explanation for understanding the molecular basis of its high affinity for GP and show that

flavopiridol is an inhibitor that stabilizes the T-state conformation as do caffeine and

glucose. We also demonstrate through kinetic studies a strong synergistic inhibition of GPb

by the pair flavopiridol/glucose.


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EXPERIMENTAL PROCEDURES

Kinetic experiments

GPb was isolated from rabbit skeletal muscle according to Fischer & Krebs (25) using

2-mercaptoethanol instead of L-cysteine and recrystallized at least four times. GPa was

prepared, and recrystallised as described (23). Protein concentration was determined from

absorbance measurements at 280 nm using an absorbance index A1%1 cm=13.2 (26).

Glucose-1-P (dipotassium salt), AMP, (oyster) glycogen, and other chemicals were obtained

from Sigma Chemical Company. Glycogen was freed of AMP by the method of Helmreich

& Cori (27). Phosphorylase activity in the direction of glycogen synthesis was measured at

pH 6.8 and 30°C with 5 µg/ml enzyme, 10 mM glucose-1-P, 1% glycogen, 1 mM AMP, and

a range of concentrations of inhibitor (s) as indicated, in 50 mM triethanolamine

hydrochloride/HCl, 100 mM KCl, 1 mM EDTA, and 1 mM DTT buffer. The enzyme was

preincubated with glycogen for 15 min at 30°C before the reaction was started by adding

glucose-1-P. Inorganic phosphate released in the reaction was measured and initial velocities

were calculated from the pseudo-first-order reaction constants as previously (20).

Crystallographic experiments

Crystallisation and data collection: Native T-state tetragonal (P43212) GPb crystals

were grown as described previously (28) with 1 mM IMP. GPb-flavopiridol and GPa-

glucose-flavopiridol complexes were cocrystallised as previously (21, 23) in a medium

consisting of 25 mg/ml enzyme, 1 mM flavopiridol, 3 mM DTT, 10 mM Bes, 0.1 mM EDTA,

0.02 % sodium azide, pH 6.7, and 1 mM spermine (GPb) or 10 mM magnesium acetate and


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50 mM glucose (GPa); as flavopiridol is insoluble under these conditions, the mixtures were

filtered out to remove precipitates. The GPb-flavopiridol crystals were transferred to a fresh

buffer solution (3 mM DTT, 10 mM Bes, 0.1 mM EDTA, 0.3 mM flavopiridol, pH 6.7) just

before room temperature data collection. The GPa-glucose-flavopiridol crystals were

transferred to a fresh buffer solution (3 mM DTT, 10 mM Bes, 0.1 mM EDTA, 10 mM

magnesium acetate, 50 mM glucose, 0.3 mM flavopiridol, 0.02 % sodium azide, pH 6.7)

containing 30% v/v glycerol for 15-30 sec prior to mounting in a loop, and flash frozen with

nitrogen gas at 100K. Data for GPb-flavopiridol complex were collected from a single

crystal, on an image plate on the beamline X31 at Hamburg (λ= 1.05 Å), at a maximum

resolution of 1.76 Å followed by data collection at a lower resolution (3.05 Å) to measure the

strong low angle reflections. Data for GPa-glucose-flavopiridol complex were collected

from a single crystal at a resolution of 2.23 Å on the same beamline station. Data for GPb-

caffeine-IMP complex were collected at a resolution of 2.30 Å from a single native GPb

crystal soaked in a solution containing 5 mM caffeine, 10 mM Bes, 0.1 mM EDTA, pH 6.7,

for 80 min, on an Image Plate RAXIS IV mounted on a Rigaku Ru-H3RHB generator with a

belt drive rotating anode (λ= 1.5418 Å), at the National Center for Scientific Research

“Demokritos” at Athens. Crystal orientation and integration of reflections were performed

using DENZO (29). Inter-frame scaling, partial reflection summation, data reduction and

post-refinement were all completed using SCALEPACK (29).

Refinement: Crystallographic refinement of the GPb-flavopiridol complex was

performed with X-PLOR version 3.8 (30) using bulk solvent corrections. All data between

29.5 and 1.76 Å were included with no sigma cut-off. The starting protein structure was the


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refined model of the room temperature GPb-glucose complex (31), with water molecules

removed. The Fourier maps calculated with SIGMAA (32) weighted (2mFo-DFc) and

(Fo-Fc) coefficients indicated tight binding of flavopiridol at the inhibitor site. A model of

flavopiridol generated using the programme SYBYL (SYBYL Molecular Modelling

Software, Tripos Associates Inc., T.A. St. Louis, 1992) was fitted to the electron density map after

small adjustments of the torsion angles of the computed model. Map interpretation was

performed using the program O (33). Several side chains of the enzyme model were

adjusted; water molecules were added to the atomic model and retained only if they met

stereochemical requirements by using WATERPICK (in X-PLOR). The final model was

refined by the conventional positional and restrained individual B-factor refinement protocol

in X-PLOR to give a final R factor value of 21.6% (Rfree=24.7%). The structure contained

residues 13-251, 259-314, 324-837 and 331 water molecules. There is no electron density

for the following residues: 252-258, 315-323, and 838-842. These residues are frequently

disordered in GPb structure. A Luzatti plot (34) determined by using XPLOR protocol

suggests an average positional error of approximately 0.24 Å.

The structure of the 100K GPa-glucose-flavopiridol complex was solved by

difference Fourier methods, starting with the published 100K GPa-glucose complex structure

(23), and refined using bulk solvent corrections and the standard building and refinement

protocols (30, 32, 33) to give R-factor = 19.5% (Rfree=25.2%). The final structure

contained residues 5-250, 260-314, 325-838, and 645 water molecules. Residues where

overall <B>-factors values (given in parentheses) exceed 60Å2 include 16-22 (71.1 Å2),


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550-556 (69.4 Å2), and 837-838 (75.9 Å2).

The structure of GPb in complex with the inhibitor caffeine and the weak activator

IMP was solved by difference Fourier methods, starting from the GPb-glucose complex (31),

with glucose and water molecules removed, and refined using bulk solvent corrections and

the standard buliding and refinement protocols (see above) to give R-factor =18.9% (Rfree

=21.9%). The final model contained residues 13-256, 261-316, 324-837, and 204 water

molecules.

Solvent-accessible areas were calculated with the programme NACCESS

(35). The structures were analysed with the graphics program O (33). Hydrogen-

bonds were assigned if the distance between the electronegative atoms was less than

3.3 Å and if both angles between these atoms and the preceding atoms were greater

than 90°. Van der Waals interactions were assigned for non-hydrogen atoms

separated by less than 4 Å. R.m.s. deviations in Cα positions and subunit rotations

were determined following the method of Sprang et al. (36) using the program

LSQKAB (37). Structural units (N-domain core, C-domain core and activation

locus) were those defined previously (36). Coordinate sets for comparison were: T-

state GPb (38) (code 2GPB), T-state GPb-glucose complex (31) (code 2GPB), and

100 K GPa-glucose complex (23) (code 2GPA). Coordinates for T-state GPb-

flavopiridol, GPb-caffeine-IMP, and 100K GPa-glucose-flavopiridol complexes

have been deposited with the RCSB Protein Data Bank (http://www.rcsb.org/) (codes

1C8K, 1GFZ, and 1E1Y, respectively).


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RESULTS

Synergistic inhibition by flavopiridol and glucose

Kinetic experiments with GPb showed that flavopiridol is an inhibitor of the enzyme

with an IC50= 15.5 ±0.3 µM, measured in the direction of glycogen synthesis, and in the

presence of saturating concentrations of glucose-1-P (10 mM), glycogen (1%) and AMP (1

mM), at 30°C (Fig. 2A). The IC50 value for glucose was calculated to be 19.7±1.3 mM.

Kaiser et al.2 found an IC50 =1 µM for flavopiridol inhibition of rabbit muscle GPb, when

tested in the direction of glycogen breakdown, in the presence of 0.8 mM AMP, 20 mM

phosphate (pH 7.2), 0.1% glycogen, and 25°C. This difference may well be a consequence of

the reaction being measured in the opposite direction of catalysis and the different substrate

concentrations used. To investigate the interaction between flavopiridol and glucose binding

to GPb, initial velocity studies were carried out by varying flavopiridol and glucose

concentrations at fixed concentrations of the substrates glucose-1-P (10 mM) and glycogen

(1%). The IC50 values for flavopiridol inhibition of the enzyme were 10.9 µM, 6.5 µM, 4.3

µM, and 2.3 µM, in the presence of 2, 4, 6, and 10 mM glucose, respectively. The kinetic data

indicate that flavopiridol inhibition of GPb is synergistic with glucose, suggesting that both

flavopiridol and glucose are able to bind to the enzyme at the same time (39). The effect of

varying glucose concentration on the IC50 value for flavopiridol is shown in Fig. 2B. At 10

mM glucose concentration, the relative IC50 value for flavopiridol was decreased by more

than 5-fold. Caffeine, a T-state inhibitor of the enzyme (with a Ki value of 0.1-0.2 mM) is


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also known to function with glucose in a synergistic mode, with each compound promoting

the binding of the other (with an interaction constant α=0.2) (40, 41).

Flavopiridol binding site

The overall architecture of the native T-state GPb with the location of the cofactor

pyridoxal 5’-phosphate (PLP), the catalytic site, the allosteric (or nucleotide) site, the

inhibitor (or nucleoside) site, and a new allosteric inhibitor site, identified recently as target

for drug interactions, is presented in Fig. 1. The inhibitor site is located at the entrance to the

catalytic site, near the domain interface, and comprises residues from both domains 1

(residues 13-484) and 2 (residues 485-842). The site binds a number of aromatic compounds

such as caffeine.

Crystallographic data collection, processing and refinement statistics for the co-

crystallized GPb-flavopiridol and GPa-glucose-flavopiridol complexes are shown in Table

I. For both complexes, the refined 2Fo-Fc electron density maps indicated that flavopiridol

(Scheme I) bound strongly at the inhibitor site (Fig. 3), consistent with the kinetic results. In

the GPa-glucose-flavopiridol complex, additional density at the catalytic site indicated

binding of glucose. The mode of binding and the interactions that glucose makes with GPa,

in the presence of flavopiridol, are almost identical with those previously reported for the

complex GPa-glucose at 100K (23). In particular, Asp283 and Asn284 of the 280s loop are

in the same position where they are found in the GPa-glucose complex. No other binding

sites were observed for flavopiridol.

Interactions of flavopiridol with inhibitor site residues


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The most characteristic feature for flavopiridol binding to GP is its stacking

interactions with the two aromatic residues, Phe285, from the 280s loop (residues 282 to 286)

and Tyr613. Flavopiridol on binding at the inhibitor site of GPb makes a few polar/polar

interactions and exploits numerous van der Waals contacts; it makes a total of 4 hydrogen

bonds to water molecules and 107 van der Waals interactions (58 nonpolar/nonpolar, 6

polar/polar and 43 polar/nonpolar) (Fig. 4A, Fig. 6A).

The benzopyran-4-one ring exploits 61 van der Waals contacts which are dominated

by the substantial contacts made to almost all atoms of Phe285 and Tyr613. In GPb-

flavopiridol, the plane of benzopyran-4-one ring lies almost parallel to the plane of the

Phe285 ring, at a distance of 3.4 Å and makes an angle of about 10° with the Tyr613 ring, at

distances varied between 3.7 Å (C4 to CG) and 3.9 Å (C2 to CZ). Carbonyl oxygen O4

makes an indirect contact to the main-chain atoms of Asp283 via a water molecule (Wat36),

and to Ile570 (O) and Ala610 (N) via another water molecule (Wat98). A water-mediated

(Wat210) link between hydroxyl O5 with Asn282 (OD1) and main-chain N of Gly612 is also

apparent.

The chlorophenyl ring is positioned in an adjacent pocket between, His571 and

Glu572, Arg770 and Phe771, Ile380 and Glu382, Tyr613, and Asn284 of the 280s loop. This

part of the molecule exploits 29 van der Waals contacts which are dominated by the

substantial contacts made to almost all atoms of Glu382. Indeed, the side-chain of Glu382

stacks against the chlorophenyl group making some 12 van der Waals contacts. CL1 is

positioned between the Tyr613 hydroxyl group and the OE2 of Glu572 at nearly the same

distance of 3.3 Å but these contacts are not of the type “carbon-bound halogen:


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electronegative atoms” identified by (42).

The 4-hydroxypiperidin-1-yl group projects into solvent and makes 2 rather long

(3.9 Å) non-polar/non-polar contacts to side-chain atoms of Phe285 and 8 contacts to water

molecules. In addition, Wat307 contacts both N1 (2.7 Å) and hydroxyl O3 (3.3 Å). This

water molecule was not previously seen in the native structure.

On forming the complex with GPb the inhibitor becomes buried. The solvent

accessibilities of the free and bound flavopiridol molecules are 560 Å2 and 191 Å2, indicating

that a surface area of 369 Å2 becomes inaccessible to water or that flavopiridol becomes 66%

buried in the enzyme complex. Both polar and nonpolar groups are buried, but the greatest

contribution comes from the nonpolar groups which contribute 337 Å2 (91%) of the surface

that becomes inaccessible. On the protein surface, a total of 249 Å2 solvent-accessible

surface area becomes inaccessible on binding flavopiridol, of which 184 Å2 (74%) is

contributed by nonpolar groups. Bigger buried surfaces have been reported for other potent

GP inhibitors such as W1807 (21) and CP320626 (24). The corresponding values for ligand

and protein buried areas in the GPb-W1807 and GPb-CP320626 complexes are 518 Å2 and

334 Å2 (W1807; Ki =1.6 nM), and 536 Å2 and 283 Å2 (CP320626; IC50 =334 nM),

respectively. The total buried surface areas (protein plus ligand), for flavopiridol (618 Å2),

CP320626 (819 Å2) and W1807 (852 Å2) binding show an approximate correlation with

affinity.


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There is no change in the conformation of the polypeptide chain on binding

flavopiridol. Superposition of the native T-state GPb with GPb-flavopiridol complex

structure over well defined residues gave r.m.s. deviation of 0.083 Å for Cα atoms. There

are small shifts in the side chain conformations of Glu382 and His571. These residues shift

to optimise interactions with the chlorophenyl ring. In the native T-state GPb structure, there

are hydrogen bonds across the domain interface in the vicinity of the flavopiridol binding site

that are important in stabilising the T-state conformation. Arg770 NH1 and NH2 groups

hydrogen bond to OE2 of Glu382, Arg569 NH2 hydrogen bonds to OD1 of Asn133 and NE2

of His571 hydrogen bonds to OD2 of Asp282. This inter-domain hydrogen bonding pattern

is also retained in the flavopiridol complex.

Comparison with GPb-caffeine complex in the T-state

In order to compare the flavopiridol binding site with the caffeine binding site in GPb

we have determined the room temperature GPb-caffeine-IMP complex structure at a 2.3 Å

resolution (Table I). IMP, which was included in the crystallisation mixture at a

concentration of 1 mM, is bound at the allosteric site and the mode of binding and the

interactions that IMP makes with GPb are similar to those observed for AMP in the T-state

GPb-AMP complex (43). Caffeine, like flavopiridol, is intercalated between the two

aromatic residues, Phe285 and Tyr613, and on binding to GPb it makes a total of 79 van der

Waals contacts (44 nonpolar/nonpolar, 4 polar/polar and 39 polar/nonpolar) and two water-

mediated links to Glu382 and Asp283, not identified in a previous study (44). The total

buried surface area (protein plus ligand) for the GPb-caffeine complex is 467 Å2.


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The GPb-flavopiridol and GPb-caffeine-IMP structures show no significant

conformational changes between the complex structures. Binding of flavopiridol and

caffeine at the inhibitor site demonstrate several similar interactions (Fig. 4B). Caffeine

binds at the inhibitor site with the purine ring occupying a location almost co-planar to the

benzopyran-4-one ring of flavopiridol but with its long axis normal to that of benzopyran-

4-one. In this location, O11 of caffeine molecule is over Tyr613 OH.


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DISCUSSION

Flavopiridol binds at the inhibitor site of GP; the inhibitor site is a hydrophobic

binding pocket of relatively low specificity. Binding at this site shows great diversity:

purines such as adenine and caffeine, nucleosides such as adenosine and inosine, nucleotides

such as AMP, IMP and ATP, NADH and certain related heterocyclic compounds such as

FMN (flavin mono-nucleotide) have been shown to bind at this site (40, 44, 45) in muscle

GPb and GPa, but liver GPa shows a more stringent selectivity for inhibitors (40). The

physiological significance of this inhibition has yet to be established but it may be used by an

unidentified compound to enhance the effects of the control of liver GPa by glucose, possibly

in response to insulin (46, 47).

The kinetic experiments on the separate and combined effects of flavopiridol and

glucose (Fig. 2) showed that flavopiridol and glucose exhibit synergy in inhibiting GPb. Our

experimental complex structures confirm these observations and show how both ligands,

glucose and flavopiridol, stabilise the less active T state through interactions with the 280s

loop (Fig. 5). Glucose, bound at the catalytic site, interacts with the side chains of Asp283

and Asn284 holding the loop in its inactive state. Flavopiridol, bound at the inhibitor site,

some 12 Å from the catalytic site, interacts with Phe285 and augments the localisation of the

280s loop in its inactive conformation. The key transition between inactive T state and active

R state GP involves a movement and disordering of the 280s loop that allows a crucial

arginine, Arg569, to enter the catalytic site in place of Asp283, and which also opens access

for glycogen to the catalytic site. The shift and disordering of the 280s loop is associated

with changes at the intersubunit contacts of the dimer that give rise to allosteric effects (14).


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By their dual action glucose and flavopiridol hold the 280s loop in the inactive conformation

and block access to the catalytic site. The effect of glucose on the potency of flavopiridol

could be an important physiological feature of a liver GPa inhibitor, as it has been suggested

for the pair CP-91149/glucose (48), because the decrease in inhibitor potency as glucose

concentrations decrease in vivo should diminish the risk of hypoglycemia.

Flavopiridol has been shown to be a potent inhibitor of CDK2 kinase activity with an

IC50 value of 0.4 µM (1). To understand how flavopiridol binds to CDK2, De Azevedo et al.

(7) have determined the x-ray structure of CDK2 in complex with the des-chloroflavopiridol

(L86-8276) at 2.33 Å resolution. The conformation of flavopiridol in the GPb-flavopiridol

complex is similar with that observed for the refined structure of the CDK2-L868276

complex but not identical. In both the GPb-flavopiridol and CDK2-L868276 structures, the

planes of benzopyran-4-one and the 3-hydroxy-1-methyl piperidinyl are almost

perpendicular with the piperidinyl ring in chair geometry. In the CDK2-L868276 structure,

the benzopyran-4-one is coplanar with the phenyl ring, but in the GPb-flavopiridol

structure, the chlorophenyl group is inclined aproximately 36° (torsion angle O1-C2-C21-

C22 = -36°) to the benzopyran-4-one. L86-8276 binds in the ATP-binding pocket of

CDK2, with the benzopyran-4-one ring occupying approximately the same region as the

purine ring of ATP but inclined about 60º relative to the adenine. The piperidinyl moiety

partially occupies the α-phosphate pocket, while the phenyl ring occupies a pocket not

observed in the ATP complex structure. The binding of L86-8276 is characterised by

predominantly hydrophobic and van der Waals interactions and specific hydrogen bonds to


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Lys33, Glu81 (main chain), Leu83 (main chain) and Asp145 and to two water molecules. In

the CDK2-L868276 complex, the buried area of L86-8276 is 301 Å2 and the buried surface

in CDK2 is 399 Å2, making a total decrease in solvent-accessible area of 700 Å2 (7)

compared to the total buried surface area in the GPb-flavopiridol complex of 618 Å2.

Furthermore, examination of the CDK2-L868276 structure suggests that the introduction of a

chlorophenyl instead of a phenyl in the L86-8276 molecule would probably increase the total

number of contacts between flavopiridol and enzyme to 61. Comparison between the

CDK2-L868276 and GPb-flavopiridol complexes show that flavopiridol makes different

interactions with the two proteins (Fig. 6AB). In CDK2 it exploits specific hydrogen bonds

that mimic those of ATP and non-polar interactions that are made mostly to aliphatic chains.

In GPb-flavopiridol complex there are no specific hydrogen bonds apart to those to water

molecules and the non-polar interactions invlolve π-π stacking with the aromatic groups of

Phe285 and Tyr613.

A compound, such as flavopiridol, that targets both enzymes, CDK2 and GP, could

have an added benefit in starving cancer cells of glycolytic intermediates at the same time

disrupting the cell cycle and sending cells into apoptosis. If flavopiridol action is to act not

only by inhibiting CDK2 but also by inhibiting GP, there needs to be comparable IC50 values

for both enzymes. In the absence of glucose there is a 40-fold difference in IC50 values

which could suggest that with doses needed to inhibit CDK2 there would be no action on GP

but in the presence of 10 mM glucose the difference in IC50 values is only 6-fold and this


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could allow concentrations at which the inhibitor could be effective with both enzymes.

Acknowledgments

This work was supported by the Greek GSRT (PENED1999, 99ED237), a Wellcome

Trust Biomedical Research Collaboration Grant (to LNJ and NGO) and EMBL Hamburg

through the HCMP Access to LIP (CHGE CT93 0040). We are grateful to Drug Synthesis &

Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment,

National Cancer Institute, Bethesda, Maryland 20892 for sending to us flavopiridol. We wish

to acknowledge the assistance of the staff at EMBL, Hamburg, for providing excellent

facilities for x-ray data collection, and Angelos Thanasoulas for help in the kinetic

experiments. We would like to thank Prof. S.-H. Kim for providing the atomic coordinates

for CDK2- des-chloroflavopiridol complex structure. We would like to thank Dr. Kim

Watson and Dr. D.D. Leonidas for discussion. Figures 1, and 3-6 were produced using

XOBJECTS, a molecular illustration programme (M.E.M. Noble, unpublished results).

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Footnotes

1 The abbreviations used are: CDK, cyclin-dependent kinase; GP, glycogen phosphorylase,

1,4-α-D-glucan:orthophosphate α-glucosyltransferase (EC 2.4.1.1); GPb, glycogen

phosphorylase b; GPa, glycogen phosphorylase a; PLP, pyridoxal 5´-phosphate; glucose, α-

D-glucose; Glucose-1-P, α-D-glucose 1-phosphate; flavopiridol, L86-8275, [(-)-cis-

5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-

benzopyran-4-one]; CP320626, 5-chloro-1H-indole-2-carboxylic acid [1-(4-

fluorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2-oxoethyl]amide; Bes, N,N-bis(2-hydroxy-

ethyl)-2-aminomethane sulphonic acid; DTT, dithiothreitol; RMSD, r.m.s. deviation, root-

mean-square deviation

2Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E.M. and Schnier, J.B., submitted for publication


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Legends for Figures

Scheme I. Flavopiridol and caffeine, showing the numbering system used.

FIG. 1. A schematic diagram of the GPb dimeric molecule viewed down the 2-fold. The

positions are shown for the catalytic, allosteric, the inhibitor site, and the new allosteric

inhibitor site. The catalytic site, marked by glucose shown in ball-and-stick representation,

is buried at the centre of the subunit accessible to the bulk solvent through a 15 Å long

channel. Glucose, a competitive inhibitor of the enzyme that also promotes the less active T

state through stabilisation of the closed position of the 280s loop (shown in white), binds at

this site. The allosteric site, which binds the allosteric activator AMP (shown), is situated at

the subunit-subunit interface some 30 Å from the catalytic site. The inhibitor site, which

binds purine compounds, such as caffeine, nucleosides or nucleotides at high concentrations

and flavopiridol (shown) is located on the surface of the enzyme some 12 Å from the

catalytic site and, in the T state, obstructs the entrance to the catalytic site tunnel. The new

allosteric inhibitor site (24), located inside the central cavity formed on association of the two

subunits, binds CP320626 molecule (shown) and is some 15 Å from the allosteric effector

site, 33 Å from the catalytic site and 37 Å from the the inhibitor site.

FIG. 2. A, Synergistic inhibition of GPb by flavopiridol and glucose. Kinetic data in the

presence of various concentrations of inhibitor flavopiridol (2, 6, 10, 15, 20 and 40 µM) in

the absence of glucose () (IC50=15.5 ± 0.3 µM) or with glucose 2 mM (•) (IC50=10.9 ±

0.3 µM), 4 mM (r) (IC50=6.5 ± 0.2 µM), 6 mM (p) (IC50=4.3 ± 0.2 µM) and 10 mM (£)


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(IC50=2.3 ± 0.1 µM). (¢) Kinetic data obtained with various concentrations of glucose (2, 6, 10,

20, 40 and 100 mM) in the absence of flavopiridol (IC50=19.7± 1.3 mM). The data are

plotted as percent inhibition vs. concentration of compound. B, The effect of glucose on the

potency of flavopiridol. IC50 values for GPb inhibition were determined in the direction of

glycogen synthesis as described in Experimental Procedures with 5 µg/ml enzyme at constant

concentrations of glucose-1-P (10 mM) and glycogen (1%), and varied flavopiridol

concentrations (2-40 µM) in the absence or presence of various concentrations of glucose.

The IC50 values were as in (A). The normalised values (obtained by dividing these values by

the IC50 value obtained in the absence of glucose) are plotted as a function of glucose

concentration.

FIG. 3. Stereo diagram of the electron density of the bound flavopiridol to GPb from a 1.76

Å simulated annealing omit map (contoured at 2.5σ level) with coefficients (Fo-Fc) map.

The refined structure of flavopiridol is shown.

FIG. 4. A, Stereodiagram of the contacts between flavopiridol and GPb, in the vicinity of the

inhibitor site shown in stereo. B, Stereodiagram showing a comparison of flavopiridol bound

to GPb with bound caffeine in the vicinity of the inhibitor site. Green: T-state GPb-

flavopiridol complex; white: T-state GPb-caffeine-IMP complex.

FIG. 5. Stabilisation of the 280s loop in its inactive conformation by glucose, bound at the

catalytic site, and flavopiridol, bound at the inhibitor site, of GPa. Coordinates of the refined

GPa-glucose-flavopiridol complex structure are displayed.


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FIG. 6. Stereodiagrams of the contacts between flavopiridol and GPb (A) and between des-

chloroflavopiridol and CDK2 (B) after superimposing the protein structures with respect to

the benzopyran-4-one rings of the two ligands.


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TABLE I. Diffraction data and refinement statistics for T-state GPb-flavopiridol, GPa-glucose-flavopiridol, and GPb-caffeine-IMP complexes.

GPb-flavopiridol complex GPa-glucose-flavopiridol complexGPb-caffeine-IMP complexSpace group P43212 P43212 P43212

Temperature RT 100K RT

No. of images (degrees) 56 (0.8° ) 50 (1°) 60 (1°)Unit cell dimensions a=b=128.4 Å, c=116.1 Å a=b=126.7 Å, c=116.0 Å a=b=128.3 Å, c=116.4 ÅResolution range 29.5 - 1.76 Å 29.9 – 2.23 Å 28.75 – 2.30 ÅNo. of observations 489,494 321,450 310,740

No. of unique reflections 95,442 40,691 43,063I/σ(I) (outermost shell)1 14.6 (2.0) 11.1 (2.4) 10.8 (2.8)

Completeness (outermost shell) 99.3% (97.5%) 87.3% (84.6%) 98.3% (94.0%)Rmerge (outermost shell)2 0.053 (0.640) 0.104 (0.433) 0.100 (0.412)

outermost shell 1.79 - 1.76 Å 2.27 - 2.23 Å 2.34 - 2.30 ÅMultiplicity 4.2 3.7 4.8Refinement (resolution) 29.5 - 1.76 Å 29.9 – 2.23 Å 28.75 – 2.30 Å

No. of reflections used (free) 90,605 (4,780) 38,621 (2,037 free) 40,861 (2,169 free)

Residues included 13-251,259-314,324-837 5-250,260-314,325-835 13-256,261-316,324-837No. of protein atoms 6571 6603 6624

No. of water molecules 331 645 204

No. of ligands atoms 15 (PLP) 15 (PLP) 15 (PLP)28 (flavopiridol) 28 (flavopiridol) 14 (caffeine)

- 12 (glucose) 23 (IMP)

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- 4 (Ser14-P) -Final R (Rfree)3 21.6% (24.7%) 19.5% (25.2%) 18.9% (21.9%)

R (Rfree) (outermost shell) 33.8% (33.6%) 25.5% (31.4%) 32.8% (36.7%)

r.m.s.d. in bond lengths (Å) 0.008 0.007 0.008

r.m.s.d. in bond angles (°) 1.34 1.24 1.31r.m.s.d. in dihedral angles (°) 24.9 23.8 25.2r.m.s.d in improper angles (°) 0.72 0.65 0.71

Average B (Å 2) for residues 13-251,259-314,324-837 5-250,260-314,325-835 13-256,261-316,324-837Overall 29.9 (28.3) 4 21.3 33.8 (31.2)4

CA,C,N,O 27.9 (26.3) 4 20.3 31.8 (29.3)4

Side chain 31.8 (30.2) 4 22.3 35.7 (33.1)4

Average B (Å 2) for ligands

PLP 17.9 12.2 19.0Flavopiridol 28.3 28.6 -

Glucose - 16.1 -Ser14-P - 24.2 -

Caffeine - - 32.1

IMP - - 82.8Water 45.1 31.4 35.7


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1σ(I) is the standard deviation of I.

2Rmerge = Σi Σh | <Ih> - Iih | / Σi Σh Iih , where <Ih> and Iih are the mean and ith measurement of intensity for reflection h, respectively.

3Crystallographic R = Σ | |Fo| - |Fc | | / Σ |Fo|, where |Fo| and |Fc| are the observed and calculated structure factor amplitudes, respectively.

Rfree is the corresponding R value for a randomly chosen 5% of the reflections that were not included in the refinement.

4Average B (Å2) (given in parentheses) for protein residues if the poorly defined residues were excluded.


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Katerina E. Tsitsanou and Louise N. JohnsonNikos G. Oikonomakos, Joachim B. Schnier, Spyros E. Zographos, Vicky T. Skamnaki,

Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site

published online August 2, 2000J. Biol. Chem.

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