The 1.76 A resolution crystal structure of glycogen phosphorylase B complexed with glucose, and CP320626, a potential antidiabetic drug.

Abstract

CP320626, a potential antidiabetic drug, inhibits glycogen phosphorylase in synergism with glucose. To elucidate the structural basis of synergistic inhibition, we determined the structure of muscle glycogen phosphorylase b (MGPb) complexed with both glucose and CP320626 at 1.76 A resolution, and refined to a crystallographic R value of 0.211 (R(free)=0.235). CP320626 binds at a novel allosteric site, which is some 33 A from the catalytic site, where glucose binds. The high resolution structure allows unambiguous definition of the conformation of the 1-acetyl-4-hydroxy-piperidine ring supported by theoretical energy calculations. Both CP320626 and glucose promote the less active T-state, thereby explaining their synergistic inhibition. Structural comparison of MGPb--glucose--CP320626 complex with liver glycogen phosphorylase a (LGPa) complexed with a related compound (CP403700) show that the ligand binding site is conserved in LGPa.

Full text

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The 1.76 A˚Resolution Crystal Structure of Glycogen
Phosphorylase B Complexed with Glucose, and CP320626,
a Potential Antidiabetic Drug
Nikos G. Oikonomakos,a,* Spyros E. Zographos,aVicky T. Skamnakia
and Georgios Archontisb
aInstitute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 48 Vas. Constantinou Avenue,
Athens 11635, Greece
bDepartment of Physics, University of Cyprus, Box 20537, CY1678, Nicosia, Cyprus
Received 29 August 2001; accepted 5 November 2001
Abstract—CP320626, a potential antidiabetic drug, inhibits glycogen phosphorylase in synergism with glucose. To elucidate the
structural basis of synergistic inhibition, we determined the structure of muscle glycogen phosphorylase b (MGPb) complexed with
both glucose and CP320626 at 1.76 A˚resolution, and refined to a crystallographic R value of 0.211 (Rfree=0.235). CP320626 binds
at a novel allosteric site, which is some 33 A˚ from the catalytic site, where glucose binds. The high resolution structure allows
unambiguous definition of the conformation of the 1-acetyl-4-hydroxy-piperidine ring supported by theoretical energy calculations.
Both CP320626 and glucose promote the less active T-state, thereby explaining their synergistic inhibition. Structural comparison
of MGPb–glucose–CP320626 complex with liver glycogen phosphorylase a (LGPa) complexed with a related compound
(CP403700) show that the ligand binding site is conserved in LGPa. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
CP320626 (Scheme 1), a potential antidiabetic drug, has
been shown to be a potent inhibitor of human liver gly-
cogen phosphorylase a (LGPa) and to produce marked
glucose lowering in diabetic ob/ob mice, without altering
plasma insulin levels.1CP320626 was also demonstrated
to be a potent inhibitor of MGPb (IC50=334 ? 10 nM)
and to act in synergism with glucose (IC50=178 ? 10
nM).2The observed synergism could be an important
physiological feature of a LGPa inhibitor, because the
decrease in inhibitor potency as glucose concentrations
decrease in vivo should minimize the risk of hypoglyce-
mia.3To understand how CP320626 binds to MGPb,
we have recently determined the X-ray structure of
MGPb in complex with CP320626 at 2.3 A˚resolution.2
CP320626 binds at a novel allosteric inhibitor site of the
enzyme, not previously observed to bind ligands. Fur-
thermore, LGPa was independently reported to bind
two related compounds, CP403700 and CP526423,
potent inhibitors of the liver isozyme.4
Given the potential importance of the detailed inter-
actions of this new class of inhibitors with GP in the
structure-assisted design and development of analogous
inhibitors, as therapeutic agents for type 2 diabetes
therapy,5we have cocrystallized MGPb with both
CP320626 and glucose and determined the structure of
the complex by X-ray crystallographic methods at 1.76
A˚resolution. The 1.76 A˚resolution structure has
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00394-7
Bioorganic & Medicinal Chemistry 10 (2002) 1313–1319
Scheme 1. 5-Chloro-1H-indole-2-carboxylic acid [1-(4-fluorobenzyl)-
2-(4-hydroxypiperidin-1-yl)-2-oxoethyl]amide
the numbering system used.
(CP320626),showing
*Corresponding author. Tel.: +30-1-727-3761; fax: +30-1-727-3758;
e-mail: ngo@eie.gr

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revealed
4-hydroxy-piperidyl moiety of the ligand as compared
to the 2.3 A˚ resolution complex structure. In order to
assess the validity of using MGPb as a model for the
LGPa in structure-assisted drug design, we have com-
pared the structure of CP320626 complexed with MGPb
with the structure of CP403700 bound to LGPa. The
structural comparison shows that the MGPb–glucose–
CP320626 complex is similar to the LGPa-1–GlcNAc–
CP403700 complex in the vicinity of the new allosteric
site.
differencesintheconformation ofthe
Results and Discussion
The overall architecture of the native T-state MGPb
with the location of the catalytic, the allosteric effector,
the inhibitor, and the new allosteric inhibitor sites is
presented in Fig. 1. Crystallographic data collection
processing and refinement statistics for the co-crystal-
lized MGPb–glucose–CP320626 complex are shown in
Table 1. The refined 2Fo–Fcelectron density map indi-
cated that CP320626 (Scheme 1) bound at the new
allosteric inhibitor site of MGPb located at the subunit
interface. Additional density at the catalytic site indi-
cated tight binding of glucose. We describe briefly the
MGPb/ligands interactions at the catalytic site and in
more detail at the new allosteric inhibitor site.
Catalytic site
The mode of binding and the interactions that glucose
makes with MGPb in the presence of CP320626 are
almost identical with those previously reported for
native MGPb.6Glucose can be accommodated at the
catalytic site of MGPb with essentially no disturbance
of the structure and the interactions it makes are similar
to those observed in the MGPb–glucose complex.
New allosteric inhibitor site
The electron density maps derived from the previous
analysis were interpreted with the 4-hydroxy-piperidyl
moiety in1C4rather than in4C1conformation. The
resolution of the present structure allowed us to define
more accurately the conformation of the 4-hydroxy-
piperidyl moiety (Fig. 2). The 4-hydroxy-piperidyl moi-
ety adopts the most stable puckered ring conformation.
In this equivalent chair conformation, the 4-hydroxy-
group (O3 in the numbering) of the 4-hydroxy-piperidyl
moiety is now axial and not equatorial.
The fitted to the electronic density map4C1chair con-
formation of the CP320626 piperidine moiety was opti-
mized by electronic structure methods described in the
Figure 1. A schematic diagram of the MGPb 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 red), is buried at the centre of the
subunit accessible to the bulk solvent through a 15 A˚ 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 in magenta), is situated at
the subunit–subunit interface some 30 A˚from the catalytic site. The
inhibitor site, which binds purine compounds, such as caffeine (shown
in green), is located on the surface of the enzyme some 12 A˚from the
catalytic site and, in the T state, obstructs the entrance to the catalytic
site tunnel. The new allosteric inhibitor site, located inside the central
cavity formed on association of the two subunits, binds CP320626
molecule (shown in orange) and is some 15 A˚
effector site, 33 A˚from the catalytic site and 37 A˚from the inhibitor
site. This figure was produced using XOBJECTS (M.E.M. Noble,
unpublished results).
from the allosteric
Table 1.
MGPb–glucose–CP320626 complex
Diffraction data and refinement statistics for T-state
Space groupP43212
No. of images (?)
Unit cell dimensions
Resolution range
No. of observations
No. of unique reflections
I/s(I) (outermost shell)b
Completeness (outermost shell)
Rmerge(outermost shell)c
58 (0.8?)a
a=b=129.0 A˚, c=116.2 A˚
26.25–1.76 A˚
483,106
93,061
15.41 (2.33)
95.8% (94.8%)
0.054 (0.696)
Outermost shell
Refinement (resolution)
No. of reflections used (free)
Residues included
No. of protein atoms
No. of water molecules
No. of ligands atoms
1.79–1.76 A˚
26.25–1.76 A˚
88,308 (4663)
13–842
6591
355
15 (PLP)
31 (CP320626)
12 (glucose)
21.1% (23.5%)
34.2% (36.6%)
0.007
1.25
24.7
0.68
Final R (Rfree)d
R (Rfree) (outermost shell)
r.m.s.d. in bond lengths (A˚)
r.m.s.d. in bond angles (?)
r.m.s.d. in dihedral angles (?)
r.m.s.d in improper angles (?)
a0.8?is the rotation range per image.
bs(I) is the standard deviation of I.
cRmerge=?i?h| <Ih>? Iih| / ?i?hIih,where <Ih> and Iihare the
mean and ith measurement of intensity for reflection h, respectively.
dCrystallographic R=? | |Fo|?|Fc| | / ? |Fo|, where |Fo| and |Fc| are
the observed and calculated structure factor amplitudes, respectively.
Rfreeis the corresponding R value for a randomly chosen 5% of the
reflections that were not included in the refinement.
1314 N. G. Oikonomakos et al./Bioorg. Med. Chem. 10 (2002) 1313–1319

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Experimental. With both HF and DFT methods the
resulting chair structures were very similar and super-
imposed very well to the X-PLOR-refined geometry.
The Cremer–Pople puckering amplitude Q7was 0.55–
0.54 for all basis sets except HF/3-21G (0.58). This is
slightly smaller than the experimental Q value of the
cyclohexane chair (0.56), and lies between the theore-
tical (HF and MP2 ab initio) values for the equatorial
(0.59–0.57) and axial (0.56–0.53) forms of piperidine.8
The configuration around the nitrogen atom of the
piperidine moiety (N3) was very similar to the peptide
bondgeometryobtained
(NMA) by MP2/6-31G* calculations.9A small devia-
tion from planarity (less than 10?) was observed in the
HF/6-31+G* and 6-311+G* basis structures. Devia-
tions from planarity (up to 10?for the O–C–N–H dihe-
dral) have been also reported in peptide bond ab initio
calculations.10The optimized structures from both
methods superimpose very well to the X-PLOR-refined
geometry for the piperidine moiety. Fitting of the entire
CP320626 ligand to the density map depends on the
configuration of the piperidine moiety, and is optimal for
anapproximately planar
around the nitrogen atom, as in the optimized structures.
forN-methyl-acetamide
piperidineconformation
The bound conformation of CP320626 appears to be
stabilized by intermolecular contacts to the protein, and
there are no intramolecular hydrogen bonds. Straight-
forward energy minimization using the program SYBYL
(SYBYL Molecular Modelling Software, Tripos Associ-
ates Inc., T.A. St. Louis, USA 1992) (with the Powell
minimiser and default parameters) resulted in a sig-
nificantly different structure. In the bound CP320626
structure the torsion angles N1–C8–C9–O1 and C7–C8–
C9–O1 are 1.6 and 179.8?, respectively, so that the con-
formation about the C8–C9 bond is significantly differ-
ent from that in the computed structure (?177.4 and
4.1?) of CP320626. In addition, the conformations about
the N2–C10, C10–C17 and C10–C11 bonds are different
in the two molecules (the torsion angles C9–N2–C10–
C11, C9–N2–C10–C17, N2–C10–C11–O2, and N2–C10–
C17–C18 are ?61.3 and ?116.7?, ?179.6 and 124.0?,
?59.9 and ?102.3?, 179.5 and ?85.7?, respectively for
the bound and the computed CP320626 molecule).
The values 179.5 and 75.7?for the dihedral angles N2–
C10–C17–C18 and C19–C17–C18–C19 in the bound
conformation correspond to frequently populated rota-
mer states of the w1and w2angles for Tyr and Phe resi-
dues, as follows by analysis of backbone-independent
and backbone-dependent rotamer libraries of protein
structures from the PDB.11
The binding of CP320626 to MGPb, in the presence of
glucose, does not promote extensive conformational
changes except for the small shifts of the atoms sur-
rounding the inhibitor, that is of residues 60, 64, and
191 that undergo conformational changes to accom-
modate the ligand. CP320626 on binding at the new
allosteric inhibitor site of MGPb makes a total of seven
hydrogen bonds and exploits 114 van der Waals inter-
actions, 41 of which are interactions between nonpolar
groups; there are in total 44 contacts to the symmetry
related subunit (Table 2). The high resolution structure
indicated six well ordered structural waters that form a
network of hydrogen bonds that link CP320626 to pro-
tein residues; these waters were not observed in the 2.3
A˚resolution structure. Thus, Wat267 mediates two
polar contacts between the indolecarboxy carbonyl O1
and main-chain O of Asn187 and Glu190 (Fig. 3).
Wat2570makes a direct hydrogen bond to OG1 of
Thr380(where the prime refers to waters or residues
from the symmetry-related subunit). There is a hydro-
gen bond from the N2 of the carboxamide to the main-
chain O of Thr380. Wat263 contacts NH1 of Arg60,
which shifts on binding of CP320626 to GPb. Wat3230
hydrogen-bonds main-chain N of Tyr1850and main-
chain N of Gly1860. These residues make van der Waals
contacts with C13 and C14 atoms of the 4-hydroxy-
piperidyl moiety. Wat3340
hydrogen bond to main-chain O of Gly1860through
another new water molecule (Wat3000). These water
molecules appear to contribute to CP320626 binding
and also stabilize the T state.
also makes an indirect
Figure 2. Stereo diagram of the electron density of the bound CP320626 to GPb from a 1.76 A˚simulated annealing omit map (contoured at 2.5s
level) with coefficients (Fo?Fc) map. The refined structure of CP320626 is shown. This figure was produced using XOBJECTS (M.E.M. Noble,
unpublished results).
N. G. Oikonomakos et al./Bioorg. Med. Chem. 10 (2002) 1313–1319 1315

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Table 2.Hydrogen bonds and van der Waals contacts between the CP320626 and residues of the new allosteric binding site of MGPba
A. Hydrogen bonds
Inhibitor atomProtein atomDistance (A˚)
N1
O1
Glu190 O
Wat267
Wat155
Thr380O
Lys191NZ
Wat155
Wat89
2.8
2.9
3.0
3.0
3.0
3.2
2.8
N2
O2
N3
O3
B. Van der Waals contacts
Inhibitor atomProtein atomNo. of contacts
CL1
C1
C2
C3
C4
N1
C5
C6
C7
C8
C9
C10
C11
O2
C12
C13
C14
O3
C16
C17
C18
C19
C20
C21
F1
Arg60 O,CG; Leu63 CB; Val64N,CA,CG2; Trp67 CE3,CZ3
Arg60 CG,NE,CD; Trp67 CE3,CZ3; Val400CG2
Arg60 CG,CD; Trp67 CZ3; Trp189 O; Pro229 CD,CG
Arg60 CD; Pro188 O; Trp189 O,C; Glu190 C,O
Arg60NE,CD,CZ,NH2; Pro188 O; Glu190 O; Lys191 CB
Arg60 CZ,NH2; Pro188 O; Glu190 C; Lys191 CA,CB; Wat267
Arg60NE,CD,CZ,NH1,NH2; Val400CG2
Arg60 CG,NE,CD; Val64 CG2; Val400CG2; Wat230
Arg60NE,CZ,NH1,NH2; Lys191 CD; Thr380O; Val400CG2
Arg60 CZ,NH1,NH2; Glu190 O; Lys191 CD
Lys191 CD; Thr380O; Wat267
Thr380O
Wat155
Lys191 CD,CE,NZ
Ala192 CB; Wat155; Wat229
Tyr1850O; Wat229; Wat3480
Gly1860CA
Wat3480
Wat155
Thr380O,CG2; Leu390CD1
His570CE1,NE2
His570CE1,NE2; Wat2110
His570CE1,NE2; Wat2110; Wat2670
Phe530CE2; Pro1880N
Phe530CE2; Gly1860O,C,CA; Asn1870C,O,N,CA;
Pro1880CA,CB,N,CG,CD
Phe530CG,CD2,CE2,CZ; Tyr1850O
Leu390CD1; His570CE1
8
6
6
6
7
7
6
6
7
5
3
1
1
3
3
3
1
1
1
3
2
3
4
2
13
C22
C23
5
2
Total 114
aWat89 is hydrogen bonded to Tyr226 OH (3.2 A˚) and to Wat154 (3.1 A˚); Wat154 is in turn hydrogen bonded to Glu190 OE1 (2.8 A˚); Wat155 is
hydrogen bonded to Ala192N (2.6 A˚) and to Wat154 (3.0 A˚); Wat267 is hydrogen bonded to both Asn187 O (3.0 A˚) and Glu190 O (3.3 A˚).
Figure 3. Interactions between CP320626 and MGPb and water structure in the vicinity of the new allosteric site. Residues from subunit 1 are shown
in green and their symmetry-related equivalents (from subunit 2) are shown in cyan. Hydrogen bonds are represented as dotted lines and water
molecules as red spheres. This figure was produced using XOBJECTS (M.E.M. Noble, unpublished results).
1316N. G. Oikonomakos et al./Bioorg. Med. Chem. 10 (2002) 1313–1319

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Mechanism of synergistic inhibition
Kinetic experiments on the separate and combined
effects of CP320626 and glucose have shown that
CP320626 and glucose exhibit synergy in inhibiting
MGPb.2Our experimental complex structure confirms
these observations and shows how both ligands,
CP320626 and glucose, stabilize the less active T state.
The extensive non-polar contacts and the seven hydro-
gen bonds from the inhibitor to the protein strengthen
the subunit–subunit interface and lock it in a con-
formation close to the T state quaternary conformation.
Thus CP320626 acts as a potent allosteric inhibitor
binding at a site distant from the catalytic site and
exerting its effects by promoting the T state conforma-
tion, consistent with the Monod–Wyman–Changeux12
model for allosteric proteins. Glucose, bound at the
catalytic site, interacts with the side chains of Asp283
and Asn284 holding the 280s loop (residues 282–286) in
its closed T-state conformation. By their dual action
CP320626 and glucose hold the 280s loop in the inactive
conformation and block access to the catalytic site.
Therefore by promoting the T state, CP320626 will also
promote synergistic glucose binding.
Comparison with LGPa-1-GlcNAc-CP403700 complex
in the T-state
Human LGPa is the more important target enzyme in
terms of treatment of type 2 diabetes because of its
direct influence on blood sugar level. The human LGPa
polypeptide chain is 846 residues long, compared with
human MGPb of 841 residues and rabbit MGPb of 842
residues. The muscle and liver enzymes are 79% iden-
tical with 100% identity at the glucose binding site (at
the catalytic site), but the two isoenzymes differ in
allosteric properties.13,14From a comparison of the
amino acid sequences of all phosphorylases, we have
found that all but one—Ala192 is serine in the human
liver isozyme—of the residues that make up the
CP320626 binding site in the rabbit MGP are highly
conserved in human LGP and, based on this observa-
tion, we suggested that the new allosteric site is likely to
be the same in the liver enzyme.2The recent crystal
structures of human T-state LGPa in complex with
CP403700 or CP526423 and 1-GlcNAc, a glucose ana-
logue inhibitor of rabbit MGP15verified our hypothesis.
The superposition of the LGPa-1–GlcNAc–CP403700
complex structure with
complex structure over well defined residues 23–249,
260–313, 326–830 gave r.m.s. deviation of 0.537 A˚ for
Caatoms, indicating that the two structures have very
similar overall conformations. Furthermore, the super-
imposition of the CP403700 complex with the CP320626
complex over 27 residues of the new allosteric inhibitor
site (370–400, 530–570, 60–67, 188–192, 1850–1880, and
229) gave r.m.s. deviations of 0.307, 0.300, and 0.978 A˚
for Ca, main chain, and side chain atoms, respectively,
indicating that the two structures superimpose well and
they closely resemble in the vicinity of the new allosteric
site (Fig. 4).
MGPb–glucose–CP320626
Figure 4. Stereodiagram showing a comparison of CP320626 bound to MGPb–glucose complex with bound CP403700 to LGPa-1–GlcNAc com-
plex (code 1exv) in the vicinity of the new allosteric inhibitor site. Green: MGPb–glucose–CP320626 complex (subunit 1); cyan: MGPb–glucose–
CP320626 complex (subunit 2); black: LGPb-1–GlcNAc–CP403700 complex. This figure was produced using XOBJECTS (M.E.M. Noble, unpub-
lished results).
Scheme 2. The molecule 1-acetyl-4-hydroxy-piperidine employed for
the geometry optimisation of the piperidine moiety of CP320626, with
the numbering system used.
N. G. Oikonomakos et al./Bioorg. Med. Chem. 10 (2002) 1313–1319 1317

Page 6

In conclusion, the higher resolution structure, and
electronic structure calculations enable for a more
accurate determination of the conformation of the
4-hydroxy-piperidyl moiety of CP320626 and water
structure. The environment of new allosteric site is
similar in the MGPb complex compared to the LGPa
complex, indicating the accuracy of MGPb as a model
in the design and optimisation of indole-2-carboxamide
inhibitors.
Experimental
Crystallization and data collection
Tetragonal (P43212) MGPb–glucose–CP320626 crystals
were grown as described previously2with 50 mM glu-
cose. Crystallographic data were collected from a single
crystal, on an image plate on the beamline X31 at Ham-
burg (l=1.05 A˚), at a maximum resolution of 1.76 A˚.
Crystal orientation and integration of reflections were
performed using DENZO.16Inter-frame scaling, partial
reflection summation, data reduction and post-refine-
ment were all completed using SCALEPACK.16
Refinement
Crystallographic refinement of the MGPb–glucose–
CP320626 complex was performed with X-PLOR ver-
sion 3.817using bulk solvent corrections. All data
between 26.25 and 1.76 A˚were included with no sigma
cut-off. The starting protein structure was the 2.3 A˚
resolution refined model of the MGPb–CP320626 com-
plex. The Fourier maps calculated with SIGMAA18
weighted (2mFo-DFc) and (Fo-Fc) coefficients indicated
tight binding of CP320626 at the new allosteric site and
glucose at the catalytic site. Map interpretation was
performed using the program O.19A few side chains of
the enzyme model were adjusted and water molecules
were added to the atomic model and retained only if
they met stereochemical requirements. The final model
was refinedbytheconventional
restrained individual B-factor refinement protocol in
X-PLOR to give a final R factor value of 21.1%
(Rfree=23.5%). The structure contains residues 13–254,
260–314, 324–837 and 355 water molecules. A Luzatti
plot20suggests an average positional error of approxi-
mately 0.23 A˚. The model displays good stereochemistry
as determined by PROCHECK21with 89.5% of resi-
dues in the most favoured regions.
positionaland
The structure was analysed with the graphics program
O.19Hydrogen-bonds were assigned if the distance
between the electronegative atoms was less than 3.3 A˚
and if both angles between these atoms and the preced-
ing atoms were greater than 90?. Van der Waals inter-
actionswereassignedfor
separated by less than 4 A˚. R.m.s. deviations in Ca,
main chain, and side-chain atoms positions were deter-
mined using the program LSQKAB.21Coordinates for
the 1.76 A˚resolution T-state MGPb–glucose–CP320626
complex have been deposited with the RCSB Protein
Data Bank (http://www.rcsb.org/) (code 1h5u).
non-hydrogenatoms
CP320626 geometry optimization
Geometry optimization of a model molecule (1-acetyl-4-
hydroxy-piperidine; Scheme
piperidine moiety of the inhibitor CP320626, was per-
formed by ab initio and Density Functional Theory
methods with the program Gaussian.22Ab initio opti-
mizations were carried out at the Hartree-Fock (HF)
level, using the 3-21G, 6-31G*, 6-31+G*, 6-311+G*
basis sets. The Density Functional Theory (DFT) cal-
culations used the Becke’s three-parametric hybrid
exchange functional23awith the Lee–Yang–Parr corre-
lation functional (B3LYP),23band the same basis sets as
for HF, except 3-21G. Frequency calculations ensured
that the optimized structures corresponded to energy
minima.
2),approximatingthe
Acknowledgements
This work was supported by the Greek GSRT
(PENED1999, 99ED237), the Joint Research and
Technology project between Greece and Cyprus (2001–
2003) (to N.G.O and G.A.), and EMBL, Hamburg
Outstation (HPRI-CT-1999-00017).
acknowledge the assistance of the staff at EMBL,
Hamburg, for providing excellent facilities for X-ray
data collection.
Wewish to
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