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Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

¹ Institute of Organic and Pharmaceutical Chemistry, ² Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, Athens 11635, Greece
³ Aventis Pharma Deutschland GmbH, a company of the Sanofi-Aventis Group, D-65926 Frankfurt am Main, Germany

Reprint requests to: Nikos G. Oikonomakos, Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, 48, Vassileos Constantinou Avenue, 11635, Athens, Greece; e-mail: ngo{at}eie.gr; fax: 30-210-7273-831.

(RECEIVED February 25, 2005; FINAL REVISION April 7, 2005; ACCEPTED April 7, 2005)

	Abstract

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC₅₀ of 2.0 µM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26Å resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.

Keywords: type 2 diabetes; glycogen phosphorylase; acyl ureas; inhibition; X-ray crystallography

Abbreviations: GP, glycogen phosphorylase, 1,4--D-glucan:orthophosphate -glucosyltransferase (EC 2.4.1.1) • rmGPb, rabbit muscle glycogen phosphorylase b • rmGPa, rabbit muscle glycogen phosphorylase a • hlGPa, human liver glycogen phosphorylase a • PLP, pyridoxal 5'-phosphate • glucose, -D-glucose • Glc-1-P, -D-glucose 1-phosphate • Glc-6-P, D-glucose 6-phosphate • W1807, (–)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarboxylate • CP320626, 5-chloro-1H-indole-2-carboxylic acid [1-(4-fluorobenzyl)- 2-(4-hydroxypiperidin-1-yl)-2-oxoethyl]amide • compound 1, 6-{2,6-dichloro-4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid • compound 2, 4-{3-chloro-4-[3-(2,4-dichloro-benzoyl)-ureido]- phenoxy}-butyric acid • compound 3, 4-{4-[3-(2,4-dichloro-benzoyl)-ureido]-2,3-dimethyl-phenoxy}-butyric acid • compound 4, 5-{3-[3- (2,4-dichloro-benzoyl)-ureido]-2-methyl-phenoxy}-pentanoic acid • r.m.s. deviation, root-mean-square deviation.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051432405.

	Introduction

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
Inhibition of glycogen phosphorylase (GP) has been proposed as a therapeutic strategy for the treatment of type 2 diabetes (Aiston et al. 2001, 2003; McCormack et al. 2001; Treadway et al. 2001) and several binding sites on the enzyme such as the catalytic, allosteric, inhibitor, and the new allosteric site (Fig. 1) have been identified as specific targets for inhibitor binding (Somsák et al. 2001, 2003; Treadway et al. 2001; Oikonomakos 2002; Kurukulasuriya et al. 2003). In some cases detailed X-ray crystallographic studies have revealed key interactions responsible for inhibitor potency and have elucidated the structural mechanisms of enzyme inhibition (for review, see Oikonomakos 2002).

View larger version (90K): [in this window] [in a new window]   Figure 1. A schematic diagram of the T-state rmGPb dimeric molecule, for residues 13 to 837, viewed down the molecular dyad, showing the positions for the catalytic, allosteric, the inhibitor, and the new allosteric binding sites. The catalytic site, which includes the essential cofactor PLP (not shown), is buried at the center of the subunit accessible to the bulk solvent through a 15-Å-long channel. Glucose (shown in black) binds at this site and promotes the less active T state through stabilization of the closed position of the 280s loop (black). The allosteric site, which binds the activator AMP, the inhibitors Glc-6-P, the Bayer compound W1807, and acyl urea compound 1 (black) is situated at the subunit–subunit interface some 30 Å from the catalytic site. The inhibitor site, which binds purine compounds such as caffeine and flavopiridol (dark gray) (Oikonomakos et al. 2000a), is situated at the entrance to the catalytic site tunnel, formed by two hydrophobic residues of Phe285 and Tyr613. The new allosteric or indole binding site, located inside the central cavity formed on association of the two subunits, binds CP320626 molecule (light gray) and is some 15 Å from the allosteric effector site, 33 Å from the catalytic site, and 37 Å from the inhibitor site (Oikonomakos et al. 2000b).

Recently acyl ureas were reported as human liver glycogen phosphorylase a (hlGPa) inhibitors, which bind to the allosteric site of the enzyme (T. Klabunde, K.U. Wendt, D. Kadereit, V. Brachvogel, H.-J. Burger, A.W. Herling, N.G. Oikonomakos, M.N. Kosmopoulou, D. Schmoll, E. Sarubbi, et al., in prep.). Here we report on the detailed analysis of four crystal structures of acyl urea inhibitors (1–4) (Scheme 1) in complex with rabbit muscle glycogen phosphorylase (rmGPb). These data show that compounds 1–4 bind at the allosteric site of the enzyme, where they occupy a position similar to that of the allosteric activator AMP. Binding of 1–4 induces significant conformational changes in the vicinity of the site, and stabilizes the T'-state conformation.

View larger version (17K): [in this window] [in a new window]   Scheme 1. Chemical structures of the acyl urea compounds 1–4, showing the numbering system used.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

View larger version (30K): [in this window] [in a new window]   Figure 2. Stereo diagrams of the 2Fo–Fc electron density maps, contoured at 1, for the bound compounds 1 (A), 2 (B), 3 (C), and 4 (D) at the allosteric site. Electron density maps were calculated using the standard protocol as implements in X-PLOR 3.8 (Brünger 1992) before incorporating ligand coordinates.

View larger version (38K): [in this window] [in a new window]   Figure 3. (A) Interactions between compound 1 (BN2) (shown in orange) and rmGPb at the allosteric site. Residues from subunit 1 are shown in green, and residues from subunit 2 are shown in cyan. (B) Comparison between the rmGPb–1 complex (shown in green) and T-state rmGPb (shown in blue) in the vicinity of the allosteric site. (C) rmGPa in the R state viewed in a similar orientation to that of the T-state rmGPb; the position of 1 is superimposed. Residues from subunit 1 are shown in light gray, and residues from subunit 2 are shown in dark gray.

Comparison with the native T-state structure
Superposition of the activation locus (residues 23–40, 41–47, 48–78, 94–103, 104–111, 118–125 from both subunits) of the structure of the native T-state rmGPb with the activation locus of structure of the rmGPb–1 complex gave an r.m.s. deviation of 0.22Å for C atoms, indicating that the two structures have very similar overall conformations within the limits of the 2.26Å resolution data. The major conformational changes on binding of 1 to rmGPb occur in the vicinity of the allosteric site. Shifts for main-chain atoms are observed for residues 47' to 49' (between 0.5 and 0.8Å ), and residues 193 to 196 (between 0.5 and 1.7Å ) that affect the subunit–subunit interface in the region between the cap' and the loop between 7 (residues 191 to 193) and 8 (residues 198 to 209) strands. The binding of 1 to rmGPb is accompanied by local conformational changes in the vicinity of the allosteric site. The greatest changes include shifts of the side-chain atoms of residues 193 to 196 by –1.0 to 3.8Å , side-chain atoms of residues 40' to 41' by –0.4 to 3.7Å , and also shifts of the side-chain atoms of residues 47' to 49' of –0.6 to 1.9Å . Similar shifts were observed previously on binding of W1807 to the allosteric site of rmGPb (Zographos et al. 1997) and rmGPa (Oikonomakos et al. 1999) and appear important in stabilizing a modified T state, denoted T', that is more tensed than the T state.

In the native T-state rmGPb structure, hydrogen bonds across the subunit interface in the vicinity of the allosteric site are thought to stabilize the dimer structure. Arg193 hydrogen bonds to the main-chain oxygens of residues Leu39' (2.9Å ) and Val40' (2.9 Å) and Glu195 hydrogen bonds to Ly41' (2.9 Å ). These contacts are present in both T- and R-state enzymes. In the complex structure with 1, the aforementioned major shift in Arg193 and minor shifts in residues 39'–40' are responsible for the disruption of the intersubunit hydrogen bonds between Arg193 and Leu39' and Val40'. However, Glu195 (OE1) is still able to hydrogen-bond to Lys41' (3.1Å ). The Tyr185'/Pro194 interaction, an important subunit/subunit contact and a major link between the cap'/2 and the tower/tower', is also retained in the 1 complex. A comparison of the two structures in the vicinity of the allosteric site is shown in Figure 3B.

Comparison with R-state GP structures
Comparison of the rmGPb–1 complex with the R-state rmGPa (Barford et al. 1991) suggests that the inhibitor is likely to have lower affinity for the R-state conformation. Superposition of the activation locus (residues 23–40, 41–78, 94–111, 118–125 from both subunits, as defined by Sprang et al. 1991) of the structure of the R-state rmGPa (subunit A) with the activation locus of structure of the rmGPb–1 complex gave an r.m.s. deviation of 1.21Å for C atoms. The transformation (obtained with LSQKAB) that allows superposition of the rmGPb–1 complex structure to the R-state rmGPa involves a rotation of one subunit by –5.2Å so as to bring the two subunits closer together at the twofold axis of the dimer. Incorporation of 1 into the allosteric site of the R-state rmGPa would result in clashes with the side chains of residues Val40', Asp42', Gln72, Tyr75, and Arg193. Movements of these residues that would enable binding of 1, as seen in the rmGPb–1 complex, appear to be suppressed by the subunit–subunit contacts that promote the R state (Fig. 3C). Similarly, superposition of the activation loci in the R-state rmGPb–AMP complex (Barford et al. 1991) and in the rmGPb–1 complex gave an r.m.s. deviation of 1.31Å for C atoms. The requirement for shifts for the residues 40', 42', 44', 67, and 193 is also apparent.

A comparison of the positions of AMP (R-state) and 1 (T'-state) bound at the allosteric site is shown in Figure 4. The ribose partially overlaps with the central acyl urea moiety of 1 (ribose C2'/N2, O3'/O1, and O2'/N1 separations are, respectively, 1.0Å , 1.6 Å , and 1.9 Å ), and the adenine partially overlaps with the dichlorophenyl group (adenine atoms C2, C4, N3, and N4 make close contacts [0.5–1.4 Å ] to O3, C13, C12, and C14 atoms of the dichlorophenyl group, respectively). In the R-state AMP complex, there is a significant shift in the side chain of Tyr75 from its T-state position, which results in van der Waals contacts from the tyrosine to the adenine. The loop region 193–196 and residues 39'–40' and 47'–49' shift on binding AMP, but these shifts are in the opposite direction to those observed with 1 binding.

View larger version (36K): [in this window] [in a new window]   Figure 4. Comparison of the positions of the inhibitor compound 1 (BN2) and the activator AMP bound at the allosteric site of rmGPb, after superimposing the rmGPb–1 protein structure onto the R-state rmGPb–AMP complex.

View larger version (40K): [in this window] [in a new window]   Figure 5. Interactions of compound 2 (BN3) (A), compound 3 (BN4) (B), and compound 4 (BN5) (C) with rmGPb in the vicinity of the allosteric site. Color code as in Figure 3A.

Overall, it is notable that, as in the case of compound 1, exactly the same conformational rearrangements are observed on binding of 2–4 to the allosteric site of rmGPb, and this indicates that binding of acyl ureas 1–4 to rmGPb stabilize similar conformations. The LSQKAB superposition of the structure of the native T-state rmGPb with the refined rmGPb–2, rmGPb–3, and rmGPb–4 complex structures over the activation loci gave r.m.s. deviations of 0.25 Å , 0.24 Å , and 0.27 Å for C atoms, respectively, indicating that the four structures have very similar overall conformations. Similarly, the LSQKAB superposition of the rmGPb–1 complex with rmGPb–2, rmGPb–3, and rmGPb–4 complex structures gave an r.m.s. deviation of 0.06 Å for C atoms.

	Conclusions

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
The X-ray crystallographic study of four rmGPb complexes with acyl urea inhibitors showed that compounds 1–4 bind at the allosteric site, at the subunit–subunit interface of the dimer. All four derivatives form three hydrogen bonds with the main-chain carbonyl of Val40', the main-chain NH of Asp42' and an ordered water molecule (through the central acyl urea moiety), and make extensive nonpolar interactions with the enzyme that involve the side chains of Trp67, Asn72, Tyr75, Arg193, and Val40', Lys41', and Val45' from the symmetry-related subunit. These interactions provide a rationale for their potency to inhibit hlGPa activity. In contrast, to previously known GP inhibitors that bind to the allosteric site, compounds 1–4 do not exploit the phosphate-recognition site, which comprises Arg309, Arg310, and a more distant arginine, Arg242. The phosphate-recognition site appears accessible by substituents positioned at the C13 or C14 atoms of the acyl urea scaffold (Scheme I). This site recognizes a variety of phosphorylated compounds, such as AMP, ATP, Glc-6-P, and the nonphysiological phosphate- mimetic W1807.

The position of inhibitors 1–4 is distinct but partially overlapping with the position of AMP bound to the relaxed state of the enzyme (Fig. 4). The binding of acyl ureas and AMP is therefore mutually exclusive. The formation of a stable rmGPb–acyl urea complex requires conformational changes in the vicinity of the allosteric site, particularly in the backbone and side chain of residues 193 to 196 and in the cap' region of the other subunit (39'–40' and 47'–49'). The ligand-induced conformational changes are characteristic of the T'-state conformation. The key rearrangement is probably the backbone displacement of the loop 193–196 that allows for van der Waals interactions with the ligand similar to those observed with W1807 (Zographos et al. 1997; Oikonomakos et al. 1999). Overall, we suggest that acyl ureas inhibit the enzyme directly by preventing binding of the allosteric activator AMP and indirectly (allosterically) by stabilizing the T' state.

The results with W1807 and several dihydropyridine diacid analogs (Zographos et al. 1997; Oikonomakos et al. 1999; Ogawa et al. 2003), phenyl diacid analogs (Lu et al. 2003), phenoxy-phthalates (Kristiansen et al. 2004), and acyl ureas (this work) show how nonphysiological compounds could be potent inhibitors of glycogenolysis. The structural results obtained with the acyl ureas can be further exploited by means of chemical optimization to yield new potent inhibitors. In fact, rational design and parallel synthesis, used to develop a series of acyl ureas, led to the discovery of hlGPa inhibitors (T. Klabunde, K.U. Wendt, D. Kadereit, V. Brachvogel, H.-J. Burger, A.W. Herling, N.G. Oikonomakos, M.N. Kosmopoulou, D. Schmoll, E. Sarubbi, et al., unpubl.), which when administered to anaesthetized Wistar rats caused a dose-dependent reduction of the glucagoninduced hyperglycemic peak.

	Materials and methods

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
The synthesis of compounds 1–4 is described in Defossa et al. (2001). RmGPb was isolated, purified, recrystallized, and assayed as previously described (Oikonomakos et al. 2002). Kinetics studies were performed in the direction of glycogen synthesis with 10 µg/mL enzyme, constant concentrations of glycogen (1% w/v), Glc-1-P (4 mM), AMP (40 µM), 1% DMSO, and various concentrations of inhibitors 1–4 (1–20 µM). The inorganic phosphate released in the reaction was determined according to Saheki et al. (1985). HlGPa was expressed and purified according to the procedures described by Rath et al. (2000). The activity of recombinant hlGPa was monitored in the direction of glycogen synthesis. The standard assay mixture (100 µL final volume) contained 30 mM HEPES (pH 7.2), 60mMKCl, 1.5 mMEDTA, 1.5 mMMgCl₂, 1 mg/mL glycogen, 1 mM Glc-1-P, 7 mU hlGPa, 1% DMSO, and the respective inhibitors at a concentration within the range 0–100 µM. The reaction was initiated by the addition of Glc-1-P and incubated for 40 min at 25°C. The inorganic phosphate released was assayed according to Drueckes et al. (1995).

RmGPb complexes with compounds 1–4 were cocrystallized in a medium consisting of 23–25 mg/mL enzyme, compounds 1–4 (at molar ratios of compound/enzyme varied from 2 to 10), 3 mM dithiothreitol, 10 mM Bes, 0.1 mM EDTA, and 0.02 % sodium azide (pH 6.7). Crystallographic data were collected from small single crystals on an image plate RAXIS IV using a Rigaku Ru-H3RHB belt drive rotating anode ( =1.5418 Å ), operating at 60 kV, 100 mA or on an image plate at EMBL-Hamburg outstation (beamline X31, =1.24 Å ). Crystal orientation, integration of reflections, interframe scaling, partial reflection summation, data reduction, and post-refinement were all performed using DENZO and SCALEPACK (Otwinowski and Minor 1997).

Crystallographic refinement of the rmGPb complexes was performed with X-PLOR version 3.8 (Brünger 1992) using bulk solvent corrections. All data were included with no cutoff. The starting model was a refined structure of the native T-state rmGPb at 1.8 Å resolution (N.G. Oikonomakos, E.D. Chrysina, D.D Leonidas, and M.N. Kosmopoulou, unpubl.), with water molecules removed. Throughout the refinement, 5% of the data were flagged for calculation of R_free. The Fourier maps calculated with SIGMAA (Read 1986) weighted (2mF_o–DF_c) and (F_o–F_c) coefficients indicated binding of compounds 1–4 at the allosteric site. Minimized conformers of compounds 1–4 generated using the program SYBYL (Tripos Associates Inc.) were fitted to the electron density map after small adjustments of the torsion angles. Map interpretation was performed using the program O (Jones et al. 1991). Several side chains of the enzyme model were adjusted; water molecules were added and retained only if they met stereochemical requirements by using WATERPICK. The final models were refined by the conventional positional and restrained individual B-factor refinement protocol as implemented in X-PLOR.

The structures were analyzed with the graphics program O (Jones et al. 1991). Hydrogen bonds and van der Waals interactions were calculated with the program CONTACT (CCP4 1994) applying a distance cutoff of 3.3 Å and 4.0 Å , respectively. The program calculates the angle O...H...N (where the hydrogen position is unambiguous) and the angle source...oxygen-bonded carbon atom. Suitable values are 120° and 90° RmGPb complex structures were superimposed using LSQKAB (Collaborative Computational Project No. 4 1994). Coordinate sets for comparison were: room temperature T-state rmGPb, R-state rmGPa (PDB code 1GPA [PDB] ), and R-state rmGPb-AMP complex (PDB code 7GPB [PDB] ). The schematic representation of the crystal structures presented in all figures were prepared with the programs MolScript (Kraulis 1991) and BobScript (Esnouf 1997) and rendered with Raster3D (Merritt and Bacon 1997). The coordinates of the new structures have been deposited with the RCSB Protein Data Bank (http://www.rcsb.org/pdb) with codes 1WUT (rmGPb–1 complex), 1WVX (rmGPb–2 complex), 1WV0 (rmGPb–3 complex), and 1WV1 (rmGPb–4 complex).

	Acknowledgments

 
This work was supported by the Greek General Secretariat for Research and Technology (GSRT) through the programs PENED-204/2001 (MNK), ENTER-EP6/2001 (EDC), Aventis Pharma Deutschland GmbH, a company of the Sanofi-Aventis Group, and the EMBL-Hamburg outstation under FP6 "Structuring the European Research Area Programme" contract no. RII3/CT/2004/5060008. The assistance of V.T. Skamnaki in protein crystallization is also acknowledged. We also acknowledge the assistance of the staff at EMBL-Hamburg outstation for providing excellent facilities for X-ray data collection.

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				C. Tiraidis, K.-M. Alexacou, S. E. Zographos, D. D. Leonidas, T. Gimisis, and N. G. Oikonomakos FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase Protein Sci., August 1, 2007; 16(8): 1773 - 1782. [Abstract] [Full Text] [PDF]

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