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doi:10.1016/j.jmb.2003.08.031 J. Mol. Biol. (2003) 333, 393–407 Structural Characterization of the GSK-3b Active Site Using Selective and Non-selective ATP-mimetic Inhibitors J. A. Bertrand1*, S. Thieffine2, A. Vulpetti1, C. Cristiani2, B. Valsasina2 S. Knapp1, H. M. Kalisz2 and M. Flocco1 1 Department of Chemistry and 2 Department of Biology Pharmacia Italia S.p.A. Discovery Research Oncology Viale Pasteur, 10, 20014 Nerviano, Italy GSK-3b is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3b may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3b, we determined crystal structures of unphosphorylated GSK-3b in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors’ interactions with GSK-3b in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3b inhibitors. q 2003 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: glycogen synthase kinase-3; selective inhibitors; signal transduction; structure-based design; X-ray crystallography Introduction Over 20 years ago, glycogen synthase kinase-3 (GSK-3) was discovered as one of many protein kinases that phosphorylate and inactivate glycogen synthase.1 Subsequently, molecular cloning of GSK-3 in mammalian tissues revealed two closely related isoforms, GSK-3a and GSK-3b,2,3 that are related by a high degree of similarity in the kinase catalytic domain (97%) but diverge at the N and C termini. Nonetheless, both GSK-3a and GSK-3b Abbreviations used: AMP-PNP, adenyl imidodiphosphate; CDK2, cyclin-dependent kinase 2; CDKs, cyclin-dependent kinases; ERK2, extracellular signal-regulated protein kinase 2; FGFR, fibroblast growth factor receptor; GSK-3b, glycogen synthase kinase 3b; I-5, 2-chloro-5-{[4-(3-chlorophenyl)-2,5-dioxo2,5-dihydro-1H-pyrrol-3-yl]amino}benzoic acid; PKA, protein kinase A; RMSD, root-mean-square deviation; SU5402, 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2yl)methylene]-2-indolinone; VEGFR, vascular endothelial growth factor receptor. E-mail address of the corresponding author: [email protected] contain a conserved N-terminal serine residue (Ser21 for GSK-3a and Ser9 for GSK-3b) whose phosphorylation is important for the regulation of the enzymatic activity.4 Despite the specificity suggested by its name, GSK-3 is involved in a diverse number of regulatory pathways by the phosphorylation of several different cellular targets. Medical interest in GSK-3 stems from its involvement in animal development, metabolic control, neurodegeneration and oncogenesis. In the phosphatidylinositide 3-kinase-dependent pathway, insulin stimulation inhibits GSK-3 by activating protein kinase B (PKB; also called Akt) which, in turn, phosphorylates GSK-3 at the conserved N-terminal serine.4 Thus, insulin stimulation inhibits GSK-3 and, in so doing, allows the dephosphorylation and activation of glycogen synthase. GSK-3 is also recognized as a key component of the Wnt signaling pathway, which is essential for pattern development during embryonic development and regulation of cell proliferation (reviewed in Refs. 5,6). In addition, GSK-3 has been implicated in Alzheimer’s disease by its potential role in the 0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. 394 Structures of GSK-3b Complexes Figure 1. Diagram showing the chemical structures of AMP-PNP, staurosporine, indirubin-3-monoxime, alsterpaullone and I-5. abnormal hyperphosphorylation of microtubuleassociated protein tau.7 Recent reviews give a more detailed description of the roles played by GSK-3 in the different cellular pathways and pathologies.8,9 Small molecule inhibitors of GSK-3 may have a therapeutic potential in a number of different human diseases such as cancer, type II diabetes, chronic inflammation processes, stroke and neurological diseases such as bipolar disorders or Alzheimer’s disease.10 To date, three groups have published structures of GSK-3b. These include two structures of the unphosphorylated apoenzyme,11,12 one structure of the Tyr216 monophosphorylated enzyme complexed with a peptide that inhibits b-catenin phosphorylation13 and one structure of the Tyr216 monophosphorylated enzyme complexed with a peptide from axin.14 Analysis of the GSK-3b structure provided insight into both its regulation of the kinase activity and its preference for pre-phosphorylated substrates. Many protein kinases are activated by the phosphorylation of residues within the activation loop. For example, ERK2 activation consists of the phosphorylation of two residues in the activation loop, Thr183 and Tyr185.15 In GSK-3b, the activation loop residue 395 Structures of GSK-3b Complexes equivalent to Tyr185 in ERK2 is Tyr216 and its phosphorylation has been reported in vivo.16 It is thought that this residue plays a role in the opening and closing of the substrate-binding site.13 In contrast to ERK2, GSK-3b does not contain a phosphorylated threonine in the activation loop. However, the site expected for the “missing” phosphothreonine is present in GSK-3b and is used for the recognition of the primed phosphorylation on substrates with the sequence motif SxxxS(P) (where S(P) represents a phosphoserine). In general, GSK-3 has a preference for substrates that contain a “priming” phosphate at position n þ 4 (n is the site of GSK-3 phosphorylation) and these substrates are phosphorylated more readily than those that lack the “priming” phosphate. In comparison with ERK2, the GSK-3 primed substrate’s n þ 4 phosphate would occupy the site generally occupied by the phosphothreonine of the activation loop. In addition, the “missing” phosphothreonine site also provides an explanation for the inhibition that results from Ser9 phosphorylation.11,12 Presumably, after Ser9 phosphorylation the N terminus of the protein is bound in the active site as a pseudo-substrate, blocking the active site. Although there are several structures of GSK-3b, no information is available concerning how ATP-mimetic inhibitors interact with the enzyme. We present here five structures of GSK-3b in complex with selective and nonselective ATP-mimetic inhibitors (Figure 1). The structural information obtained from these complexes provides a useful tool for the design of second-generation GSK-3b specific inhibitors. Results and Discussion General structure The five structures of GSK-3b presented here were solved by the difference Fourier method using the coordinates of the unphosphorylated GSK-3b.11 As expected, the overall structures of the enzyme (Figure 2) are similar to that of the apoenzyme. In fact, the RMSD calculated for 333 structurally equivalent atoms in the apoenzyme (PDB code 1H8F) and the inhibitor complexes range from 0.3 Å to 0.6 Å. Several sections of the structure lacked interpretable electron density, indicating a high degree of disorder. These include the N-terminal segment 1– 34, the loop 120 –124 and the C-terminal segment 386 –420. In addition, the electron density was weak for the region 285 – 295. The crystals contain two molecules of GSK-3b in the asymmetric unit and, in general, the two molecules are treated as identical unless specified otherwise. The chemical structures of the ATPmimetic inhibitors co-crystallized with GSK-3b are shown in Figure 1 and the corresponding X-ray data collection and refinement statistics are in Table 1. AMP-PNP complex The 2.4 Å crystal structure of GSK-3b with the non-hydrolyzable ATP analog adenyl imidodiphosphate (AMP-PNP) provides a logical starting point for understanding how ATP-mimetic inhibitors interact with GSK-3b. Difference electron density shows the AMP-PNP molecule and the two magnesium ions bound in the GSK-3b active site (Figure 3(a)). Interestingly, all three nucleotide phosphate groups and the two magnesium ions are all well defined in the binary complex. As observed in the previous structures of protein kinases with ATP-analogs, AMP-PNP binds in the cleft formed between the N- and C-terminal lobes of GSK-3b, with the adenine group making hydrogen bonds with the hinge residues Asp133 and Val135 (Figures 2 and 4(a)). In addition to the polar interactions, the adenine group also makes hydrophobic interactions with GSK-3b residues Ile62, Val70, Ala83, Val110, Leu132, Tyr134 and Leu188. The ribose group of AMP-PNP interacts with GSK-3b through a single hydrogen bond between O3 and the carbonyl oxygen of Glu185. No direct hydrogen bonds are observed between O2 and GSK-3b residues. This is in contrast to what was observed for several other protein kinase 0 0 Figure 2. Stereo view showing the a-carbon trace of the GSK-3b complex with AMP-PNP and magnesium. Every 15th a-carbon and the magnesium ions are shown as spheres and AMP-PNP is shown as ball-and-stick. 396 Structures of GSK-3b Complexes Table 1. Crystal structure data and refinement statistics Data collection GSK-3b complex Space group Cell parameters (Å) a b c X-ray source Resolution (Å) No. observations Total Unique Completeness (%) Rsym I=sI Refinement Resolution range (Å) No. reflections Working set (%) Test set (%) Rcryst =Rfree RMSD Bond lengths (Å) Bond angles (8) AMP-PNP P21 21 21 Staurosporine P21 21 21 Indirubin-3-monoxime P21 21 21 Alsterpaullone P21 21 21 I-5 P21 21 21 82.69 85.21 178.14 ID14eh2 ESRF 2.40 83.73 87.05 177.80 ID14eh4 ESRF 2.20 83.43 86.44 178.82 ID14eh2 ESRF 2.10 82.67 85.99 178.19 ID14eh2 ESRF 2.30 84.17 86.66 178.65 ID14eh4 ESRF 2.77 206,766 50,028 98.9 (100.0) 0.076 (0.518) 15.9 (3.1) 185,970 63,705 95.4 (97.9) 0.060 (0.419) 12.6 (1.8) 334,335 76,152 99.8 (100.0) 0.060 (0.423) 21.3 (3.6) 194,732 57,037 99.6 (99.9) 0.060 (0.512) 18.1 (2.4) 106,100 33,530 98.5 (99.6) 0.093 (0.569) 10.8 (1.9) 30 –2.40 30–2.20 30–2.10 30–2.30 30 –2.77 47,034 2,468 0.206/0.233 60,456 3,206 0.230/0.252 72,232 3,844 0.229/0.245 54,132 2,846 0.225/0.248 31,837 1,660 0.212/0.251 0.010 1.5 0.010 1.4 0.012 1.5 0.010 1.4 0.009 1.4 Numbers in parenthesis correspond to the shell of data at highest resolution. complexes with nucleotides, in which a hydrogen bond is formed with a conserved residue shortly after the hinge. In GSK-3b, this residue is Thr138 and although it seems possible for a threonine to make this type of hydrogen bond, it is not the case in this structure. Instead, the Og of Thr138 is directed away from the ribose group, making a hydrogen bond with the backbone nitrogen of Arg141. The position and interactions made by the AMPPNP phosphate groups are indicative of a productive binding-mode. Indeed, a superposition of the active phosphorylated structure of CDK2 with AMP-PNP and peptide substrate17 (PDB accession code 1QMZ) onto the GSK-3b structure reveals that all the critical residues for phosphoryl-transfer occupy similar positions in the two structures. Lys85 hydrogen bonds to the aand b-phosphates of AMP-PNP and also forms a salt-bridge with a kinase-conserved glutamate (Glu97) from the C-helix. Mg1 coordinates four oxygen atoms; one from each of the a- and g-phosphates and one from each of the kinase conserved residues Asn186 and Asp200. Mg2 shows octahedral coordination of six oxygen atoms; one from each of the b- and g-phosphates, both carboxylate oxygen atoms from Asp200 and two water molecules. In addition, the kinase conserved residue Lys183 (catalytic loop) extends up towards the g-phosphate making hydrogen bonds with a g-phosphate oxygen and a carboxylate oxygen of another kinase-conserved residue Asp181 (catalytic loop). No direct interactions are observed between the AMP-PNP phosphate groups and the nucleotide-binding loop of GSK-3b. Instead the loop interacts with the phosphate groups through bridging water molecules and shields the phosphate groups from the bulk solvent. It is also noteworthy to mention that Og of Ser66, one of the residues from the nucleotide-binding loop, hydrogen bonds to a carboxylate oxygen of Asp264 from the other GSK-3b molecule in the asymmetric unit. Consequently, the position of the loop may be partially influenced by the intermolecular contact. However, the variation in its position in the different complexes presented here suggests that the loop adapts to the inhibitors. This adaptation appears to occur independently from the intermolecular contact, depending only on the inhibitor. A superposition of the other reported GSK-3b structures11 – 13 onto the GSK-3b/AMP-PNP complex reveals several significant differences. Firstly, the complex with AMP-PNP is the most “closed” structure with respect to the N- and C-terminal lobes. Indeed, a rotation that closes the interdomain angle by 4.48 for the two other unphosphorylated structures (PDB accession numbers 1H8F and 1IO9) and 7.28 for the active phosphorylated structure (PDB accession number 1GNG) is required to fully superimpose these structures onto the GSK-3b/AMP-PNP complex. Secondly, all the other structures contain a sulfate or phosphate group in the vicinity of Val214. However, no tetrahedral shaped molecules are observed in the vicinity of Val214 in any of the structures with inhibitors bound in the ATP-site (this work). Instead, two water molecules are located in the same region; one water bridges between the backbone nitrogen and Arg180 and the other extends Structures of GSK-3b Complexes 397 Figure 3. Electron density maps of the inhibitors bound to GSK-3b. The Fo 2 Fc omit map is contoured at 3.3s. The hinge region that interconnects the N- and C-terminal domains is shown to the left of the bound inhibitors and Gln185 in the lower right in green. Magnesium ions are shown in dark blue and water molecules in red. Inhibitors are shown in pink. (a) AMP-PNP, (b) staurosporine, (c) indirubin-3-monoxime, (d) alsterpaullone and (e) I-5. 398 Structures of GSK-3b Complexes Figure 4. Diagram showing the binding of (a) AMP-PNP, (b) staurosporine, (c) indirubin-3-monoxime and (d) alsterpaullone to GSK-3b. Residues playing a direct or indirect role in inhibitor binding are shown in a ball-andstick representation. Inhibitor molecules are shown in tan; water molecules and Mg2þ are shown in red and yellow, respectively. The Ca trace of GSK-3b is shown in blue and green for the N- and C-terminal domains, respectively. To avoid “cluttering” in the AMP-PNP diagram, the two water molecules coordinating Mg1 are omitted and Asp200, the residue coordinating Mg1 and Mg2, is unlabelled. out into the solvent region, making hydrogen bonds only with the first water molecule. A more detailed comparison of the AMP-PNP complex and the activated, Tyr-phosphorylated structure (1GNG) reveals other differences that are presumably related to the activation. In the AMPPNP complex, the aromatic ring of Tyr216 is rotated inwards so that it makes hydrophobic interactions with Val214. However, in the activated structure Tyr216 is rotated outward and the phosphate group interacts with Arg220 and Arg223. Nevertheless, the main-chain paths for residues 213– 223 in both the unactivated and the activated structures are similar, with the largest deviation being observed for Tyr216. Staurosporine complex Staurosporine is a potent inhibitor of GSK-3b, with a reported IC50 value of 15 nM.18 The 2.2 Å co-crystal structure of staurosporine with GSK-3b reveals how the inhibitor binds in the ATP-binding site (Figures 3(b) and 4(b)). The hinge interactions include hydrogen bonds from N1 of staurosporine to the Asp133 carbonyl oxygen and from O5 of staurosporine to the backbone nitrogen of Val135. These hinge interactions mimic those observed for the adenine ring of AMP-PNP. Surprisingly, these are the only direct hydrogen bonds observed between GSK-3b and staurosporine. The other polar interaction is a water-mediated interaction 399 Structures of GSK-3b Complexes from the methylamino nitrogen (N4) of the glycosidic ring to the carbonyl oxygen of Gln185. This water-mediated interaction is in contrast to the direct interaction observed in the other structures of protein kinases in complex with staurosporine.19 – 23 In addition, the structures of CDK2, Chk1, Lck and PKA in complex with staurosporine also contain a hydrogen bond to a conserved residue shortly after the hinge (Thr138 in GSK-3b). No interaction of this type is observed in the GSK-3b complex with staurosporine. As observed in the AMP-PNP complex, Thr138 makes hydrogen bonds to the backbone nitrogen of Arg141 and to a conserved water molecule. In the staurosporine complex, this water molecule is part of a hydrogen-bonding network that starts with Og of Thr138, passes through four water molecules and ends with the carbonyl oxygen of Val135 (Figures 3(b) and 4(b)). Interestingly, this water network fills the cavity that exists under the fuzed carbazole moiety of staurosporine. Staurosporine also makes a significant number of hydrophobic interactions with GSK-3b, especially through the fuzed carbazole moiety. Indeed, formation of the GSK-3b/staurosporine complex buries 891 Å2 of surface area. Residues that contribute to this surface include Ile62, Gly63, Gly65, Val70, Ala83, Asp133, Tyr134, Gln185, Asn186, Leu188, Cys199 and Asp200. A superposition of the AMP-PNP and the staurosporine complexes with GSK-3b using the C-terminal domain reveals several differences with respect to inhibitor binding. One difference is the angle of binding in the active site. The adenine plane of AMP-PNP comes in to interact with the hinge-region of GSK-3b in the “classical” way for a protein kinase. However, the plane of the fuzed carbazole moiety of staurosporine interacts with a significantly different angle. A comparison of the two planes reveals a difference in angle of approximately 158. The difference in angle also explains why there are no direct interactions between GSK3b and the glycosidic-ring of staurosporine. This is because the glycosidic-ring is located slightly above the ribose-binding pocket. This lack of occupancy of the ribose pocket for a protein kinase in complex with staurosporine appears to be unique for GSK-3b. In the other complexes with staurosporine, the fuzed carbazole moieties are coplanar with the adenine ring plane and the glycosidic rings are bound in the ribose pocket.19 – 23 Another difference that can be observed from the superposition of the GSK-3b complexes with AMPPNP and staurosporine is the position of the N-terminal domain. In the complex with staurosporine, the N-terminal domain has rotated back roughly 108 from its position in the AMP-PNP complex, increasing the interdomain angle to allow greater access to the ATP binding-site. Presumably, staurosporine binding has induced the N-terminal domain movement in order to occupy a preferred binding mode. However, it is unclear why this binding mode is preferred over the more classical mode of staurosporine binding observed in the other protein kinase/staurosporine complexes.19 – 23 Indirubin-30 -monoxime Indirubins have been reported as potent inhibitors (IC50 values in the 5– 50 nM range) of GSK-3b.18 Within the series, the two most potent GSK-3b inhibitors are indirubin-30 -monoxime (IC50 ¼ 22 nM) and 5-iodoindirubin-30 -monoxime (IC50 ¼ 9 nM). Indirubins are also described as potent inhibitors (IC50 values in the 50– 100 nM range) of cell cycle regulating cyclin-dependent kinases (CDKs).24 This finding led to crystal structures of both active and inactive CDK2 in the presence of substituted indirubins; CDK2 (inactive) with indirubin-30 -monoxime, CDK2 (inactive) with indirubin-5-sulphonic acid and CDK2/CyclinA (active) with indirubin-5-sulphonic acid.24,25 Interestingly, these two compounds highlight the differences in specificity within the class for GSK3b and the CDKs.18 While the 30 -monoxime substituent is extremely potent in GSK-3b (IC50 ¼ 22 nM) it is significantly less potent against CDK2/CyclinA (IC50 ¼ 440 nM). However, the opposite pattern is reported for the 5-sulphonic acid substituent, with the compound being more potent for CDK2/CyclinA (IC50 ¼ 35 nM) than GSK-3b (IC50 ¼ 280 nM). To determine the basis of this selectivity pattern, indirubin-30 -monoxime was co-crystallized with GSK-3b. The 2.1 Å structure reveals a donor – acceptor –donor series of hydrogen bonds between indirubin-30 -monoxime and the hinge residues of GSK-3b (Figures 3(c) and 4(c)). The N1 and O2 atoms form hydrogen bonds with the carbonyl oxygen of Asp133 and the backbone nitrogen of Val135, respectively. These interactions are similar to those observed in the complexes with AMP-PNP and staurosporine. However, indirubin-30 -monoxime also makes a third hydrogen bond with N1 and the carbonyl oxygen of Val135. This pattern of hinge interactions was also observed in the structures of CDK2 in complex with indirubin-30 -monoxime and indirubin-5-sulphonic acid.24,25 In addition to the hinge interactions with indirubin-30 -monoxime, GSK-3b also interacts with the inhibitor through a bridging water molecule; the oxime oxygen hydrogen bonds with a water molecule that, in turn, hydrogen bonds with the backbone oxygen of Gln185 (Figure 4(c)). Another water molecule, that is also hydrogen bonded to the Og of Thr138, helps to position the bridging water molecule. Interestingly, both of these water molecules were also observed in the staurosporine complex. A final feature that explains the potency of indirubin-30 -monoxime is its complementary shape that allows it to bury its apolar ring system in GSK-3b. Formation of the GSK-3b/indirubin-30 -monoxime complex buries 683 Å2 of surface area in a tight-fitting pocket. The pocket sandwiches the inhibitor between Ile62, 0 400 Val70, and Ala83 on the top and Leu188 on the bottom. The complementary shape of the GSK-3b hinge segment Leu132-Asp133-Tyr134-Val135Pro136 forms another portion of the pocket. Finally, Arg141 forms the final piece of the pocket by orienting its side-chain to shield the edge of the inhibitor’s aromatic ring from the bulk solvent. In the case of GSK-3b, modification of the indirubin scaffold to obtain indirubin-30 -monoxime gives a significant (27-fold) improvement in inhibitor potency.18 Based on the structure, this improvement can be attributed to the bridging water molecule that links the inhibitor’s hydroxyl group to the carbonyl oxygen of Gln185. In addition, the combination of the monoxime group and the bridging water provide a partial occupancy of the ribose pocket. Unfortunately, coordinates for the CDK2 (inactive) indirubin-30 monoxime complex were not available for comparison with GSK-3b.24 However, the minor change in CDK2/CyclinA potency (fivefold) with respect to modifying indirubin to obtain indirubin-30 monoxime suggests that CDK2 does not benefit from the same type of water interaction. Superposition of CDK2/CyclinA with indirubin5-sulphonic acid25 (PDB accession number 1E9H) onto GSK-3b with indirubin-30 -monoxime provides an understanding of the potencies with respect to indirubin modifications in the 5-position.18,24 In the case of CDK2/CyclinA, the inhibitor obtains a large increase in potency by the addition of the sulphonic acid group in position 5, going from an IC50 value of 2200 nM for indirubin to 35 nM for indirubin-5-sulphonic acid (63-fold improvement). In contrast, for GSK-3b, the inhibitor obtains a significantly smaller increase in potency, going from an IC50 value of 600 nM for indirubin to 280 nM for indirubin-5-sulphonic acid (twofold improvement). Moreover, in the case of indirubin-30 monoxime the addition of a sulphonic acid group in the 5-position actually causes a fourfold decrease in affinity (IC50 ¼ 22 nM for indirubin-30 monoxime and IC50 ¼ 80 nM for indirubin-30 monoxime-5-sulphonic acid). In the structure of CDK2/CyclinA with indirubin-5-sulphonic acid, the sulphonic acid group is directed away from the hinge and towards the DFG segment (CDK2 residues 145– 147 and GSK-3b residues 200– 202). CDK2 residue Lys33 (Lys85 in GSK-3b) bridges between one of the inhibitor’s sulphoryl oxygen atoms and the side-chain of Glu51 (Glu97 in GSK3b). Another sulphoryl oxygen makes a hydrogen bond with the backbone nitrogen of Asp145 (Asp200 in GSK-3b). The combination of the open shape of the pocket and the complementary charge in the region of the inhibitor’s sulphonic acid help to explain CDK2’s affinity for indirubin-5sulphonic acid. Analysis of the corresponding region in GSK-3b reveals that a similar pocket exists with the correct charge distribution for accommodating a sulphonic acid group. However, a minor modification of the shape of the pocket in GSK-3b and the resulting steric conflict may Structures of GSK-3b Complexes explain the difference in affinity for indirubin-5sulphonic acid. This is due to a single residue change in the amino acid composition of the pocket. In CDK2, Ala144 helps to form the bottom of the pocket and makes a van der Waals contact with an oxygen of the sulphonic group (3.4 Å). This is the same oxygen that hydrogen bonds with the backbone nitrogen of Asp145 (see above). However, in GSK-3b the corresponding residue is Cys199 and the side-chain Sg is directed up into the pocket. As a result, the pocket contains an indentation that could potentially clash with a bulky substituent, such as a sulphonic acid group, in the 5-position of indirubin. In addition, having a cysteine side-chain in this position could potentially block the formation of the hydrogen bond between the sulphonic acid’s oxygen atom and the adjacent residue’s backbone nitrogen (Asp145 in CDK2 and Asp200 in GSK-3b). Presumably, the combination of the indentation in the pocket and the loss of the hydrogen bond would result in a shift of the inhibitor to favorably reposition a bulky 5-position substituent. However, the shift in position would provide a new environment with modified characteristics for the substituent. Further analysis of the GSK-3b IC50 values with respect to substitution of the indirubin 5-position reinforces this interpretation. Although a sulfonic acid group is poorly supported in this position a slight modification in charge and hydrogen bonding capabilities to sulfonamide makes a favorable substituent for GSK-3b (IC50 ¼ 40 nM for indirubin-5-sulfonamide). Alsterpaullone Paullones have been reported as potent ATPcompetitive inhibitors of CDKs with promising antitumoral properties.26 In addition, a subsequent modeling study of the paullones bound in the CDK1 active site helped to explain the relation between inhibitor modifications and experimental IC50 values.27 More recently, paullones were also shown to be potent inhibitors of GSK-3b and CDK5/p25.28 Alsterpaullone (9-nitropaullone), the most potent GSK-3b inhibitor of the series (GSK3b IC50 value of 4 nM), was shown to compete with ATP for binding in the GSK-3b active site. In addition, an investigation of the selectivity of alsterpaullone using 25 purified kinases has shown the compound to be extremely selective for GSK-3a/b and CDK1/2/5.28 The combination of high potency and selectivity for GSK-3b led us to determine the structure of GSK-3b in complex with alsterpaullone. The 2.3 Å co-crystal structure shows alsterpaullone bound in the GSK-3b active site (Figures 3(d) and 4(d)). Alsterpaullone’s hinge interactions include two direct hydrogen bonds with Val135 and one water mediated interaction with Asp133; the N5 and carbonyl oxygen atoms of alsterpaullone make a pair of hydrogen bonds with the backbone nitrogen and carbonyl oxygen of Val135, respectively, and the water molecule 401 Structures of GSK-3b Complexes bridges between the carbonyl oxygen atoms of alsterpaullone and Asp133. In addition to the hinge interactions, alsterpaullone also makes polar interactions between the nitro group in position 9 and the side-chain amino group of Lys85. The final polar interaction between alsterpaullone and GSK-3b occurs through a bridging water molecule; the water hydrogen bonds with N12 of alsterpaullone and the carbonyl oxygen of Gln185. A water molecule in this position was already observed in the GSK-3b structures with staurosporine and indirubin-30 -monoxime and, in all three cases, the water is oriented by a hydrogen bond network that starts with Thr138 Og. One feature that makes alsterpaullone unique is the positioning of its coupled ring systems. In the complex with GSK-3b, the inhibitor’s sevenmembered ring is situated slightly below the adenine-binding pocket and the pucker of ring B directs the attached ring systems (Figures 1 and 4(d)). Ring A extends downward, following the path of the hinge towards the guanidine group of Arg141. Rings C and D extend away from the hinge, passing through the ribose pocket and up towards the side-chain amino group of Lys85. The shape of alsterpaullone also allows it to bury itself in the GSK-3b active site. Indeed, formation of the alsterpaullone/GSK-3b complex buries 676 Å2 of surface area. The coupled ring systems blanket over residues Thr138, Leu188, and Cys199. The complementary shape of rings A and B follows the contour of the hinge residues Asp133-Tyr134Val135-Pro136 and the side-chain of Arg141 caps the end of the pocket. Finally, Asp200 and the side-chains of Lys85 and Leu132 form the surface around alsterpaullone’s NO2 group. One significant difference from the other complexes with GSK-3b is the absence of van der Waals interactions from the N-terminal residues Ile62 and Val70. In the complex with alsterpaullone, the nucleotide-binding loop is shifted up slightly so that no direct interactions are made with the inhibitor. Not surprisingly, several residues from the nucleotide-binding loop are poorly defined in the electron density as a result of disorder. values of less than 50%. Also, although a number of the other GSK-3b inhibitors have been reported as selective, the majority also inhibit the CDKs at a similar level. However, the most potent 3-anilino4-arylmaleimides are not inhibitors of CDK2/ cyclin A.31 This finding led us to investigate the 3-anilino-4-arylmaleimides as inhibitors of GSK3b. For this purpose, 2-chloro-5-{[4-(3-chlorophenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]amino}benzoic acid (Figure 1), hereinafter referred to as I-5, was synthesized using the protocols of Smith and co-workers.31 In order to take advantage of commercially available starting materials, the 3-chloro substituent of ring B (GSK-3a IC50 ¼ 76 nM) was chosen over the more potent 2-NO2 (GSK-3a IC50 ¼ 28 nM) or 3-NO2 (GSK-3a IC50 ¼ 26 nM) ring B substituents. Although Smith and co-workers reported a GSK-3a IC50 value for I-5, no value has been reported for GSK-3b. Thus, in addition to solving the 2.8 Å co-crystal structure of I-5 and GSK-3b, IC50 (160 nM) and isothermal titration calorimetry (Figure 5; KD ¼ 40 nM) measurements were made on GSK-3b using I-5. Finally efforts were made to further investigate the selectivity of I-5. The crystal structure of I-5/GSK-3b shows the inhibitor bound in the active site, making an elaborate number of interactions with residues from both the N- and C-terminal lobes (Figures 3(e) and 6). Not surprisingly, the hinge The 3-anilino-4-arylmaleimide inhibitor I-5 Inhibitors containing a maleimide core structure have been shown to be potent inhibitors of GSK3b.29 – 31 Initial reports on GSK-3b inhibition by maleimides focused on the bisindoylmaleimide derivatives of staurosporine that were being widely used as inhibitors of protein kinase C.30 However, the lack of selectivity of these compounds made them less appealing for GSK-3b inhibitor development. More recently, 3-anilino-4arylmaleimides have been reported as potent, selective inhibitors of GSK-3a/b.31 In the same report, the selectivities of several compounds in the class were evaluated using a panel of 25 kinases. At an inhibitor concentration of 10 mM, the majority of the kinases showed inhibition Figure 5. Titration experiment showing the binding of I-5 to unphosphorylated GSK-3b. The isothermal titration calorimetry data were measured in PBS buffer with 1 mM DTT. The thermodynamic parameters determined by least-square fitting were, n ¼ 1:02; KB ¼ 2:5 ^ 0:4 £ 107 Mol21 ðKD ¼ 40 nMÞ; DHobs ¼ 12:2 ^ 0:2 kcal=mol; TDS ¼ 22:5 kcal=mol; DG ¼ 29:7 kcal=mol: 402 Structures of GSK-3b Complexes Figure 6. Stereo view showing I-5 bound in the GSK-3b active site. Residues playing a direct or indirect role in inhibitor binding are shown in a ball-and-stick representation. I-5 is shown in light brown; chlorine atoms are in green and water molecules are in red. The Ca trace of GSK-3b is shown in blue and green for the N- and C-terminal domains, respectively. For the sake of clarity, Leu132 and Tyr134 are unlabelled and Val110 has been omitted from the Figure. interactions between I-5 and GSK-3b are similar to those observed for staurosporine; N1A of I-5 to the carbonyl oxygen of Asp133 and O2A of I-5 to the backbone nitrogen of Val135. Also, ring A of I-5 is sandwiched between Ala83, on top, and Leu188, on the bottom. However, besides the ring A interactions, I-5 makes no other interactions that resemble those of staurosporine. Instead, the inhibitor takes advantage of the lack of interconnectivity between the rings to rotate rings B and C out of plane from ring A. Ring B is rotated out of the plane from ring A to allow maximum interactions with Val70, Lys85 and Cys99. Ring C is also rotated out of plane from the ring A plane, allowing it to make extensive interactions with the nucleotide-binding loop. Substituents on rings B and C contribute to I-5’s binding affinity for GSK3b with several specific interactions. The chlorine atom of ring B extends up towards the amino group of Lys85, making van der Waals interactions. As for ring C, the chlorine atom is directed towards the nucleotide-binding loop, making interactions with backbone atoms from Gly63 and Asn64. Ring C’s carboxylate group makes the most intricate series of interactions; one carboxylate oxygen hydrogen bonds with Nh1 and Nh2 of Arg141 and the other with the Ne2 of Gln185 and a water molecule (Wat1). Interestingly, Wat1 is held in place by hydrogen bonds to Og of Thr138, as observed in several other complexes, and to Oe2 of Gln185. The three-dimensional form of I-5 allows it to bury itself in the GSK-3b active site. In all, formation of the GSK-3b/I-5 complex buries 779 Å2 of surface area. Residues contributing to this surface include Ile62, Gly63, Asn64, Gly65, Val70, Ala83, Lys85, Val110, Leu132, Val135, Thr138, Arg141, Gln185, Asn186, Leu188, Cys199, and Asp200. One significant difference from the other complexes is the detailed number of interactions made with the nucleotide-binding loop. In the GSK-3b/I-5 complex, the loop folds down towards the inhibitor and, in so doing, forms pockets around rings B and C. A superposition of the AMP-PNP and I-5 complexes using the C-terminal domain shows how GSK-3b adapts to the inhibitor. The reorganization of the nucleotide-binding loop in the I-5 complex begins with the segment Ile62Gly63-Asn64 shifting up slightly to accommodate the chlorine substituent of ring C. After Asn64, the loop shifts downwards towards Asp200, with Gly65-Ser66 occupying a similar position to that of the b-phosphate group of AMP-PNP. Subsequently, the loop shifts gradually back up through the segment Phe67-Gly68. In addition to the rearrangement of the nucleotide-binding loop, two residues in the active site, Arg141 and Gln185, also shift positions to accommodate I-5. In the AMP-PNP complex, Arg141 makes charge– charge interactions with both side-chain oxygen atoms of Glu137 and the carbonyl oxygen of Gln185 interacts with the 30 -OH group of AMP-PNP. However, in the I-5 complex the side-chains of Arg141 and Gln185 both rotate around to interact with the inhibitor’s carboxylate oxygen atoms. In order to evaluate I-5’s level of selectivity, the inhibitor was run against an internal kinase selectivity screen (KSS) that includes 30 different kinases. Two motivating factors for running I-5 through the KSS were (1) the fact that no information was available concerning the selectivity of I-5 and (2) the fact that the KSS includes several kinases that were not present in the panel used by Smith and co-workers to evaluate four different Interestingly, in 3-anilino-4-arylmaleimides.31 addition to GSK-3b (IC50 ¼ 160 nM), I-5 also inhibits two other protein kinases, VEGFR3 (also known as Flt-4; IC50 ¼ 560 nM) and FGFR1 403 Structures of GSK-3b Complexes (IC50 ¼ 1,200 nM). These results were somewhat surprising given the level of selectivity reported for the other 3-anilino-4-arylmaleimides.31 One potential explanation could be due to the fact that different compounds were tested in different screens. However, this explanation seems unlikely considering the slight differences between the compounds tested in the screens; I-5 contains a chlorine atom in position 3 of ring B while the most similar compound tested by Smith and co-workers contains a nitro group in the same position. A more likely explanation for the difference in selectivity would be due to the differences in the number and types of protein kinases tested in the screens. In effect, no members of the VEGFR or FGFR subfamily of kinases were included in the selectivity screen run by Smith and co-workers. Therefore, it seems logical that the 3-anilino-4-arylmaleimides, in general, inhibit VEGFR3 and FGFR1. The selective inhibition of GSK-3b, VEGFR3 and FGFR1 by I-5 can be explained on a structural basis using sequence and structural comparisons between the three enzymes. Table 2 shows the residues in GSK-3b that interact with I-5, along with the corresponding residues in the other kinases. In addition, a superposition of the kinase domain structures of GSK-3b (I-5 complex) and FGF1R32 (PDB accession number 1FGI) allows us to dock I-5 into the FGF1R active site (Figure 7). Not surprisingly, based on the IC50 value of 1.2 mM, I-5 fits well into the ATP-site of FGFR1. Only a minimum amount of rearrangement of the nucleotide-binding loop appears necessary to accommodate the inhibitor. A comparison of the positions of the interacting atoms in 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5402), the oxindolinone inhibitor present in the FGFR1 structure,32 and those of I-5 reinforce Table 2. Amino acid compositions in the ATP-binding pocket GSK-3ba CDK2 FGFR1 VEGFR3 Ile62b Gly63b Asn64b Val70 Ala83 Lys85 Val110 Leu132 Asp133b Tyr134 Val135b Thr138 Arg141 Gln185 Leu188 Cys199 Asp200 † † Glu † † † † Phe Glu Phe Leu Asp Lys † † Ala † Leu † Glu † † † Ile Val Glu † Ala Asn Glu Arg † Ala † Leu † Tyr † † † † Val Glu Phe Cys Asn Asn Arg † † † a Listed are GSK-3b residues that interact (interatomic distance ,3.9 Å) with I-5. b Residues that interact with I-5 using only main-chain atoms. †, indicates the same residue as in GSK-3b. the docking results. Clearly, the hinge interactions made by I-5 would be similar to those made by the oxindolinone ring of SU5402. However, the surprising result is the similarity in the positions of the carboxylate groups in the two inhibitors. Based on the interactions observed between SU5402’s carboxylate group and the side-chain nitrogen of FGFR1’s Asn568, we would expect the carboxylate of I-5 to make a similar interaction with Asn568. Actually, closer analysis of the superimposed structures reveals that the position occupied by FGFR1 atom Nd2 of Asn568 corresponds to Wat1 in the GSK-3b structure. Hence, Asn568 in FGFR1 replaces the combination of Thr138 and Wat1 in GSK-3b that interact with the carboxylate of I-5. In addition to Wat1, Arg141 and Gln185 also interact with I-5’s carboxylate group in the GSK-3b/I-5 complex. The corresponding residues in FGFR1 are Glu571 and Arg627, respectively. Based on the position of these residues in the FGFR1/SU5402 structure, we would not expect these residues to behave differently in a complex with I-5; Glu571 hydrogen bonds with Nd2 of Asn568 and makes charge– charge interactions with the guanidino group of Arg570 while Arg627 extends out away from Asn568. The lack of an available structure for VEGFR3 makes it impossible to perform a similar docking with I-5. However, using the sequence similarities between the kinase domains of VEGFR3 and FGFR1 (Table 2) and recognizing the reported similarities in structure between FGFR1 and VEGFR233 we can draw conclusions about the VEGFR3/I-5 complex. Firstly, we would expect I-5 to fit well into the active site and make similar hinge interactions. Secondly, VEGFR3 would take advantage of the Asn in the position equivalent to Thr138 in GSK-3b to interact with I-5’s carboxylate group. Finally, we would expect I-5 to fit slightly better in the VEGFR3 active site than in the FGFR1. This conclusion is based on the fact that VEGFR3 also has a cysteine in the position that corresponds to Cys199 in GSK-3b. Therefore, VEGFR3 would be able to make van der Waals interactions with I-5’s ring B that would not be possible with the alanine in FGFR1. I-5 inhibits GSK-3b, VEGFR3 and FGFR1 but, at an inhibitor concentration of 10 mM, does not inhibit CDK2/cyclinA. This finding is likely due to the fact that CDK2 contains an acidic residue (Asp86) in the position that corresponds to Thr138 in GSK-3b (Table 2). As mentioned above for FGFR1 and VEGFR3, an asparagine or, in this case, an aspartic acid could potentially replace the interaction made by Thr138 and Wat1. However, at neutral pH an aspartic acid would not be able to form a similar hydrogen bond with the I-5’s carboxyl group. By having an acidic group in this position, I-5 obtains selectivity against those kinases that contain an acidic residue in the equivalent position, a population that corresponds to roughly 50% of the known kinases. Of course, 404 Structures of GSK-3b Complexes Figure 7. Stereo view showing I-5 docked into the active site of FGFR1 by the superposition of Ca atoms in the ATPbinding sites (Table 2) of GSK-3b and FGFR1. I-5 is shown in light brown and SU5402 in blue. For the sake of clarity, Gly485, Glu486 and Val561 are unlabelled and Ile545 has been omitted from the Figure. for those kinases having non-acidic residues in this position there are additional factors to consider in order to obtain a reasonable level of inhibition. Obviously, a logical way to determine the importance of I-5’s carboxyl group for the selectivity against CDK2 would be to analyze a similar 3-anilino-4-arylmaleimide that does not contain a carboxyl group in the 3-position. In the report by Smith and co-workers,31 two compounds satisfying these criteria were analyzed for percent inhibition using the 25 kinases, but unfortunately, no values were reported for CDK2/cyclinA with these compounds. Interestingly, Mohammadi and co-workers used a similar explanation for why SU5402 is specific for FGFR1.32 Apparently, SU5402 also takes advantage of its carboxyl group to obtain selectivity for FGFR1 and against both the insulin and PDGF receptors. This observation and other similarities between the two inhibitors raise the question of whether SU5402 would also inhibit GSK-3b. In conclusion, the structures of five GSK-3b complexes with inhibitors provide, for the first time, information concerning how the enzyme interacts with selective and non-selective inhibitor scaffolds. Staurosporine, a non-selective inhibitor, interacts with GSK-3b in a unique manner that utilizes a water network to adapt to the enzyme. Indirubin30 -monoxime benefits from both the complementarity between the inhibitor and the enzyme and the conserved water molecules (Wat1 and Wat2). Alsterpaullone uses the form of the coupled ring systems to direct the interacting groups of the inhibitor towards the hinge, Lys85 and the conserved water molecules (Wat1 and Wat2). I-5 takes advantage of its ability to independently rotate the attached ring systems, allowing it to position substituents that interact with the nucleotide-binding loop, Lys85, Arg141, Gln185 and Wat1. On the basis of selectivity, indirubin-30 -monoxime and alsterpaullone can be grouped together because both inhibitors have similar problems for selectivity with respect to the CDKs. In contrast, I-5 does not have selectivity problems with the CDKs but does have issues with respect to the tyrosine kinases VEGFR3 and FGFR1. I-5 obtains selectivity versus the CDKs by taking advantage of sequence differences just outside of the ATP-binding cleft. Unfortunately, in this case, the strategy of going outside the ATP-binding cleft to obtain specificity led to new selectivity issues. However, future efforts could potentially take into account other differences between GSK-3b, VEGFR3 and FGFR1 to obtain selective GSK-3b inhibitors. Materials and Methods Construct design, expression and purification of GSK-3b DNA encoding for full length GST was amplified by PCR, incorporating an N-terminal Xba I restriction site and C-terminal stop codon, a sequence corresponding to the Prescission site and Bam HI restriction site. The product was digested with Xba I and Bam HI ligated into appropriately digested pVL 1392 (Pharmingen, San Diego, CA), creating the pVL-GST vector. DNA encoding for full length GSK-3b was amplified by PCR, incorporating an N-terminal Bgl II restriction site and C-terminal stop codon and Bgl II restriction site. The product was digested with Bgl II and ligated into pVL-GST digested by Bam HI. Spodoptera frugiperda insect cells, SF21, were transfected with the pVL-GST-GSK-3b plasmid using the baculogold transfection system (Pharmingen) as described by the manufacturer’s protocol. One liter of High Fivee cells (Invitrogen, San Diego, CA) in suspension at 2 £ 106 cells/ml was inoculated with 10 ml of amplified GSK-3b virus and incubated at 27 8C for 48 hours. Cells were harvested by centrifugation at 1500g for 15 minutes. Cells were frozen by submersion in dry ice/alcohol and then thawed when needed for purification. 405 Structures of GSK-3b Complexes For the purification of GST-GSK-3b, cells were re-suspended in 100 ml of buffer A (50 mM Hepes (pH 7.2), 10% (v/v) glycerol, 20 mM DTT, 150 mM NaCl, protease inhibitor cocktail (1 tablet/25 ml of buffer; Roche Biochemicals)) and mechanically homogenized. The lysate was centrifuged at 25,000g for 45 minutes to remove insoluble material. The supernatant was passed over a 0.8 mm vacuum filter and the filtrate was loaded onto a Glutathione Sepharose column (Amersham Biosciences Biosciences, Piscataway, NJ). After loading, the column was washed with five column volumes of buffer A followed by five column volumes of buffer A with only 10 mM DTT, and finally five column volumes of buffer B (50 mM Hepes (pH 7.2), 10% glycerol, 3 mM DTT, 150 mM NaCl). The protein was cleaved on the column by adding half a column volume of buffer B and Prescission enzyme (30 ml/ml of resin; Amersham Biosciences Biosciences) for 16 hours at 4 8C. After cleavage, the protein was eluted with 4 £ 1/2 column volumes of buffer B and fractions containing GSK-3b, as analyzed by SDS-PAGE, were pooled and dialyzed against 50 mM Hepes (pH 7.2), 10% glycerol, 3 mM DTT, 80 mM NaCl. After dialysis, GSK-3b was loaded onto Mono S column (Amersham Biosciences) and the column was washed for 15 column volumes. Next, a linear gradient was applied from 0 to 100% of elution buffer (50 mM Hepes (pH 7.2), 10% glycerol, 3 mM DTT, 500 mM NaCl) for 100 column volumes. GSK-3b was thereby separated into three peaks corresponding to the different phosphorylation forms (unphosphorylated, monophosphorylated and diphosphorylated). Fractions containing the different phosphorylated forms of GSK3b, as analyzed by SDS-PAGE, Western blot and ES-MS, were pooled and concentrated to approximately 12 mg/ ml in 10% glycerol, 185 mM NaCl and 100 mM Hepes (pH 7.2). A typical final yield for each liter of High Fivee culture was 1 – 2 mg of purified GSK-3b. ATP-mimetic inhibitors The inhibitors AMP-PNP, staurosporine, indirubin and alsterpaullone were obtained commercially. I-5, the 3-anilino-4-arylmaleimide inhibitor, was synthesized as described in the protocols reported by Smith and co-workers.31 crystals. A seed stock solution was prepared by crushing a single GSK-3b crystal in 20 ml of reservoir solution. At the time of crystallization, the seed stock solution was vortexed and 0.1 ml was added to the 1 ml of protein and reservoir solutions. X-ray data were collected at the ESRF beamlines ID141 and ID14-4 (Grenoble, France). Data were processed using DENZO and SCALEPACK.35 Bound ligands in the active site were identified by the difference Fourier method using the phases of the apoenzyme GSK-3b structure.11 Model building of the protein and inhibitors were performed with O36 and the structures were refined with CNX37 using non-crystallographic symmetry restraints for residues of the C-terminal lobes in the two monomers. Graphic Figures were made using MolScript version 2.138 and Raster3D version 2.4.39 Surface areas were calculated by the Lee and Richards algorithm40 using the software package CNX.37 A summary of the diffraction data and the refinement statistics are shown in Table 1. Activity assay FGFR1, GSK-3b and VEGFR2 were expressed as GSTfusion proteins and purified using a Glutathione Sepharose column. Each kinase was assayed for its ability to phosphorylate the appropriate biotin labeled peptide/protein substrate using radioactively labeled ATP. Reactions were stopped by the addition of a concentrated EDTA solution þ Streptavidin coated Scintillation Proximity Assay beads. The beads were then captured by CsCl flotation, counted by liquid scintillation and IC50 values were generated by fitting data using a sigmoidal dose-response (variable slope) model. Protein Data Bank accession numbers Atomic coordinates for the GSK-3b complexes with AMP-PNP, staurosporine, indirubin, alsterpaullone and I-5 have been deposited with the RSCB Protein Data Bank (accession codes 1PYX, 1Q3D, 1Q4I, 1Q3W, and 1Q4L, respectively). Crystallographic studies Acknowledgements All crystals of unphosphorylated GSK-3b were grown at 20 8C by vapor diffusion in hanging drops. Inhibitor concentrations of 1 mM in the crystallization drops were used for all crystallization trials. Crystals of the AMPPNP/Mg complex were grown by mixing 1 ml of protein solution with 1 ml of reservoir solution (16– 24% (w/v) PEG 3350 monodisperse, 5% glycerol, 20 mM magnesium chloride and 100 mM Hepes (pH 7.0)). These crystals are isomorphous to the apo GSK-3b crystals,11,12,34 with two molecules in the asymmetric b ¼ 85:2 A and unit and cell dimensions of a ¼ 82:7 A; Prior to data collection, a single crystal of c ¼ 178:1 A: GSK-3b-AMP-PNP was serially transferred into cryoprotectant solutions containing increasing levels of glycerol (final concentration 25%) and frozen by plunging into liquid nitrogen. In order to avoid the serial transfer step, subsequent crystal complexes were grown using 10% glycerol in the reservoir solution. 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