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The Schools of 1 Biosciences and 2 Medicine, The University of Birmingham, Birmingham, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because inflammatory diseases of the gastrointestinal tract predispose to the development of cancer and because COX-2 expression is elevated during the progression of colon carcinoma, it has become widely accepted that the anti-inflammatory and antineoplastic actions of NSAID are interrelated and mediated by COX-2 inhibition (1 , 2 , 15 , 16) . However, several lines of evidence indicate that the therapeutic actions of NSAID may also include targets other than COX. For example, the doses of aspirin required to treat chronic inflammatory diseases are greater than those required to inhibit COX. Also, aspirin derivatives that are not efficient COX inhibitors remain anti-inflammatory (17, 18, 19) . Similarly, the NSAID doses required to demonstrate antineoplastic activities in vitro have almost always been greater than those required for mere inhibition of either COX-1 or COX-2 (reviewed in Ref. 20 ). In addition, it has been demonstrated that a spectrum of COX-2-selective and nonselective NSAID displayed invariant antiproliferative and proapoptotic actions against transformed embryo fibroblasts, irrespective of whether the cells were derived from wild-type, COX-1-/-, COX-2-/-, or COX-1-/- and COX-2-/- mice (4) . These observations combine to indicate strongly that a second non-COX target exists in the antineoplastic actions of NSAID.
Besides inhibiting COX-1 and COX-2, NSAID also inhibit members of the NAD(P)H-dependent aldo-keto reductase (AKR) family, including the human enzyme AKR1C3, also known variously as 3
-hydroxysteroid dehydrogenase type 2 (21)
, 17ß-hydroxysteroid dehydrogenase type 5 (22)
, and PGD2 11-ketoreductase (23)
. AKR1C3 has a broad tissue distribution, including tissues where NSAID have been shown to protect against cancer. In terms of Km and kcat/Km, PGD2 represents a strong candidate for a physiologically relevant in vivo substrate (23)
. AKR1C3 converts PGD2 to PGF2 (23)
, which in the adipocyte model has been shown to block differentiation by indirect antagonism of peroxisome proliferator-activated receptor (PPAR) 
(24)
. When not metabolized to PGF2, PGD2 is nonenzymatically converted to PGJ2 and thence stepwise to 15-deoxy-
12,14-PGJ2, a natural activating ligand for PPAR (25)
. Thus, the relative PGD2 11- ketoreductase activity of AKR1C3 is likely to be a key determinant of PPAR activity within cells.
We and others have shown that the human acute myeloid leukemia cell lines HL-60 and KG1 express AKRIC3 (26
, 27)
and that treatment of HL-60 cells with AKR1C3 inhibitors [including indomethacin (IMN)] results in increased sensitivity to the antiproliferative and prodifferentiative actions of all-trans-retinoic acid and 125-dihydroxyvitamin D3 (27
, 28)
. Similarly, exposure of HL-60 cells to an excess of PGD2 mimics the action of AKR1C3 inhibitors in promoting all-trans-retinoic acid-induced differentiation. Reciprocally, overexpression of AKR1C3 in HL-60 cells increases resistance to all-trans-retinoic acid and 125-dihydroxyvitamin D3 (29)
. Importantly, we recently observed that the capacity of PGD2 and the AKRIC3- inhibiting NSAID IMN to each promote the differentiation of HL-60 acute myeloid leukemia cells was negated by the PPAR antagonist GW9662. Furthermore, a large body of recent work has determined that PPAR is a key regulator of proliferation differentiation and apoptosis in diverse cells and that both synthetic and natural PPAR ligands exert antineoplastic activity in diverse in vitro and animal models of neoplasia (for recent examples, see Refs. 30, 31, 32, 33, 34
). We have therefore proposed that AKR1C3 provides a plausible target for non-COX-dependent antineoplastic activities activities of NSAID (29)
.
The possibility that the chemoprotective actions of NSAID against cancer are mediated via a non-COX mechanism may provide new clinical in roads to the management of these diseases. At present, the use of NSAID is limited by their COX-associated toxicities. It may therefore be beneficial to derive cancer drugs that are more directed against AKR1C3 and that limit these toxicities. Thus, to explore the mechanism of NSAID inhibition of AKR1C3 and enable the future development of non-COX-selective inhibitors with potential antitumor and other clinically beneficial effects, we have initiated a structure-function study of recombinant AKR1C3 and report here on the high-resolution crystal structures of ternary complexes containing AKR1C3, NADP+, and one of acetate, flufenamic acid (FLF), or IMN (Fig. 1)
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MATERIALS AND METHODS |
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Production of AKR1C3 Expression Vector.
The AKR1C3 open reading frame was amplified from a cDNA clone (KIAA0119; Kazusa DNA Research Institute, Chiba, Japan) by PCR using primers based on the 5' end (5'-CAGCATATGGATTCCAAACAGCAG-3') and the 3' end (5'-CAGCTCGAGATATTCATCTGAATATGGTAT-3') that introduced an NdeI site that overlapped the start codon and a downstream XhoI cloning site (excluding the stop codon). The resulting PCR fragment was cloned into pGEMT-Easy vector (Promega). After digestion with NdeI and XhoI, the fragment was gel purified and ligated into pET21b(+) (Novagen), yielding pET21b-AKR1C3. The direction and nucleotide sequence of the cloned AKR1C3 gene in the pET21b vector were confirmed by DNA sequencing. The plasmid pET21b-AKR1C3 was designed to introduce a COOH-terminal His6 tag to aid purification.
Expression and Purification of Recombinant AKR1C3.
Overnight cultures of Escherichia coli BL21(DE3) cells (Novagen) expressing the pET21b-AKR1C3 construct in LB medium containing 100 µg·ml-1 ampicillin were seeded (0.1%) into 400-ml batches of fresh LB medium containing ampicillin and then incubated at 37°C for 16 h, with shaking at 220 rpm. Expression of the protein occurred without the need for induction with isopropyl-ß-D-thiogalactopyranoside. Bacterial cultures were pelleted by centrifugation (3,800 x g, 15 min), and the cells were disrupted by resuspension in BugBuster reagent (Novagen) containing Benzonase DNase (Novagen), using 10 ml lysis reagent/400 ml original culture. After incubation at room temperature with shaking (150 rpm) for 25–30 min, the cell debris was pelleted by centrifugation (48,000 x g, 30 min, 4°C). The cleared lysate was mixed with nickel-nitrilotriacetic acid His-bind resin (Ni-NTA; Novagen) using 0.5 ml resin/400 ml original culture, followed by gentle shaking at 4°C for 30 min. The mixture was loaded into an empty column, and unbound protein was removed by washing with 8 ml of 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl. After an additional wash with 20 mM imidazole in the same buffer, the recombinant protein was eluted in 1.5 column volumes of 100 mM imidazole. After the addition of DTT to a concentration of 2 mM, the protein was further purified by fast protein liquid chromatography gel filtration on a Superdex 200 HR10/30 column using 10 mM potassium phosphate, 1 mM EDTA, and 1 mM DTT (pH 7.0) at a flow rate of 0.4 ml·min-1. Protein was monitored by absorbance at 280 nm, and purity of the fractions (1 ml) was assessed by SDS-PAGE. NADP+ was added to a final concentration of 2 mM, and the protein was concentrated by centrifugation (3,000 x g, 4°C) on a Vivaspin concentrator (Mr 30,000 cutoff; Vivascience) to varying concentrations between 15 and 45 mg·ml-1. Approximately 20 mg of highly pure, His-tagged recombinant protein suitable for crystallography could be purified from 1 liter of cell culture.
Measurement of Steady-State Kinetic Parameters.
Enzyme activity was measured as either reduction of 5-dihydrotestosterone or androsterone or oxidation of 3-androstanediol. Reduction reactions were monitored in 1-ml volumes containing 5–100 µM 5-dihydrotestosterone or 2–50 µM androsterone in 3% (v/v) acetonitrile, 150 µM NADPH, and 10 mM potassium phosphate buffer (pH 7.0) at 30°C. Initial velocities were measured by observing the rate of change of absorbance of pyridine nucleotide at 340 nm (
6270 M-1·cm-1) in 1 ml, with a 1-cm light path. Calculation of kcat and Km values used the Leonora program (35)
, yielding estimates of the kinetic constants and their associated SEs (Table 1)
. For inhibition studies, IMN and FLF were added at several different concentrations to give final concentrations varying between 0.5 and 50 µM, and steroid substrate concentrations were then varied as indicated above. The type of inhibition and the inhibition constants were calculated using the Leonora program. All reactions were initiated by the addition of enzyme.
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-androstanediol. The fluorescence emission of NADPH at 450 nm (5-nm slit width) with excitation at 340 nm (15-nm slit width) was monitored at 30°C. Each cuvette contained a 3-ml reaction mixture with 10 mM potassium phosphate buffer (pH 7.0), 200 µM NADP+, and 3-androstanediol (1–40 µM) with 3% DMSO as solvent. A standard curve was constructed by monitoring fluorescence changes with incremental additions of NADPH. FLF and IMN were used in inhibition studies, and data were analyzed as described above.
Crystallization and Data Collection.
Crystals were grown by the hanging-drop vapor-diffusion method in 6-µl drops. Form I crystals were obtained from a 1:1 mixture of purified protein [16 mg·ml-1, in a buffer containing 10 mM potassium phosphate buffer (pH 7.0), 1 mM DTT, 1 mM EDTA, and 2 mM NADP+] and a reservoir solution containing 25% (w/v) polyethylene glycol 4000 (Fluka), 100 mM sodium citrate (pH 6.0), 2.5% (v/v) 2-methyl-2,4-pentanediol, and 800 mM ammonium acetate. Crystals were soaked in artificial mother liquor supplemented with 15% (v/v) 2-methyl-2,4-pentanediol shortly before flash cooling to 100 K in a cryostream. Form I crystals were approximately 0.6 x 0.3 x 0.2 mm, taking 6 days to grow to maximum size, and diffracted beyond 1.1 Å resolution. A complete data set was collected to 1.20 Å on beam line ID14-1 (
= 0.93 Å) at the European Synchrotron Radiation Facility using an Area Detector Systems Corporation (ADSC) Quantum 4 CCD detector. Form II crystals were grown in similar conditions, except that only 400 mM ammonium acetate was used. Crystals grew to a maximum size of 0.4 x 0.3 x 0.2 mm over 4 days and diffracted to 
2.0 Å resolution. A complete data set was collected to 2.1 Å resolution on beam line ID29 ( = 0.93 Å) at the European Synchrotron Radiation Facility using an ADSC Q210 detector. For the FLF and IMN inhibitor soaks, crystals were placed into a cryobuffer containing 25% (w/v) polyethylene glycol 4000, 100 mM sodium citrate (pH 6.0), 10% (w/v) ethylene glycol, 10% (w/v) DMSO, 800 mM NaCl, and 5 mM of either FLF or IMN, for a period of 20 min before flash cooling in the cryostream. Data were collected on beam line ID14-2 ( = 0.93 Å) at the European Synchrotron Radiation Facility using an ADSC detector. All diffraction data were integrated and scaled using either Mosflm/Scala (36)
or Denzo/Scalepack (Ref. 37
; Table 2
).
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; Fig. 5
] as a search model. A clear solution (top peak in both rotation and translation functions) was seen with a monitor value of 0.64 and a packing coefficient of 0.53. The structure was initially refined using the rigid body, simulated annealing, and geometry minimization protocols against a maximum likelihood target with the program CNS before modeling in amino acid differences. This refined structure was then used to solve acetate complex I (crystal form I, native) using CNS molecular replacement. Crystals soaked with FLF or IMN were isomorphous to the native crystals (Table 2)
. 2mFo-DFc SIGMAA-weighted (40)
electron density maps were calculated after partial refinement of the native structure against structure factor amplitudes for the soaked crystals to check for incorporation of the inhibitor. The structures were initially refined using CNS against a maximum likelihood target and later refined using Refmac5 (41)
incorporating maximum likelihood and TLS refinement (42)
. Molecular models were inspected, and manual adjustments were made using the graphics package TURBO-FRODO (43)
using 2mFo-DFc and mFo-DFc SIGMAA-weighted electron density maps as a guide. Correct modeling of ligands was checked using CNS simulated annealing omit maps (44)
. Finally, all models were validated using the program Procheck (45)
. The final statistics are given in Table 2
. Structure superimpositions were calculated using the "magic fit" and "improve fit" features of the program Swiss PDB Viewer (46)
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RESULTS |
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-androstanediol and reduction of androsterone. These substrates were chosen in preference to PGD2 for the pragmatic reasons that they and their products are more chemically stable and that the kinetics of the enzyme have been more extensively analyzed in the past with these substrates. These tests confirmed that the ability of His6-tagged recombinant AKR1C3 protein to reduce 5-dihydrotestosterone or androsterone and oxidize 3-androstanediol yielded kinetic constants similar to those published previously (47
, 48)
for untagged recombinant protein (Table 1)
.
Inhibition by the NSAID FLF and IMN.
When tested in the oxidative direction with varying concentrations of 3-androstanediol, both FLF and IMN displayed potent competitive inhibition against the steroid substrate with Ki values of 0.14 ± 0.01 and 0.27 ± 0.01 µM, respectively. However, when tested in the reductive direction against varying concentrations of androsterone, the inhibitors were less potent, and the data for both showed a better fit to a mixed type of inhibition. The Ki values corresponding to the competitive components were calculated to be 3.1 ± 0.5 and 2.1 ± 0.4 µM for FLF and IMN, respectively, with uncompetitive components of 4.4 ± 0.3 and 4.6 ± 0.5 µM.
The Three-Dimensional Structure of AKR1C3.
Two crystal forms were obtained from purified protein, differing only in the concentration of ammonium acetate required for crystal growth. Despite the similarity in growth conditions, crystal form I diffracts to significantly higher resolution (Table 2)
, and consequently, most of our analysis and studies are concentrated on this form. The crystal form I structure (Fig. 2)
consists of amino acids 6 to 320, NADP+, 1 molecule each of acetate and 2-methyl-2,4-pentanediol, and 434 water molecules and represents an enzyme:NADP+:acetate ternary complex (Figs. 3A
and 4A
), hereafter referred to as acetate complex I. The refined crystal form II structure (acetate complex II) consists of amino acids 6 to 321, NADP+, one molecule each of acetate and 2-methyl-2,4-pentanediol, and 286 water molecules. Overall, the electron density maps for both models were extremely clear and well defined. The COOH-terminal His6 tag was disordered in both models and completely absent in the electron density maps. There was also some weak electron density in the hydrophobic, substrate-entry channel, indicating disordered solvent molecules that could not be modeled in acetate complexes I and II.
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8ß8 superbarrel, capped at the NH2-terminal end with an antiparallel hairpin loop (Figs. 2
, 4D
, and 5
). At the COOH-terminal end of the barrel, the loops connecting each ß-strand to the following -helix contribute amino acid side-chains into the active site (Fig. 2)
. Two of these loops are particularly extended: amino acids 116–143 (loop A); and amino acids 217–238 (loop B). Two additional helices, H1 and H2, precede helix 7 and follow helix 8, respectively. Following helix H2, the COOH-terminal tail, also known as loop C, forms an extended, meandering coil, occupying the space near to the active site between loops A and B and forming interactions with both.
The Active Site.
The active site (Fig. 2)
is located at the COOH-terminal end of the central ß-barrel. The NADP+ is in an extended conformation, so that atom C2 of the nicotinamide ring is positioned above the center of the barrel with the A side (as defined by Ref. 49
) facing the barrel at an angle of 45° to the barrel axis and stacked against the side-chain of Tyr216 from strand ß7 (Figs. 2
and 4A
). The B face of the nicotinamide ring is exposed to the active site, with the C4-pro-R position available for hydride transfer. The NADP+ diphosphate moiety straddles a gap formed between the ends of ß-strands 7 and 8 and forms H-bonds to Ser217N, Ser217OG, Leu219N, Ser221N, Gln222NE2, and Lys270N. The 2'-phosphate group is salt bridged to Lys270 and Arg276 and H-bonded to Ser271OG and Tyr272N, presumably to favor NADP(H) over NAD(H), thereby avoiding a costly transhydrogenation cycle between the NAD(H) and NADP(H) pools. The adenine ring is sandwiched between the hydrophobic part of the Arg276 side-chain (atoms CG and CD) on one side and the aliphatic side-chains of Leu219, Leu236, and Ala253 on the other, while forming several H-bonds around the ring edge. The substrate-binding site is located on the other side of the nicotinamide ring from the central ß-barrel and consists mainly of hydrophobic, aromatic amino acid side-chains. In both acetate complexes, the acetate molecule is in an identical position, close to and in an approximate stacking arrangement with the nicotinamide ring (Fig. 4A)
. The methyl group of the acetate molecule points into a hydrophobic pocket comprising Tyr24, Tyr55, Leu54, Trp227, and Phe306. The carboxylate carbon is 3.17 Å away from the nicotinamide C4 position on the pro-R side. Oxygen OXT of the acetate is H-bonded to His117NE2 (2.93 Å) and Tyr55OH (2.47 Å), which is itself H-bonded to Lys84NZ (2.89 Å). Lys84NZ is H-bonded to Asp50OD2 (2.66 Å) and Ser51O (2.87 Å). The four amino acids Asp50, Tyr55, Lys84, and His117 are strongly conserved across the AKR family and have been proposed to form a catalytic tetrad that catalyzes the oxidation of alcohol or reduction of ketone functional groups via a "push-pull mechanism" (50)
. Briefly, in the reduction of carbonyls, it is proposed that the hydride ion equivalent on the pro-R C4 position of the NADPH nicotinamide attacks the substrate carbonyl to form a transitory tetrahedral oxyanion, which abstracts the proton from Tyr55OH to form tyrosinate-55 anion and the product alcohol. The tyrosinate anion is stabilized by the adjacent, positively charged Lys84NZ (50)
.
The acetate complexes I and II are structurally very similar over the entire protein fold (Table 3)
, despite different crystal packing arrangements. When comparing the rotamer angles of acetate complexes I and II, only 38 of a total of 321 amino acids have a difference in chi1 angle of more than 20°.
NSAID Complexes.
The ternary complexes with either FLF or IMN bound to AKR1C3:NADP+ were obtained from soaking experiments using form I crystals after replacing the ammonium acetate in the crystal stabilization solution (artificial mother liquor) with sodium chloride. The FLF ternary complex consists of amino acids 6 to 320, NADP+, 2 molecules of FLF, and 237 water molecules, whereas the IMN ternary complex consists of amino acids 6 to 320, NADP+, IMN, DMSO, and 363 water molecules. Strong and clearly defined electron density in the active site region allowed the unambiguous fitting of a molecule of either FLF (FLF1) or IMN (Fig. 3)
, interacting with amino acid side-chains from ß-strands 4, 5, 6, and 7, as well as loops B and C (Fig. 4, B and C)
. Of the 17 amino acid side-chains with at least one atom within 4.0 Å of FLF1 and/or IMN (marked in Fig. 5
), 8 are aromatic, and 9 are invariant among AKR1C1–AKR1C4. During the latter stages of refinement, it became apparent that a second molecule of FLF (FLF2) was bound next to the ß-hairpin loop at the NH2 terminus (Figs. 3C
and 4D
).
In the active site, FLF1 binds next to the nicotinamide ring, with the carboxylate group occupying a similar position to the acetate group in acetate complexes I and II, and with a similar H-bonding pattern (Fig. 4, A and B)
. There is little perturbation of the active site structure on binding FLF1. Phe311 (loop C) is affected the most, with its CA atom displaced by 1.2 Å toward the inhibitor and an 100° rotation about its CA–CB bond to allow the aromatic side-chain to interact better with the trifluoromethyl-benzene ring. The indole ring of Trp227 is also tilted by 15° to enlarge the active site on binding of the inhibitor. The dihedral angle between the two rings of FLF1, defined as C7-C6-C1'-C7' is -113° (Figs. 1A
and 4B
). The majority of interactions between FLF1 and its nearest neighbors are van der Waal contacts, but there is a bifurcated H-bond from FLF1N to FLF1O1 (2.85 Å, predicted D–H:A angle = 123°) and from FLF1N to the amide oxygen of the nicotinamide ring (2.91 Å, predicted D–H:A angle = 134°). In addition, there is an H-bond between FLF1F1 and Tyr216OH (2.97 Å).
At the second FLF-binding site, there is clear electron density to position the two aromatic rings and also the carboxylic acid group of FLF2. The electron density for the trifluoromethyl group is very weak and poorly defined, suggesting that FLF2 is bound with a lower occupancy and possibly in two alternative conformations, with the trifluoromethyl group either cis or trans to the carboxylic acid with dihedral angles (see above) of 48° or -133°, respectively. Fig. 4D
shows FLF2 in its hydrophobic binding pocket: ring 1 (C1-C6) interacts with the side-chains of Val8, Val18, Leu261, Gly264, and Val266; ring 2 (C1'--C6') packs against Gln6, atoms CB to CD of Arg258, Gln262, and Phe284. Arg301 from a crystallographic symmetry-related molecule is positioned close to FLF2 ring 1, so that the plane of the Arg301 guanidinium group is parallel to and approximately 3.5 Å away from the plane of the FLF2 carboxylate group.
With IMN, the chlorobenzoyl group is in the cis conformation, with respect to the indole ring, as defined earlier (see Fig. 1B
). The planes of the two ring systems are almost perpendicular to each other. With the indole ring positioned in the plane of Fig. 1
, the chlorobenzene ring points toward the viewer, with a C*- N- C9- C10 dihedral angle of 44° [cf. -59.16° in the IMN:COX-2 complex, PDB entry 4cox (51)
]. There are few H-bonds between IMN and the enzyme:NADP+ complex; the remaining contacts are van der Waal interactions (Fig. 4C)
. The indole ring, which has forced the side-chain of Phe306 to rotate 120° about the CA-CB bond, is approximately perpendicular to the NADP+ nicotinamide ring, with the shortest distance (3.3 Å) between atom C4 of the nicotinamide and IMNC5. The bridging carbonyl is at an angle of 40° to the plane of the nicotinamide ring, with the C and O atoms 4.0 and 4.2 Å away from the nicotinamide C4 atom, respectively. The carbonyl oxygen is too far away from Tyr55OH to H-bond directly. Instead, there is an unidentifiable solvent peak (Figs. 3D
and 4C
) occupying the same position as the acetate OXT (Fig. 4A)
, and it is 2.0, 2.6, and 2.8 Å from the IMN carbonyl oxygen, Tyr55OH, and His117NE2, respectively. The carboxylate group points toward and interacts with the oxygen atoms NO1 and NO2 from the nicotinamide half of the NADP+ diphosphate moiety, forming three H-bonds, and an additional H-bond is formed between IMNO2 and Gln222N.
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DISCUSSION |
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8ß8 architecture, where differences in the active site loops determine specificity of substrate and inhibitor binding. The four structures presented here, the first of AKR1C3, are very similar to each other (Table 3)
and homologous to other members of the AKR family, particularly the closely related AKR1C family members (Fig. 5)
. However, there are important differences between AKR1C3 and the other AKR1C family members in the active site and in loop A, which must be responsible for the differences in activities (47)
. Although AKR1C3 is structurally most similar to AKR1C2, loop A of AKR1C3 is shifted toward loop C (Asn134CA to Asn134CA distance of 6.2 Å in the superimposed structures of PDB:1J96_A and acetate complex I) and is structurally more similar to loop A of aldose reductase (PDB entries 1AH0 and 1AH4).
Acetate in the Active Site.
The electron density in the active site of acetate complex I clearly shows a molecule of acetate, used as an additive during the crystallization process. The presence of acetate in the protein drop significantly improved the growth and quality of crystals. Analysis of all available AKR structures in the PDB databank is particularly revealing; eight structures, representing nine polypeptide chains, have a carboxylic acid-containing compound bound in the active site. In all of them, the carboxylate group is bound next to the conserved tyrosine and histidine residues in the active site. There are three additional chains with bound acetate in the active site. In one example, PDB entry 1J96, an acetate molecule has bound in the active site in preference to testosterone, an inhibitor, which is also present and bound in the active site entrance channel (52)
. When superimposing all of the protein structures with an active site-bound carboxylate, it can be seen that the carboxylate adopts one of two possible orientations. Seven of the structures (from a total of 12) have carboxylate groups superimposing with the acetates in the two acetate complexes presented here. However, in all of the structures, one of the carboxylate oxygens is H-bonded to both atoms OH of the active site tyrosine and NE2 of the histidine. In our acetate complex I structure, one of the acetate oxygens, labeled OXT, is H-bonded to Tyr55OH with a short distance of 2.47 Å. An oxyanion hole, analogous to that in serine proteases, has been proposed for the AKR family, but instead of main-chain amides stabilizing the build up of charge on the oxygen, the hole is proposed to consist of the conserved active site tyrosine (Tyr55), histidine (His117), and nicotinamide ring. Analysis of the ligand-bound AKR structures deposited in the PDB reveals five structures with short H-bonds (
2.60 Å) to atom OH of the active site tyrosine and three structures with short H-bonds between the carboxylate oxygen and atom NE2 of the active site histidine. In addition, there is one structure of an inhibitor-bound complex (PDB entry 1AFS; Table 3
) where the carbonyl oxygen (atom O3) of testosterone is <2.6 Å from the active site tyrosine OH group in both of the non-crystallographic symmetry (NCS)-related structures (labeled chains A and B). It should be noted that the accuracy of bond lengths and interatomic distances is very dependent on the resolution of the X-ray structure. Most of the AKR X-ray structures deposited in the PDB have a resolution lower than 2.0 Å (dmin 
2.0 Å). However, at a resolution of 1.20 Å in the acetate complex I, the overall estimated coordinate error is estimated at ±0.037, ±0.034, or ±0.028 Å, based on R value, free R value, or maximum likelihood, respectively.
NSAID Inhibition.
Analysis of the two NSAID ternary crystal structures shows that FLF and IMN bind at the active site. The carboxylate of FLF1 occupies the acetate-binding site, as expected, with oxygen atom O1 occupying the proposed oxyanion hole (Figs. 4, A and B)
. The H-bond between Tyr55OH and atom FLF1O1 is also short (2.6 Å). The other end of FLF1 H-bonds via the atom F1 to Tyr216OH, which ring stacks with the NADP+ nicotinamide ring and thus forms a "molecular clamp" around the nicotinamide. IMN also contains a carboxylate, but surprisingly, this does not bind in the acetate-binding pocket (Fig. 4C)
. Instead, it H-bonds to atoms NO1 and NO2 of the NADP+ diphosphate moiety, indicating that the carboxylate group exists predominantly in the neutral, protonated form. The IMN carbonyl group (atoms C9 and O1) is pointing in toward the oxyanion hole. The hole itself is occupied by a solvent peak, which is only 2.0 Å from IMNO1 and 2.6 and 2.8 Å from Tyr55OH and His117NE2, respectively. To test whether solvent peak 1 was actually covalently linked to the carbonyl oxygen, we modeled in a hydroperoxide derivative of IMN and subjected this to several cycles of test refinement using the program Refmac5 (41)
. The resultant Fo-Fc difference electron density clearly showed a negative peak between IMNO1 and solvent peak 1. Also, there were no peaks in an anomalous (F+ - F-) map corresponding to this position. In the absence of any further data, we cannot identify the origin of this solvent peak. The FLF carboxylate group has a lower pKa (3.9 in free solution) than IMN (4.5 in free solution) and therefore is more likely to be charged, even in the solvent-protected active site.
In this study, we observed a 10–20-fold increase in the values of Ki for the inhibitors when inhibiting reductive compared with oxidative reactions. Both drugs were potent competitive inhibitors against 3-androstanediol in the oxidative direction, in agreement with the drugs binding to the active site of the enzyme:NADP+ complex as observed in the crystal structures. However, the drugs were less potent inhibitors of the reduction of androsterone, suggesting that the affinity of the drugs for the enzyme:NADPH complex may be lower than that for the enzyme:NADP+ complex. The inhibition was clearly of a mixed type, suggestive of a second inhibitor-binding site on the enzyme. Interestingly, equilibrium dialysis studies on the binding of NSAID to the related AKR1C9 from rat liver (53)
revealed the presence of two complexes, a high-affinity ternary complex corresponding to enzyme:NAD+:IMN (Kd = 1–2 µM for IMN) and a low-affinity binary complex corresponding to enzyme:IMN (Kd = 22 µM). It seems likely that a similar effect is being seen in these studies, and this is supported by the presence of a second FLF molecule observed next to the ß-hairpin loop at the bottom of the TIM barrel in the crystal structure (Figs. 2
and 4D
). The higher average B-factor for FLF2, compared with those for FLF1 and the protein, indicates that this binding site has lower affinity for FLF. However, no second molecule of IMN could be observed in the corresponding hydrophobic pocket in the AKR1C3:IMN complex structure.
The kinetic data also suggest that it is possible that the conformation of IMN and FLF1 in the active site of AKR1C3 is dependent on the oxidation state of the nucleotide. We have attempted to model IMN in alternative conformations with the carboxylate group superimposed on the acetate of acetate complex I (data not shown). In these models, steric hindrance exists between IMN and the aromatic side-chains that make up the active site, although these clashes could potentially be avoided by displacement of the side-chains. Due to the plasticity of the active site side-chains [observed in this study and proposed by Penning et al. (47) ], we cannot rule out other binding modes of IMN. Additional studies are required.
A Comparison of IMN Inhibition of AKR1C3 and COX.
NSAID inhibitors are better known for their ability to inhibit COX. It is interesting to compare the IMN ternary complex with the structures of IMN-bound COX-2, determined to 2.9 Å resolution (51)
. As with AKR1C3, IMN is bound to COX-2 at the bottom of a hydrophobic channel and blocks substrate access to the active site. The carboxylate group salt bridges to a conserved Arg120 side-chain. The bound IMN is also in a cis conformation, as defined earlier, but the chlorobenzoyl group is rotated about the N-C9 bond by 100° with respect to IMN-bound AKR1C3, so that with the indole ring in the plane of the paper (Fig. 1B)
, it is pointing away from the viewer. Structures also exist for iodo-IMN-bound COX-1 (54)
, but due to the limited resolution of that study (4.5 Å), the authors could not unambiguously position IMN into electron density, and so two models, cis and trans, were fitted. A detailed comparison of the IMN-bound complexes of AKR1C3 and COX-2 should enable the development of NSAID that are more AKR1C3 or COX-2 selective.
Targetting AKR1C3 in Cancer.
There is current widespread interest in exploiting PPAR not only in hematological malignancies but also in solid tumors and diabetes. As a result, there have been great efforts to develop pharmacologically active synthetic PPAR ligands. Regrettably, the early-generation drugs have proven severely hepatotoxic to humans (55)
. Thus, AKR1C3 provides not only a strong candidate for the COX-independent target of NSAIDs but also a means of therapeutically targeting PPAR using established drugs of known toxicology. Furthermore, the structural data reported here should help enable the development of novel NSAID-like drugs with improved properties and better selectivity against COX. It remains to be seen whether drugs can be developed with sufficient selectivity against other members of the AKR1C family. Indeed, many AKR enzymes are inhibited by NSAID. But, the sequence differences among the AKR1C family are mostly in the three loops (loops A, B and C; Fig. 5
) that define the active site entrance channel and ligand-binding pockets, and there are also observed differences in the activity and substrate profiles of this family (23
, 47) , suggestive of structure differences that could be exploited in a drug design.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Notes: A. L. Lovering was a recipient of a Ph.D. scholarship from the School of Biosciences, University of Birmingham. Present address for A. L. Lovering: The Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Requests for reprints: Scott A. White, The School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom B15 2TT. Phone: 44-121-414-7534; Fax: 44-121-414-5925; E-mail: S.A.White{at}bham.ac.uk
Received 9/ 9/03. Revised 11/28/03. Accepted 1/ 6/04.
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in rheumatoid synovial cells. J. Pharmacol. Exp. Ther., 302: 18-25, 2002.-hydroxysteroid dehydrogenases. J. Biol. Chem., 270: 20162-20168, 1995.-hydroxysteroid dehydrogenase isoform (AKR1C3) that exhibits high prostaglandin D-2 11-ketoreductase activity. J. Biochem. (Tokyo), 124: 940-946, 1998.. J. Biol. Chem., 273: 1855-1858, 1998. inhibits pancreatic cancer invasion and decreases expression of tissue plasminogen activator. Surgery, 134: 206-212, 2003.[CrossRef][Medline]
during thyroid carcinogenesis. Cancer Res., 63: 5274-5280, 2003. by a novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest and apoptosis in breast cancer cells. Cancer Res., 63: 5926-5939, 2003.-dependent pathway. Int. J. Cancer, 107: 837-843, 2003.[CrossRef][Medline]
-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP+ and ursodeoxycholate. Biochemistry, 40: 10161-10168, 2001.[CrossRef][Medline]
-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem. J., 351: 67-77, 2000.[CrossRef][Medline]
-hydroxysteroid dehydrogenase reveals a "push-pull" mechanism for proton transfer in aldo-keto reductases. Biochemistry, 37: 3538-3548, 1998.[CrossRef][Medline]
-hydroxysteroid dehydrogenase type 3 in complex with testosterone and NADP at 1.25-Å resolution. J. Biol. Chem., 276: 42091-42098, 2001.-hydroxysteroid dehydrogenase: evidence for binary and ternary dead-end complexes containing nonsteroidal antiinflammatory drugs. Biochem. J., 278: 835-841, 1991.
-hydroxysteroid dihydrodiol dehydrogenase, a member of the aldo-keto reductase superfamily. Proc. Natl. Acad. Sci. USA, 91: 2517-2521, 1994.-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP. Biochemistry, 35: 10702-10711, 1996.[CrossRef][Medline]
-hydroxysteroid dihydrodiol dehydrogenase. Structure, 5: 799-812, 1997.[Medline]
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(A, B, and D) or 0.8 
and the given resolution] is superimposed with the bias-free simulated annealing omit map (colored in blue) calculated using the established protocol in CNS 