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Originally published In Press as doi:10.1074/jbc.M608883200 on January 23, 2007

J. Biol. Chem., Vol. 282, Issue 13, 9740-9747, March 30, 2007
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Structure of Human Eg5 in Complex with a New Monastrol-based Inhibitor Bound in the R Configuration*Formula

Isabel Garcia-Saez{ddagger}, Salvatore DeBonis{ddagger}, Roman Lopez§, Fernando Trucco§, Bernard Rousseau§, Pierre Thuéry, and Frank Kozielski{ddagger}1

From the {ddagger}Laboratoire des Moteurs Moléculaires, IBS, Institut de Biologie Structurale Jean-Pierre Ebel, CNRS-Commissariat à l'Energie Atomique (CEA)-Université Joseph Fourier, 41 rue Jules Horowitz, F-38027 Grenoble, the §Groupe de Chimie Combinatoire et Criblage à haut débit, CEA-Saclay, Service de Marquage Moléculaire et de Chimie Bioorganique, Bat 547, 91191 Gif-sur-Yvette, and Service de Chimie Moléculaire DSM/DRECAM, CEA/Saclay 91191 Gif-sur-Yvette, France

Received for publication, September 15, 2006 , and in revised form, January 10, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Drugs that target mitotic spindle proteins have been proven useful for tackling tumor growth. Eg5, a kinesin-5 family member, represents a potential target, since its inhibition leads to prolonged mitotic arrest through the activation of the mitotic checkpoint and apoptotic cell death. Monastrol, a specific dihydropyrimidine inhibitor of Eg5, shows stereo-specificity, since predominantly the (S)-, but not the (R)-, enantiomer has been shown to be the biologically active compound in vitro and in cell-based assays. Here, we solved the crystal structure (2.7Å) of the complex between human Eg5 and a new keto derivative of monastrol (named mon-97), a potent antimitotic inhibitor. Surprisingly, we identified the (R)-enantiomer bound in the active site, and not, as for monastrol, the (S)-enantiomer. The absolute configuration of this more active (R)-enantiomer has been unambiguously determined via chemical correlation and x-ray analysis. Unexpectedly, both the R- and the S-forms inhibit Eg5 ATPase activity with IC50 values of 110 and 520 nM (basal assays) and 150 nM and 650 nM (microtubule-stimulated assays), respectively. However, the difference was large enough for the protein to select the (R)- over the (S)-enantiomer. Taken together, these results show that in this new monastrol family, both (R)- and (S)-enantiomers can be active as Eg5 inhibitors. This considerably broadens the alternatives for rational drug design.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mitotic spindle is a recognized target in cancer chemotherapy to inhibit cell proliferation by blocking microtubules (MTs).2 Nowadays some proteins of several other superfamilies, also associated to the spindle, have moved to the spotlight, including kinases like the Auroras (1) and Polo (2), or Eg5, a member of the kinesin superfamily that belongs to the kinesin-5 family (3). Several structurally unrelated chemical compounds have been identified (4) that inhibit Eg5 in vitro by locking the motor in a conformation that cannot release ADP from the active site (5–7). In cell-based assays this results in a failure of cells to separate the duplicated centrosomes (8) leading to mitotic arrest and eventually apoptotic cell death (9–11). Monastrol (Fig. 1A), the first Eg5 inhibitor identified (8), serves as a model to understand the mechanism in molecular detail of how a motor is inhibited (5–7, 12–16). This small molecule is used for "chemical genetics" to understand the function of Eg5 or to reveal basic questions of cell division (17) but also serves as a basis for the development of more potent monastrol-based drugs (18) that might be of therapeutic value for cancer chemotherapy in the future. In cell-based assays the effect of racemic monastrol is rather weak with an IC50 of roughly 50–60 µM (5, 6, 9) but has been improved by chemical optimization (18). There is stereo-selectivity, since the (S)-enantiomer of monastrol binds preferentially to Eg5 (5, 6), whereas the (R)-enantiomer shows significantly reduced inhibitory activity. The crystal structures of the Eg5 motor domain alone (19) and in complex with (S)-monastrol (20) have been solved allowing a detailed atomic analysis of the interactions that occur in the inhibitor-binding pocket and reflected an important advancement in the elucidation of the inhibition mechanism. In addition, the crystal structure allows rational design of new monastrol analogues that might result in improved effectiveness of the inhibitor compared with the original compound. There are several good reasons to further develop more potent monastrol-based compounds as drug candidates. First, the parallel synthesis of new analogues using combinatorial chemistry is easily accessible through the one-pot Biginelli three-component cyclocondensation (21). Second a highly enantioselective Biginelli asymmetric synthesis for dihydropyrimidines (DHPMs) is now available (22) allowing the enriched synthesis of one enantiomer over the other.


Figure 1
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  		FIGURE 1.
Chemical structures of monastrol (A) and mon-97 (B).

 
We have recently prepared a library of about 140 monastrol analogues (DHPM core structure) using combinatorial chemistry techniques for structure-activity-relationship studies and tested their inhibition of Eg5 ATPase activity as well as induction of monoastral spindles in HeLa cells, which led to the identification of more potent monastrol analogues (23).3 Surprisingly, a subgroup of compounds effectively inhibited Eg5, although the inhibition could not be explained by modeling these compounds into the inhibitor-binding pocket, based on the current model of the Eg5-(S)-monastrol complex. The first likely possibility is that the structure of the inhibitor-binding pocket changes to better accommodate the inhibitor analogue. A second possibility is that the conformational change induced by the presence of a phenyl group in this new Eg5 inhibitor analogue imposes a different positioning with respect to monastrol to be accommodated by the Eg5 inhibitor-binding pocket.

Here we report the crystal structure of human Eg5 in complex with a new monastrol analogue named (R)-mon-97, belonging to the new potent ketodihydropyrimidine family (Fig. 1B). We show that while monastrol binds to Eg5 as the (S)-enantiomer, it is the (R)-enantiomer of mon-97 that binds to the same site. Surprisingly the structure of the inhibitor-binding pocket as well as the overall Eg5 structure is very similar to that of the Eg5-(S)-monastrol complex. These results show that rational drug design of monastrol-type analogues that is only based on the structure of the Eg5-(S)-monastrol complex should therefore be taken cautiously, since some important active analogues might simply be missed. Future work on developing more effective inhibitor analogues should certainly consider these results broadening the possibilities to find even more potent DHPM-based inhibitors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and Purification—The motor domain of human Eg5 (residues 1–386) was initially cloned into the His tag vector pET28a, expressed in Escherichia coli BL21(DE3)pLys (Novagen) and purified with a 5-ml HisTrap HP column using standard fast protein liquid chromatography procedures (Amersham Biosciences). Failure to crystallize the His-tagged protein led us to modify the original vector by site-directed mutagenesis to shorten the protein construct to 368 residues (19) and to remove the expression of the histidine tag by using the QuikChange site-directed mutagenesis kit (Stratagene) and the following forward and reverse primers: Eg5-1, 5'-CCT GAA GTG AAT CAG AAA TGA ACC AAA AAA GCT TTG ATT AAG G-3'; Eg5-2, 5'-C CTT AAT CAA AGC TTT TTT GGT TCA TTT CTG ATT CAC TTC AGG-3'. The modified plasmid was transformed into E. coli BL21(DE3)Star (Invitrogen). Further purification steps follow the protocol described by Turner et al. (19) with minor modifications. Three liters of bacterial culture were grown at 37 °C in 2x yeast tryptone medium to an A600 nm of approximately 0.7 and induced overnight with 0.5 mM isopropyl beta-D-thiogalactopyranoside (MP Biomedicals). Harvested cells were suspended in 25 ml of buffer A (50 mM PIPES, pH 6.8, 2 mM MgCl2, 1 mM ATP, 1 mM tris(2-carboxyethyl)phosphine, 1 mM EGTA) supplemented with 1/2 tablet of a mixture of protein inhibitors (Complete EDTA-free, Roche Applied Science) and 2.5 mg of lysozyme and disrupted by four cycles of freeze-thaw. After bacterial lysis, 1 mM DNase I and 10 mM MgCl2 were added and incubated in gentle centrifugal mixing during 1 h at 4°C (all the following steps were performed at this temperature). The cell lysate was centrifuged at 40,000 rpm for 1 h, and the supernatant was mixed with a pre-equilibrated ion-exchange cellulose DE-52 (Whatman) in buffer A (approximately 5 ml of equilibrated resin for 6 ml of supernatant). Resin and sample were mixed gently for a few minutes, loaded onto a plastic syringe, and the flow-through and two column washes were collected. The pooled sample was applied to a 5-ml SP-Sepharose HP column (Amersham Biosciences) previously equilibrated with buffer A. The protein was eluted by using a KCl gradient (buffer B: buffer A + 250 mM KCl) at approximately 140 mM. Eg5 fractions were identified by SDS-PAGE, pooled, and diluted with buffer A to 50 mM KCl. The protein sample was loaded onto a pre-equilibrated 5-ml Q-Sepharose HP column (Amersham Biosciences) with buffer A, and fractions were collected immediately and analyzed by SDS-PAGE. Purified Eg5 (about 24 mg of protein) was concentrated to 10 mg/ml, frozen in liquid nitrogen, and stored at –20 °C.

Crystallization of the Eg5-(R)-mon97 Complex—Crystals obtained with mon-97 appeared after 5 days in hanging drops by mixing 1 µl of protein (7.7 mg/ml), 1-µl reservoir containing 20% polyethylene glycol-4000, 0.2 M K2HPO4, 0.1 M MES, pH 5.6–6.5, 0.2 µl of 5 mM racemic mon-97, and 0.2 µl of 64 mM AMPPCP, in VDX plates (Hampton Research) at 4 °C. To obtain good diffracting crystals, each condition was repeated two to four times due to a problem of reproducibility that could not be solved by macro- or microseeding. A nice looking plate obtained with the described conditions at pH 5.6 was immersed in cryoprotectant solution (24% polyethylene glycol-4000, 0.2 M K2HPO4, 0.1 M MES, pH 5.6, 20% glycerol), flash-frozen, and stored in liquid nitrogen for further crystallographic analysis, since crystals were not stable in the crystallization drops. Under these conditions, the complex crystallized in the orthorhombic space group P212121 with unit cell parameters a = 69.37 Å, b = 79.90 Å, c = 159.86 Å and 2 molecules per asymmetric unit. Crystals of native Eg51–368 (7.7 mg/ml) were obtained with 18% polyethylene glycol-3350, 0.1 M MES, pH 5.6, 0.2 M NaNO3, and 8% erythritol, after 2 days using vapor diffusion by hanging drops (1 µl of protein and a 1-µl reservoir) in 24-well VDX plates at 20 °C. The crystal tested was monoclinic P21 with unit cell parameters a = 52.36 Å, b = 77.76 Å, c = 93.06 Å and beta = 93.76° and 2 molecules per asymmetric unit as described previously (19).

Data Collection, Structure Determination, and Refinement— The Eg5-mon-97 crystals were measured on beamline BM30A at the European Synchrotron Radiation Facility (Grenoble, France). Data were processed at 2.7 Å with XDS and scaled with XSCALE (25). Since this crystal had the same space group and similar unit cell parameters as the crystal of Eg5 in complex with (S)-monastrol (Ref. 20; PDB code 1Q0B), this structure was used without inhibitor, Mg2+ADP, or solvent molecules, as a model for rigid-body refinement using RIGID from CNS (26). The refinement yielded a Rfree of 31.2%. An initial electron density map inspection showed the presence of Mg2+ADP in the nucleotide-binding site plus an extra density around loop L5 that indicated the presence of the inhibitor. The model was rebuilt manually using TURBO-FRODO (27) and refined by successive runs of density modification by solvent flipping, energy minimization, and B-factor refinement. Mg2+ADP was included in early stages of refinement, and water molecules were added progressively. The (R)-mon-97 structure deduced from the x-ray data of the corresponding R,R-diastereoisomer (see below) was fitted in the last steps of refinement into the electron density.

The final Rworking and Rfree for the Eg5-(R)-mon-97 model were 23.4 and 28.1%, respectively; more data collection and refinement statistics are shown in Table 1. The quality of the final model was assessed with PROCHECK (28) and WHAT IF (29) by uploading the structure to the Biotech Validation Suite for Protein Structures server.


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  		TABLE 1
X-ray data collection and structure refinement of Eg5-(R)-mon-97

 
The Ramachandran plot showed 90.3% of residues in most favored regions and 9.7% in additional allowed regions.

A data set of native Eg5 to 2.5 Å was collected on ID14-3 at the European Synchrotron Radiation Facility (Grenoble, France), to have an in-house reference model in case modifications in purification, etc. would occur and affect the Eg5 structure in comparison with the formerly published structure. We also wanted to be able to compare the electron densities of native and inhibitor-bound structures and not only rely on the structure coordinates. Data were processed with DENZO/SCALEPACK (30) and SCALA from CCP4 (31). The structure was solved by molecular replacement using AMoRe (32) and the subunit A of the native Eg5 structure (Ref. 19; PDB code 1II6 [PDB] ), as a model. A two-molecule/asymmetric unit search yielded a correlation coefficient of 60.8% and R-factor of 38.3%. Cycles of manual building using TURBO-FRODO (27), energy minimization, B-factor refinement, and automatic water molecule picking at latter stages of refinement using CNS (26) gave a final model with Rworking 23.01% and Rfree 26.9%. After confirming that our in-house structure and PDB code 1II6 [PDB] were almost identical, we used PDB code 1II6 [PDB] for structural superpositions, since this structure was reported to have a higher resolution (2.1 Å).

Structural superpositions were calculated using the server SuperPose Version 1.0 (33).

Preparation of mon-97—Racemic mon-97 was prepared using a modification of the procedure by Dondoni et al. (34). To a stirred solution of 3-hydroxybenzaldehyde (3.7 g, 30 mmol, 1.0 eq) we added benzoylacetone (4.9 g, 30 mmol, 1.0 eq), methylthiourea (0.93 g, 90 mmol, 3.0 eq), and Ytterbium triflate (0.93 g, 1.5 mmol, 0.05 eq). The mixture was refluxed overnight and evaporated. Ethyl acetate was added, and the insoluble material was removed by filtration. The filtrate was washed with brine, dried over MgSO4, and concentrated in vacuo. The crude solid obtained was crystallized from a ether:hexane mixture and yielded 4.25 g (42%) of mon-97 as a white solid.

Separation of mon-97 Enantiomers by Chiral HPLC—The two enantiomers were separated by chiral high performance liquid chromatography (ChiralPack AS, 250 x 4.6 mm), elution hexane/ethanol, 86:14 v/v; flow rate, 1.0 ml/min; detection, 220 nm.

Preparation of the Mosher Ester—2.4 g of imidazole (34.7 mmol, 2.2 eq) and 2.6 g of tert-butyldimethylsilyl chloride (17.4 mmol, 1.1 eq) were added to a stirred solution of 5.35 g mon-97 (15.8 mmol, 1.0 eq) in 50 ml of dry N,N-dimethylformamide and stirred overnight at room temperature. The mixture was diluted with water and extracted with Et2O(4 x 50 ml). The combined organic phases were washed with a CuSO4 solution (2 x 50 ml) followed by brine, dried over anhydrous MgSO4, and evaporated under vacuum. Purification by crystallization with hexane:ether yielded 2.6 g (37%) of pure silylated aldehyde as a pale yellow solid. 164 mg (0.36 mmol) of this silylated compound in 2 ml CH2Cl2 was added to a mixture of (R)-(+)-Mosher acid (127 mg, 0.54 mmol, dicyclohehylcarbodiimide (134 mg, 0.65 mmol), and dimethylaminopyridine (16 mg, 0.13 mmol)) in 4 ml of dry dichloromethane. The resulting mixture was stirred for 48 h at room temperature. The precipitate obtained by adding 25 ml of diethylether was removed by filtration. The filtrate was washed successively with diluted aqueous HCl solution (2 x 10 ml), water (15 ml), aqueous saturated NaHCO3 (2 x 10 ml), and finally brine. The organic layer was dried over MgSO4 and concentrated under vacuum. Purification of the crude product by flash chromatography on silica gel (eluent Et2O-hexane 1:2 to 1:1) gave 45 mg of each of the two diastereoisomers (less polar, Rf = 0.67, ether:hexane 3:7) and (more polar, Rf = 0.48, ether:hexane 3:7) with a total yield of 38%. X-ray analysis showed the R,R configuration for the more polar diastereoisomer.

Confirmation of Absolute Configuration of the More Active Enantiomer—A 10-ml solution containing 35 mg of the (R,R)-diastereoisomer (0.05 mmol) in 10% KOH in H2O:MeOH (3:1) was stirred for 2 h at room temperature. After solvent evaporation, 10 ml of ethyl acetate was added, and the mixture was neutralized with diluted HCl and then washed with water. The organic layer was dried over MgSO4 and evaporated in vacuum. Purification by flash chromatography on silica gel (Et2O-hexane 1:1) yielded 3.2 mg (17%, white solid) of enantiopure (R)-mon-97. Chiral HPLC analysis showed a retention time (tR = 39.6 min) identical to the (+)-enantiomer resulting from chiral HPLC resolution.

The crystallographic data for (R)-mon-97 coupled with the (R)-Mosher acid were collected at 100 K on a Nonius Kappa-CCD area detector diffractometer using graphite-monochromated Mo-K{alpha} radiation ({lambda} = 0.71073 Å). The data were processed with DENZO/SCALEPACK (30). The structure was solved by direct methods with SHELXS-97 and subsequent Fourier-difference synthesis and refined by full-matrix least squares on F2 with SHELXL-97 (35). Absorption effects were corrected with SCALEPACK (30). All non-hydrogen atoms were refined with anisotropic displacement parameters. The hydrogen atoms were introduced at calculated positions and were treated as riding atoms with an isotropic displacement parameter equal to 1.2 (CH) or 1.5 (CH3) times that of the parent atom. Thanks to the presence of anomalous scatterers, the absolute configuration could be determined. 2788 Friedel pairs were measured, and the correct enantiomorph was determined from the value of the Flack parameter, refined together with all other parameters, –0.10(8) (Ref. 36).

Crystal Data and Refinement Details—C35H39F3N2O4SSi, M = 668.83, orthorhombic, space group P212121, a = 9.9917 (2), b = 14.3572 (6), c = 23.5783 (9) Å, V = 3382.4 (2) Å3, Z = 4, Dc = 1.313 g cm–3, µ = 0.188 mm–1, F (000) = 1408. Refinement of 424 parameters on 6407 independent reflections out of 63,567 measured reflections (Rint = 0.064) led to R1 = 0.038, wR2 = 0.097, S = 1.120, {Delta}rhomax = 0.19, {Delta}rhomin =–0.20 e Å–3.

Basal and MT-stimulated ATPase Activity Tests—The inhibition of basal and MT-stimulated Eg5 ATPase activity by (R)- and (S)-enantiomers of mon-97 was measured as described previously (6).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structure of the Protein-Inhibitor Complex—The crystal structure of Eg5 in complex with mon-97 (Fig. 2) contains 2 molecules per asymmetric unit (named A and B). Each Eg5 molecule has one Mg2+ADP located in the nucleotide-binding site, one molecule of the inhibitor mon-97 and a total of 214 water molecules. We used racemic mon-97 for crystallization, expecting to identify, as in the case of monastrol, the (S)-enantiomer in the inhibitor-binding pocket. However, our first attempts to fit a generated (S)-mon-97 form into the electron density map failed, and we decided to build mon-97 without any constraints and assumptions into the density (supplemental Fig. 1). We constructed an inhibitor conformation that nicely fitted into the electron density. After applying the CahnIngold-Prelog system to unambiguously determine the absolute configuration of bound mon-97, we found to our surprise that the (R)-, but not the (S)-, enantiomer was present in the crystal structure. (R)-mon-97 is located in a cavity delimited by loop L5, {alpha}2, {alpha}3, and beta4 (Fig. 2). The residues that are in direct contact with the inhibitor are: Glu-116, Gly-117, Glu-118, Arg-119, Trp-127, and Ala-133 (loop L5), Leu-160 (strand beta4), and Tyr-211, Glu-215, and Arg-221 (helix {alpha}3). There are slight differences between the inhibitor found in the two molecules of the asymmetric unit: in molecule A there are additional hydrophobic contacts with Pro-137 (helix {alpha}2), Leu-214 and Ala-218 (helix {alpha}3) (distance ≤ 3.9 Å) (Fig. 3A and Fig. 4A). The overall folding of Eg5-(R)-mon-97 is almost identical to that of the complex of Eg5 with (S)-monastrol (main chain r.m.s.d. 0.3 Å). Structural comparison with the native model (PDB code 1II6 [PDB] ) showed that the areas affected by the presence of the inhibitor are {alpha}0, {alpha}3, {alpha}4, {alpha}5, L1, L5, L8, L10, switch I, switch II, and the neck linker region (main chain r.m.s.d. 4.1 Å) (Movie 1 in supplemental material). However, despite the induced fitting by (R)-mon-97 and conformational changes in the protein that seem to be almost identical in both cases, (S)-monastrol has a different residue environment in its pocket than mon-97 (Fig. 4).


Figure 2
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  		FIGURE 2.
Overall structure of the Eg5 motor domain with bound (R)-mon-97 in the inhibitor-binding pocket. Mg2+ADP in the active site and (R)-mon-97 are shown as ball-and-stick models. Residues that are in contact with the inhibitor are shown in violet. Figs. 2, 3, 5B, and 7 were generated using PyMOL (44).

 
Confirmation of the Absolute Inhibitor Configuration—To further confirm this unexpected result we determined the absolute configuration of the more active (R)-mon-97 enantiomer by x-ray analysis and chemical correlation techniques (Fig. 5). First both mon-97 enantiomers were separated by chiral HPLC (CHIRALPAK AS) of racemic mon-97 (Fig. 5, path A). The first enantiomer was eluted at tR 32.4 min and the second one at tR 39.6 min. The purity of separated enantiomers was checked by analytical HPLC and found to be >99% for each. ([{alpha}]D =–52°, c = 3.0 EtOH, first (–)-enantiomer and ([{alpha}]D =+53°, c = 3.0 EtOH, second (+)-enantiomer). Second we performed chemical correlation as follows (Fig. 5, path B): after silylation, racemic mon-97 was converted into its two diastereoisomeric Mosher esters (37), (R,S)- and (R,R)-, which were separated by conventional chromatography. Subsequent x-ray analysis and structure determination to 0.8 Å resolution showed the more polar ester to have the R,R configuration (fig. 5B). The (R,R)-diastereoisomer was then deprotected to enantiopure (R)-mon-97, the retention time of which was shown by chiral HPLC to be identical to the more active Eg5 inhibitor obtained from racemic mon-97, i.e. the (R)-enantiomer of mon-97. Eg5 inhibition was also higher for this "chemically resolved" enantiomer (data not shown). This result also showed that (R)-mon-97 alone and in complex with Eg5 has almost identical conformations.


Figure 3
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  		FIGURE 3.
Stereo plot of residues involved in inhibitor interactions. Inhibitors are shown in green, water molecules in red, and protein residues forming the binding pocket are colored in pale pink. The sulfur atom is depicted in yellow. A, Eg5-(R)-mon-97 complex. Note that the dihydropyrimidine ring is non-planar. B, Eg5-(S)-monastrol.

 


Figure 4
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  		FIGURE 4.
Schematic view of the contacts between (R)-mon-97 (A) and (S)-monastrol (B) and residues of the Eg5 inhibitor-binding pocket using the program LIGPLOT (24). Hydrophobic contacts (≤3.9 Å) and hydrogen bonds are depicted with red and green dashed lines, respectively. The ethyl group of (S)-monastrol points toward the solvent, whereas in the (R)-mon-97 structure the phenyl group that substitutes the ethyl group is buried in the hydrophobic pocket.

 


Figure 5
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  		FIGURE 5.
A, scheme that shows the identification of the absolute configuration of the more potent mon-97 enantiomer by chemical correlation. B, molecular structure of mon-97 coupled with the (R)-Mosher acid and its systematic name.

 


Figure 6
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  		FIGURE 6.
Inhibition of Eg5 ATPase activity by mon-97. A, inhibition of the basal Eg5 ATPase activity by (R)-mon-97 (filled circles) or (S)-mon-97 (filled squares). B, inhibition of the MT-stimulated Eg5 ATPase activity by (R)-mon-97 (filled circles) or (S)-mon-97 (filled squares).

 
Inhibition of Basal and MT-stimulated Eg5 ATPase Activities—Subsequently, we measured the inhibition of the basal and MT-stimulated Eg5 ATPase activity. In both cases the (R)-enantiomer of mon-97 is the more active agent with IC50 values of 110 nM (basal) and 150 nM (MT-stimulated) compared with 520 nM (basal) and 650 nM (MT-stimulated) for the (S)-enantiomer (Fig. 6). That is in contrast with the reported activity for monastrol, where predominantly the (S)-enantiomer shows good inhibition properties.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Diverse Biological Roles of Dihydropyrimidines—DHPMs represent a surprisingly diverse family of biologically active compounds (21). They are known to act as calcium channel modulators for the treatment of cardiovascular diseases, as potential {alpha}1a adrenoreceptor-selective antagonists used to relieve the symptoms of benign prostatic hyperplasia and as inhibitors of the kinesin superfamily member Eg5, becoming potential lead compounds for drug development in cancer chemotherapy. The crystal structure of the Eg5-(S)-monastrol complex was the first structure of a DHPM in complex with its protein target (20). Our new structure of Eg5, in complex with a new, more potent monastrol analogue in its R configuration, provides novel insights into the biochemical mechanism of inhibition of Eg5 by antimitotic agents.


Figure 7
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  		FIGURE 7.
Surface rendering plot of the Eg5 inhibitor-binding pocket. The loop L5 region is colored in orange and adjacent regions in pale pink. Mg2+ADP is shown as a ball-and-stick model, whereas (S)-monastrol and (R)-mon-97 are shown as space filling models to highlight the optimal occupancy of the cavity by (R)-mon-97 (A) compared with (S)-monastrol (B). Note that the yellow sphere corresponds to the sulfur atom.

 
Structural Comparison between (S)-Monastrol and (R)-mon-97 Complexes—Structural comparison between Eg5-(R)-mon-97 and the complex with (S)-monastrol shows almost identical overall conformational modifications to Eg5 compared with the native structure. This strongly suggests that they have a similar mode of action. In addition both compounds bind in the same pocket formed by loop L5/{alpha}2 and {alpha}3. Therefore, minor differences in the inhibitor-binding pocket must be responsible for the enormous differences in affinities for the two compounds: (S)-monastrol inhibits the basal Eg5 ATPase activity with an IC50 value of 1.7 µM, whereas (R)-monastrol displays an IC50 value of about 8.2 µM (data not shown). (R)-mon-97 and (S)-mon-97 have IC50 values of 110 and 520 nM, respectively, which is about 17 times better. The improved affinity can be explained by the different binding modes of the two DHPM inhibitors. Overall, the complex with (R)-mon-97 shows more hydrophobic contacts with residues of loop L5/{alpha}2 and {alpha}3 (Fig. 4A). In the Eg5-(S)-monastrol complex, the ethyl ester group points toward the solvent and makes contact with a water molecule (Figs. 3B and 4B). In contrast, the phenyl group in (R)-mon-97, which substitutes the ethyl group in (S)-monastrol, fits nicely in the hydrophobic pocket filling the cavity by exerting hydrophobic interactions with residues Glu-116, Leu-160, Leu-214, Glu-215, Arg-221, (Figs. 3A, 4A, and 7A), while in the complex with (S)-monastrol this cavity remains empty (Fig. 7B). It is noteworthy that Gly-117 (loop L5), Leu-160, located in strand beta4, Glu-215, and Arg-221 ({alpha}3) do not interact in the complex with (S)-monastrol. In contrast (R)-mon-97 does not contact Ile-136 that is involved in binding of the sulfur atom in the (S)-monastrol complex. In the (R)-mon-97 complex it is Glu-118 that contacts the sulfur atom and there is no water molecule implicated.

Comparison with other Eg5-Inhibitor Complexes—Structural comparisons of Eg5-(R)-mon-97 have been performed with native Eg5 (PDB code 1II6 [PDB] ) and Eg5 in complex with structurally different inhibitors found to date in the PDB: (S)-monastrol (PDB code 1Q0B), two 3,5-diaryl-4,5-dihydropyrazole analogues (PDB codes 1YRS and 2G1Q), three 2,4-diaryl-2,5-dihydropyrrole analogues (PDB codes 2FKY, 2FL2, and 2FL6), a 4-phenyltetrahydroisoquinoline analogue (PDB code 2FME), and a pyrrolotriazine-4-one analogue (PDB code 2GM1) (19, 20, 38–42). Structural differences between native Eg5 and complexed with (S)-monastrol have been identified previously (20). These mainly concern loop L5, the switch I and switch II region, and the neck linker. The distal differences are conserved throughout all Eg5-inhibitor complexes without exception: switch I, which is a featureless loop in the binary Eg5-ADP complex, turns into a short helix in the ternary Eg5-ADP-inhibitor complex, and the helix {alpha}4 displays a 20° wider opening (switch II helix up) when compared with native Eg5. It is also noteworthy that in our inhibitor-bound structure the onset of switch helix {alpha}4 is two turns shorter than in native Eg5. Interestingly it has been shown for Kif1A that the length of this helix depends on the nucleotide state (43). In all cases, the neck linker shows a rotation of about 120° and is in a docked conformation (Movie 1 in supplemental material). In the Eg5-ADP-mon-97 structure, Eg5 appears to simulate an ATP-like state as previously shown for the complex with (S)-monastrol.

Proximal structural changes in the inhibitor-binding pocket of Eg5 are as conserved as the distal changes: all inhibitors bind to the same pocket formed by loop L5/{alpha}2 and {alpha}3. With the unique exception of Eg5 in complex with the bulkier pyrrolotriazine-4-one analogue, all inhibitors bind by forming a structurally very similar binding pocket. The differences observed in comparison with PDB code 2GM1 (overall main chain r.m.s.d. 1.24 and 0.96 Å for the loop L5 area), where the bulky pyrrolotriazine analogue causes considerable changes in the induced fit conformation of loop L5, indicate that this loop is also capable of adapting to different inhibitor compound conformations. There are also noteworthy proximal similarities in the inhibitor-binding pocket formed by the two adjacent elements, loops L5/{alpha}2 and {alpha}3. All inhibitors, for which structural information is available, interact systematically with both adjacent secondary structure elements, never with one alone. The conserved residues involved in inhibitor binding in all the structures analyzed: Glu-116, Glu-118, Arg-119, and Pro-137 (L5/{alpha}2) and Leu-214 ({alpha}3) are located in these areas. As already shown for the (R)-mon-97 complex, residue Leu-160 of strand beta4 is also implicated in the interaction in almost all Eg5-inhibitor complexes except for PDB code 2FME and the structure obtained with (S)-monastrol (PDB code 1Q0B). In conclusion, we observed that despite the type of inhibitor, the allosteric pocket re-arranges around the different classes of inhibitors in a very similar manner (with the exception of the bulky pyrrolotriazine derivative).

Implications for Rational Drug Design—Rational drug design uses the knowledge gained from structural information of protein-inhibitor complexes to develop improved and more effective inhibitor analogues. The fact that the (S)-enantiomer of monastrol and the (R)-enantiomer of the similar mon-97 analogue is selected by the enzyme for complex formation leading to different binding modes, and the slightly different conformations of (R)-mon-97 found in the two Eg5 molecules of the asymmetric unit, are indicative of the compounds flexibility and shows the difficulty of any in silico drug design prediction for this type of molecules. For the development of more potent lead compounds by rational drug design based on the DHPM scaffold, it will be necessary to take into account the possibility that for certain analogues either the (R)- or the (S)-enantiomer, or both can bind to the inhibitor-binding pocket as we have shown for this N-methylketoderivative DHPM, mon-97. Although it complicates the drug design task, it offers the chance of finding more potentially effective DHPM inhibitors for Eg5 in the future.


   FOOTNOTES
 
* This work was supported by grants from Association pour la Recherche sur le Cancer (Contract number 3973), Alliance des Recherches sur le Cancer and Structural Proteomics in Europe (Contract number QLG2-CT-2002-00988). Crystallographic data for the structural analysis of (R)-mon-97 coupled with the (R)-Mosher acid have been deposited at the Cambridge Crystallographic Data Centre, CCDC number 630372. 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Movie 1.

The atomic coordinates and structure factors (code 2IEH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

1 To whom correspondence should be addressed: Institut de Biologie Structurale (CEA-CNRS-UJF), Laboratoire de Moteurs Moléculaires, 41 rue Jules Horowitz, 38027 Grenoble, France. Tel.: 33-4-3878-4024; Fax: 33-4-3878-5494; E-mail: frank.kozielski{at}ibs.fr.

2 The abbreviations used are: MT, microtubule; AMPPCP, adenylyl 5'-(beta,{gamma}-methylene)diphosphonate; DHPM, dihydropyrimidines; MES, 4-morpholinoethanesulfonic acid; PIPES, 1,4-piperazinebis (ethanesulfonic acid); PDB, Protein Data Bank; HPLC, high performance liquid chromatography; r.m.s.d., root mean square deviation. Back

3 R. Lopez, H. Comas, F. Trucco, B. Rousseau, R.-L. Indorato, S. DeBonis, I. Garcia-Saez, D. A. Skoufias, and F. Kozielski, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Richard H. Wade for useful discussions and corrections. We acknowledge the technical assistance of Laurence Serre at beamline BM30A and Hanna-Kristi Leiros at ID14-3 (European Synchrotron Radiation Facility (Grenoble, France)). Goulven Merer and David-Alexandre Buisson are acknowledged for chiral HPLC resolution performed at Service de Marquage Moléculaire et de Chimie Bioorganique.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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