Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / SKOUFIAS Dimitrios


Eg5: the kinesin involved in the formation of the bipolar spindle

Human kinesin Eg5, a member of the BimC family, is involved in the formation of the bipolar spindle during early stages of mitosis (prometaphase). Eg5 inhibition provokes mitotic arrest showing a very typical pattern, the monoastral phenotype (Figure 1), characteristic of cells that cannot separate the bipolar spindle. Since it is specific to cell division and its inhibition can lead to apoptosis, it has become the most studied kinesin to be used as a target for screening new potential chemotherapeutic drugs.

Looking for new inhibitors that target Eg5

Our multidisciplinary approach of the study of Eg5 included screening of available small molecules libraries (1,2), test of potential inhibitors on cell lines (mitotic arrest phenotypes in HeLa cells) and structural studies (X-ray crystallography, electron microscopy, hydrogen/deuterium exchange mass spectrometry). The molecule monastrol is the historical Eg5 inhibitor, but since its finding more structurally different molecules have been found to inhibit Eg5, e.g. Gossypol, flexeril, phenothiazines and S-trityl-L-cysteine (STLC). This last one has been found in the laboratory after a screening of a small molecule library from the NCI and was found to be 36 times more effective than monastrol in cell based assays. STLC is a reversible, tight binding inhibitor of Eg5 that specifically blocks mitotic progression (3). Studies using hydrogen/deuterium exchange mass spectrometry and mutagenesis revealed the STLC binding region in Eg5. This region is identical to that found for monastrol and showed that, similarly, there is a mechanism of induced-fitting (4,5). Combinatorial chemistry has also been performed to improve the inhibition of STLC and monastrol (6,7).

Phénotype monoastral obtenu avec STLC
Figure 1: Monoastral phenotype obtained upon inhibition of Eg5 with STLC(3)

Structure of the motor domain of Eg5 in complex with (R)-mon-97

onastrol, the 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. We solved the crystal structure (2.7A) 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 (Figure 2). 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 S-forms inhibit Eg5 ATPase activity with IC(50) values of 110 and 520 nM (basal assays) and 150 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 (8).

Figure 2: 3D Structure of the motor domain of Eg5 in complex with (R)-mon-97 (in green). Residues in contact with the inhibitor are shown in violet.

Figure 3: Comparaison entre la structure A) avec (R)-mon-97 (8) et B) la structure avec le (S)-monastrol. A noter le mécanisme de "induced-fit" qui accommode les molécules dans le site de fixation.


1. DeBonis, S., Skoufias, D., Lebeau, L., Lopez, R., Robin, G., Margolis, R.L., Wade, R.H., Kozielski, F. (2004) In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol.Cancer Ther. 3, 1079-90

2. Kozielski, F., DeBonis, S., Skoufias, D.A. Chapter book: Methods in Molecular Medecine; Microtubules Protocols, 2007 189-207

3. Brier, S., Lemaire, D., DeBonis, S., Forest, E., Kozielski, F. (2004). Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5. Biochemistry 43, 13072-82

4. Brier, S., Lemaire, D., DeBonis, S., Forest, E., Kozielski, F. (2006). Molecular dissection of the inhibitor binding pocket of mitotic kinesin Eg5 reveals mutants that confer resistance to antimitotic agents. J.Mol.Biol. 360, 360-76

5. Skoufias, D.A., DeBonis, S., Saoudi, Y., Lebeau, L., Crevel, I., Cross, R., Wade, R.H., Hackney, D., Kozielski, F. (2006) S-trityl-L-cysteine is a reversible, tight binding of the human kinesin Eg5 that specifically blocks mitotic progression. J. Mol. Biol. 281, 17559-69

6. Klein, E., DeBonis, S., Thiede, B., Skoufias, D.A., Kozielski, F., Lebeau, L. (2007). New chemical tools for investigating human mitotic kinesin Eg5. Bioorg. Med. Chem. 15, 6474-88

7. DeBonis, S., Skoufias, D.A., Indorato, R.-L., Liger, F., Marquet, B., Laggner, C., Joseph, B., Kozielski, F. (2007). SAR and docking investigations on a series of STLC analogues as inhibitors of the HsEg5. J.Med.Chem. In press.

8. Garcia-Saez, I., DeBonis, S., Lopez, R., Trucco, F., Rousseau, B. Thuéry, P., Kozielski, F. (2007). Structure of human Eg5 in complex with a new monastrol-based inhibitor bound in the R configuration. J. Biol. Chem. 282, 9740-7.

CENP-E: a kinetochore-associated kinesin

The human kinetochore is a highly complex macromolecular structure that connects chromosomes to spindle microtubules (MT) in order to facilitate accurate chromosome segregation. CENP-E (Centromere-Associated Protein E), a member of the kinesin superfamily, is an essential component of the kinetochore since it is required to stabilize the attachment of chromosomes to spindle MTs, to develop tension across aligned chromosomes, to stabilize spindle poles and to satisfy the mitotic checkpoint. The 312 kDa protein comprises four different domains. In this study we have focused on the N-terminal motor domain, which includes the ATP binding site and a region for microtubule-binding.

Crystals of the CENP-E motor domain have been obtained by high-throughput crystallization screening using the automated TECAN crystallization robot available in the laboratory (Figure 1). The crystals (737 x 132 x 79 µm) belong to the space group P21 with cell parameters a = 49.35 Å, b = 83.70 Å, c = 94.16 Å and a monoclinic angle ß = 103.05°.

Figure 1

We solved the crystal structure of the motor domain and linker region of human CENP-E with MgADP bound in the active site to 2.5 Å resolution (Figure 2). This structure displays subtle but important differences compared to the structures of human Eg5 and conventional kinesin. Our structure reveals that the CENP-E linker region is in a “docked” position identical to that in the human plus-end directed conventional kinesin. CENP-E has many advantages as a potential anti-mitotic drug target and this crystal structure of human CENP-E would provide a starting point for high-throughput virtual screening of potential inhibitors.

Figure 2

The structures of CENP-E and tubulin have been docked into a cryoEM map to investigate the mode of interaction between this kinesin and microtubules (Figure 3) (in collaboration with E. Neumann). The CENP-E-microtubule interface found in this study has been identified by measuring hydrogen/deuterium exchange with mass spectrometry (in collaboration with S. Brier and D. Lemaire) using the marine natural product Adociasulfate-2, a kinesin inhibitor that has been shown to compete with microtubules. This molecule was used as a tool to identify the microtubule binding regions of two kinesins, CENP-E and Eg5.

Figure 3


1. Garcia-Saez, I., Blot, D., Kahn, R. & Kozielski, F. (2004). Crystallization and preliminary crystallographic analysis of the motor domain of human kinetochore-associated protein CENP-E, using an automated crystallization procedure. Acta Crystallogr. D Biol. Crystallogr. 60, 1158-60

2. Garcia-Saez, I., Yen, T., Wade, R.H. & Kozielski, F. (2004). Crystal structure of the motor domain of the human kinetochore protein CENP-E. J. Mol. Biol. 340, 1107-16

3. Demande de brevet d’invention en Europe. Patent n° 04 290 800.4. Deposition date: 25-3-2004 (CEA). Title: Crystal of kinetochore-Associated molecular motor protein CENP-E and their use for drug screening

4. Neumann, E., Garcia-Saez, I., DeBonis, S., Wade, R.H., Kozielski, F. and Conway, J.F. (2006). Human kinetochore-associated kinesin CENP-E visualized at 17 Å resolution bound to microtubules. J. Mol. Biol. 362, 203-11

5. Brier, S., Carletti, E., DeBonis, S., Hewat, E., Lemaire, D. and Kozielski, F. (2006). The marine natural product Adociasulfate-2 as a tool to identify the MT-binding region of kinesins. Biochemistry 45, 15644-53