Abstract
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 (2.7A) 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. 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 IC(50) 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.
Highlights
EXPERIMENTAL PROCEDURESProtein 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)
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, 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]
The difference was large enough for the protein to select the (R)- over the (S)-enantiomer. These results show that in this new monastrol family, both (R)- and (S)-enantiomers can be active as Eg5 inhibitors
Summary
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). 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 ϭ 69.37 Å, b ϭ 79.90 Å, c ϭ 159.86 Å and 2 molecules per asymmetric unit. Space group Molecular/asymmetric unit Maximum resolution (Å) No of unique reflections Overall completeness (%) Last shell completeness (%) Multiplicity Rsymc cI/(I)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
