Development and Biological Evaluation of Potent and Specific Inhibitors of Mitotic Kinesin Eg5

Gärtner, Michael; Sunder-Plassmann, Nils; Seiler, Jeanette; Utz, Mathias; Vernos, Isabelle; Surrey, Thomas; Giannis, Athanassios

doi:10.1002/cbic.200500005

Cited by 150 publications

(81 citation statements)

References 26 publications

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“…This bipolar structure facilitates the ability of Kinesin-5 to interact with and slide adjacent microtubules during mitosis, thus contributing to the plus-end-directed force that is necessary to form and maintain the mitotic spindle (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Due to the importance of Kinesin-5 for mitosis, several studies have identified Kinesin-5 inhibitors (31)(32)(33)(34)(35)(36)(37)(38)(39)(40) that can be used in the treatment of cancer and other diseases of abnormal mitotic index (reviewed in refs 41 and 42).…”

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Pathway of ATP Hydrolysis by Monomeric Kinesin Eg5

2006

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Kinesin-5 family members including human Eg5/KSP contribute to the plus-end-directed force necessary for the assembly and maintenance of the bipolar mitotic spindle. We have used monomeric Eg5-367 in the nucleotide-free state to evaluate the role of microtubules at each step in the ATPase cycle. The pre-steady-state kinetic results show that the microtubule-Eg5 complex binds MgATP tightly, followed by rapid ATP hydrolysis with a subsequent slow step that limits steady-state turnover. We show that microtubules accelerate the kinetics of each step in the ATPase pathway, suggesting that microtubules amplify the nucleotide-dependent structural transitions required for force generation. The experimentally determined rate constants for phosphate product release and Eg5 detachment from the microtubule were similar, suggesting that these two steps are coupled with one occurring at the slow rate after ATP hydrolysis followed by the second step occurring more rapidly. The rate of this slow step correlates well with the steady-state k cat , indicative that it is the rate-limiting step of the mechanism.Kinesins are cytoskeletal motor proteins that utilize the energy from the ATPase cycle to perform mechanical work along microtubules. Conversion of the nucleotide state at the motor active site triggers conformational changes in the motor, which dictates the affinity of the motor for its filament and ultimately results in force generation. Although the specific structural events linking the ATP hydrolysis cycle to molecular motion are unknown, a mechanism for the pathway of conformational change that is responsible for amplifying small movements at the nucleotide binding site to large-scale changes in distant regions of the motor is now emerging.Kinesins share an ~350 amino acid motor domain that contains the nucleotide binding site and the microtubule binding region (reviewed in refs 1 and 2 and also see the kinesin homepage: http://www.proweb.org/kinesin/). A third region that is conserved within kinesin subfamilies is the ~14-20 amino acid sequence adjacent to the motor domain called the neck linker. Together, the motor domain and the neck linker make up the core domain, and numerous studies have investigated the ATPase mechanism of different kinesin monomers in the presence of microtubules including Kinesin-1/conventional kinesin (3-8), Kinesin-5/Eg5/BimC (9-11), and Kinesin-14/Ncd/Kar3/(12-15). The structural differences that arise between various kinesin subfamilies have fine-tuned the rate and equilibrium constants that govern the overall mechanochemical cycles. Therefore, each kinesin motor elicits a different work output that is † This work was supported by NIH Grant GM54141 from NIGMS and NIH K02-AR47841 Career Development Award from NIAMS to S.P.G. * Corresponding author. . E-mail: spg1@pitt.edu. ‡ Present address: Department of Chemistry, Dartmouth College, Hanover, NH 03755.1 Abbreviations: Eg5-367, human Eg5/KSP motor domain containing the N-terminal 367 residues followed by a C-terminal His6 tag; Eg5-43...

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Pathway of ATP Hydrolysis by Monomeric Kinesin Eg5

2006

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“…The function of Eg5 in the mitotic spindle has been shown to be indispensable. Perturbation of its function prior to anaphase B by either antibody (1,11) or small molecule inhibitors (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) causes collapse of the bipolar spindle into a monoaster and leads to apoptosis in cells with intact spindle checkpoint machinery (24,25). As a result Eg5 has garnered substantial interest as a potential chemotherapeutic target in cancer treatment.…”

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Dimeric Eg5 Maintains Processivity through Alternating-site Catalysis with Rate-limiting ATP Hydrolysis

Krzysiak

Gilbert

2006

Journal of Biological Chemistry

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Eg5/KSP is a homotetrameric, Kinesin-5 family member whose ability to cross-link microtubules has associated it with mitotic spindle assembly and dynamics for chromosome segregation. Transient-state kinetic methodologies have been used to dissect the mechanochemical cycle of a dimeric motor, Eg5-513, to better understand the cooperative interactions that modulate processive stepping. Microtubule association, ADP release, and ATP binding are all fast steps in the pathway. However, the acidquench analysis of the kinetics of ATP hydrolysis with substrate in excess of motor was unable to resolve a burst of product formation during the first turnover event. In addition, the kinetics of P i release and ATP-promoted microtubule-Eg5 dissociation were observed to be no faster than the rate of ATP hydrolysis. In combination the data suggest that dimeric Eg5 is the first kinesin motor identified to have a rate-limiting ATP hydrolysis step. Furthermore, several lines of evidence implicate alternating-site catalysis as the molecular mechanism underlying dimeric Eg5 processivity. Both mantATP binding and mantADP release transients are biphasic. Analysis of ATP hydrolysis through single turnover assays indicates a surprising substrate concentration dependence, where the observed rate is reduced by half when substrate concentration is sufficiently high to require both motor domains of the dimer to participate in the reaction.Eg5 is a member of the homotetrameric, Bim C/Kinesin-5 family. Members of this family function during mitosis and provide a plus-end directed force that has become associated with bipolar spindle assembly, spindle maintenance, and microtubule (MT) 2 flux (1-10). The function of Eg5 in the mitotic spindle has been shown to be indispensable. Perturbation of its function prior to anaphase B by either antibody (1, 11) or small molecule inhibitors (12-23) causes collapse of the bipolar spindle into a monoaster and leads to apoptosis in cells with intact spindle checkpoint machinery (24, 25). As a result Eg5 has garnered substantial interest as a potential chemotherapeutic target in cancer treatment.Mechanistically, the ATPase cycle of monomeric Eg5 motor domains are fairly well understood both free in solution and bound to . Off the MT, monomeric Eg5 demonstrates weak ATP binding and has a propensity to form a nonproductive Eg5⅐ATP complex (31). However, on the MT, ATP binding is tight, substrate productively proceeds through ATP hydrolysis, and the rates of all the individual steps in the mechanochemical cycle are accelerated (30). The MT-activated ATPase cycle concludes with a conformational change of the motor domain in relation to the MT, termed "rolling" (29), followed by the rate-limiting event in the cycle, the coupled action of phosphate (P i ) release, and motor detachment from the MT (29,30,33).In vivo the individual Eg5 motor domains probably do not function independently; therefore, analysis of a higher order oligomeric structure is necessitated. Indeed, previous analysis of dimeric Eg5, Eg5-513, has...

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“…Over the last decade, the effects of various Eg5 inhibitors on the proliferation of cancer cells have been investigated, and the mechanisms of action of several Eg5 inhibitors have been studied [7][8][9][10][11] . Dimethylenastron is a cell-permeable quinazoline-thione compound that acts as a potent inhibitor of Eg5 [12] . We have demonstrated previously that dimethylenastron inhibits pancreatic tumor growth by suppressing cell proliferation and resulting in robust apoptosis [11] .…”

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Dimethylenastron suppresses human pancreatic cancer cell migration and invasion in vitro via allosteric inhibition of mitotic kinesin Eg5

et al. 2011

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Aim:The mitotic kinesin Eg5 plays a critical role in bipolar spindle assembly, and its inhibitors have shown impressive anticancer activity in preclinical studies. This study was undertaken to investigate the effect of dimethylenastron, a specific inhibitor of Eg5, on the migration and invasion of pancreatic cancer cells. Methods: Human pancreatic cancer cell lines PANC1, EPP85, BxPC3, CFPAC1, and AsPAC1 were used. Eg5 expression was examined using immunofluorescence microscopy. Cell migration and invasion were analyzed with wound healing and transwell assays. Cell proliferation was examined using sulforhodamine B and MTT assays. The binding of dimethylenastron to Eg5 was analyzed with a molecular modeling study, and the ADP release rate was examined with the MANT-ADP reagent. Results: Eg5 expression was 9-16-fold up-regulated in the 5 pancreatic cancer cell lines. Treatment of PANC1 pancreatic cancer cells with dimethylenastron (3 and 10 μmol/L) for 24 h suppressed the migratory ability of the cancer cells in a concentration-dependent manner. The invasion ability of the cancer cells was also reduced by the treatment. However, treatment of PANC1 cells with dimethylenastron (3 and 10 μmol/L) for 24 h had no detectable effect on their proliferation, which was inhibited when the cancer cells were treated with the drug for 72 h. Molecular modeling study showed that dimethylenastron could allosterically inhibit the motor domain ATPase of Eg5 by decreasing the rate of ADP release. Conclusion: Dimethylenastron inhibits the migration and invasion of PANC1 pancreatic cancer cells, independent of suppressing the cell proliferation. The findings provide a novel insight into the mechanisms of targeting Eg5 for pancreatic cancer chemotherapy.

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Development and Biological Evaluation of Potent and Specific Inhibitors of Mitotic Kinesin Eg5

Cited by 150 publications

References 26 publications

Pathway of ATP Hydrolysis by Monomeric Kinesin Eg5

Pathway of ATP Hydrolysis by Monomeric Kinesin Eg5

Dimeric Eg5 Maintains Processivity through Alternating-site Catalysis with Rate-limiting ATP Hydrolysis

Dimethylenastron suppresses human pancreatic cancer cell migration and invasion in vitro via allosteric inhibition of mitotic kinesin Eg5

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