Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aneuploidy may result from abnormalities in the biochemical pathways and cellular organelles associated with chromosome segregation. Monastrol is a reversible, cell-permeable, non-tubulin interacting inhibitor of the mitotic kinesin Eg5 motor protein which is required for assembling and maintaining the mitotic spindle. Monastrol can also impair centrosome separation and induce monoastral spindles in mammalian somatic cells. The ability of monastrol to alter kinesin Eg5 and centrosome activities and spindle geometry may lead to abnormal chromosome segregation. Mouse oocytes were exposed to 0 (control), 15, 30, and 45 microg/ml monastrol in vitro for 6 h during meiosis I and subsequently cultured for 17 h in monastrol-free media prior to cytogenetic analysis of metaphase II oocytes. A subset of oocytes was cultured for 5 h prior to processing cells for meiotic I spindle analysis. Monastrol retarded oocyte maturation by significantly (P < 0.05) decreasing germinal vesicle breakdown and increasing the frequencies of arrested metaphase I oocytes. Also, significant (P < 0.05) increases in the frequencies of monoastral spindles and chromosome displacement from the metaphase plate were found in oocytes during meiosis I. In metaphase II oocytes, monastrol significantly (P < 0.05) increased the frequencies of premature centromere separation and aneuploidy. These findings suggest that abnormal meiotic spindle geometry predisposes oocytes to aneuploidy.
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PMID:Transient exposure to the Eg5 kinesin inhibitor monastrol leads to syntelic orientation of chromosomes and aneuploidy in mouse oocytes. 1506 83

Kinesins are a group of related molecular motor proteins that have great potential as targets for antimitotic drug development. We have developed two novel assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) generated by microtubule-activated kinesin adenosine triphosphatase activity. The assays were validated using the mitotic Eg5 kinesin-specific inhibitor, monastrol. A panel of nine kinesin motor domain proteins, representing 8 of the 14 classes of kinesins, was screened. The coefficient of variation for both assays was determined to be 4-14% depending on the panel member. Using the Eg5 kinetic assay with monastrol the IC50 value was 12 microM, which agrees well with previously published results. Two other closely related mitotic kinesins (AnBimC and MKLP1) were found to have IC50 values in the millimolar range. The other panel members (kinesin heavy chain, chromokinesin KIF4A, KIF3C, CENP-E, MCAK, and KIFC3) were not significantly inhibited by millimolar levels of monastrol. It is anticipated that screening of the nine-member panel of kinesins in these assays will serve as a platform for the discovery and development of specific kinesin modulators.
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PMID:Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators. 1513 68

The human kinetochore is a highly complex macromolecular structure that connects chromosomes to spindle microtubules (MTs) in order to facilitate accurate chromosome segregation. Centromere-associated protein E (CENP-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. Here we report the 2.5A resolution crystal structure of the motor domain and linker region of human CENP-E with MgADP bound in the active site. 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 will provide a starting point for high throughput virtual screening of potential inhibitors.
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PMID:Crystal structure of the motor domain of the human kinetochore protein CENP-E. 1523 70

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.
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PMID:Mechanistic analysis of the mitotic kinesin Eg5. 1524 93

Eg5 is a kinesin-like motor protein required for mitotic progression in higher eukaryotes. It is thought to cross-link antiparallel microtubules, and provides a force required for the formation of a bipolar spindle. Monastrol causes the catastrophic collapse of the mitotic spindle through the allosteric inhibition of Eg5. Utilizing a truncated Eg5 protein, we employ difference infrared spectroscopy to probe structural changes that occur in the motor protein with monastrol in the presence of either ADP or ATP. Difference FT-IR spectra of Eg5-monastrol-nucleotide complexes demonstrate that there are triggered conformational changes corresponding to an interconversion of secondary structural elements in the motor upon interaction with nucleotides. Notably, conformational changes elicited in the presence of ADP are different from those in the presence of ATP. In Eg5-monastrol complexes, exchange of ADP is associated with a decrease in random structure and an increase in alpha-helical content. In contrast, formation of the Eg5-monastrol-ATP complex is associated with a decrease in alpha-helical content and a concomitant increase in beta-sheet content. One or more carboxylic acid residues in Eg5 undergo unique changes when ATP, but not ADP, interacts with the motor domain in the presence of monastrol. This first direct dissection of inhibitor-protein interactions, using these methods, demonstrates a clear disparity in the structural consequences of monastrol in the presence of ADP versus ATP.
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PMID:Disparity in allosteric interactions of monastrol with Eg5 in the presence of ADP and ATP: a difference FT-IR investigation. 1528 21

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore-microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.
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PMID:The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK. 1530 53

Human Eg5, a member of the kinesin superfamily, plays a key role in mitosis, as it is required for the formation of a bipolar spindle. We describe here the first in vitro microtubule-activated ATPase-based assay for the identification of small-molecule inhibitors of Eg5. We screened preselected libraries obtained from the National Cancer Institute and identified S-trityl-L-cysteine as the most effective Eg5 inhibitor with an IC50 of 1.0 micromol/L for the inhibition of basal ATPase activity and 140 nmol/L for the microtubule-activated ATPase activity. Subsequent cell-based assays revealed that S-trityl-L-cysteine induced mitotic arrest in HeLa cells (IC50, 700 nmol/L) with characteristic monoastral spindles. S-trityl-L-cysteine is 36 times more potent for inducing mitotic arrest than the well-studied inhibitor, monastrol. Gossypol, flexeril, and two phenothiazine analogues were also identified as Eg5 inhibitors, and we found that they all result in monoastral spindles in HeLa cells. It is notable that all the Eg5 inhibitors identified here have been shown previously to inhibit tumor cell line growth in the NCI 60 tumor cell line screen, and we conclude that their antitumor activity may at least in part be explained by their ability to inhibit Eg5 activity.
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PMID:In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. 1536 2

During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.
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PMID:The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5. 1545 52

Human Eg5, a mitotic motor of the kinesin superfamily, is involved in the formation and maintenance of the mitotic spindle. The recent discovery of small molecules that inhibit HsEg5 by binding to its catalytic motor domain leading to mitotic arrest has attracted more interest in Eg5 as a potential anticancer drug target. We have used hydrogen-deuterium exchange mass spectrometry and directed mutagenesis to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors, in particular for S-trityl-l-cysteine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays. The binding of this inhibitor modifies the deuterium incorporation rate of eight peptides that define two areas within the motor domain: Tyr125-Glu145 and Ile202-Leu227. Replacement of the Tyr125-Glu145 region with the equivalent region in the Neurospora crassa conventional kinesin heavy chain prevents the inhibition of the Eg5 ATPase activity by S-trityl-l-cysteine. We show here that S-trityl-l-cysteine and monastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix alpha3-strand beta5 and loop L5-helix alpha2, and both inhibitors trigger similar local conformational changes within the interaction site. It is likely that S-trityl-l-cysteine and monastrol inhibit HsEg5 by a similar mechanism. The common inhibitor binding region appears to represent a "hot spot" for HsEg5 that could be exploited for further inhibitor screening.
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PMID:Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5. 1547 1

Kinesin motor proteins utilize the energy from ATP hydrolysis to transport cellular cargo along microtubules. Kinesins that play essential roles in the mechanics of mitosis are attractive targets for novel antimitotic cancer therapies. Monastrol, a cell-permeable inhibitor that specifically inhibits the kinesin Eg5, the Xenopus laevis homologue of human KSP, can cause mitotic arrest and monopolar spindle formation. In this study, we show that the extent of monastrol inhibition of KSP microtubule-stimulated ATP hydrolysis is highly dependent upon ionic strength. Detailed kinetic analysis of KSP inhibition by monastrol in the presence and absence of microtubules suggests that monastrol binds to the KSP-ADP complex, forming a KSP-ADP-monastrol ternary complex, which cannot bind to microtubules productively and cannot undergo further ATP-driven conformational changes.
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PMID:Mechanism of inhibition of human KSP by monastrol: insights from kinetic analysis and the effect of ionic strength on KSP inhibition. 1556 18


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