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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinesin molecular motor "walks" processively along microtubules, touching down with alternate motor domains and transiently bridging between sites spaced 8 nm apart axially. To allow bridging, the coiled coil tail of kinesin would need to unzip a region immediately adjacent to the heads, and the tail region sequence at this point indeed contains potentially destabilising interruptions in the regular hydrophobic heptad repeat. We noticed that such interruptions are substantially absent from the coiled coil tails of Eg5, a slow kinesin homologue, and ncd, a reverse-directed homologue, and we wondered if this precluded their processivity. We measured the temperature dependence of kcat/K50% MTs, an index of the chemical processivity of a motor, the number of ATPs split per productive diffusional encounter of motor with microtubule. We found two-headed ncd (GSTMC5) and two-headed Eg5 (E437GST) constructs to be slightly if at all processive in solution over the range 4 degrees C to 30 degrees C. By contrast, two-headed kinesin constructs K401 and K430 were processive, and became substantially more so with increasing temperature. Arrhenius plots for the solution ATPase were linear for all three motors. Arrhenius plots for MT gliding assays were linear and essentially parallel for E437GST and GSTMC5 (Ea = 61 and 63 kJ mol-1) but for K430 the plot was biphasic, with a break at 17 degrees C, corresponding to a 30% reduction in Ea from 84 to 57 kJ mol-1. The data indicate that ncd and Eg5 are only slightly if at all processive, and suggest that this may be related to structural differences in their coiled coil neck regions.
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PMID:Kinetic evidence for low chemical processivity in ncd and Eg5. 936 54

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.
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PMID:A small-molecule inhibitor of skeletal muscle myosin II. 1174 24

Monastrol, a cell-permeable inhibitor of the kinesin Eg5, has been used to probe the dynamic organization of the mitotic spindle. The mechanism by which monastrol inhibits Eg5 function is unknown. We found that monastrol inhibits both the basal and the microtubule-stimulated ATPase activity of the Eg5 motor domain. Unlike many ATPase inhibitors, monastrol does not compete with ATP binding to Eg5. Monastrol appears to inhibit microtubule-stimulated ADP release from Eg5 but does not compete with microtubule binding, suggesting that monastrol binds a novel allosteric site in the motor domain. Finally, we established that (S)-monastrol, as compared to the (R)-enantiomer, is a more potent inhibitor of Eg5 activity in vitro and in vivo. Future structural studies should help in designing more potent Eg5 inhibitors for possible use as anticancer drugs and cell biological reagents.
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PMID:Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5. 1232 73

The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, a central coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, a small cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimeric Eg5 constructs have been examined in order to define the minimal monastrol binding domain on HsEg5. NMR relaxation experiments show that monastrol interacts with all of the Eg5 constructs used in this study. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by binding directly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating that monastrol does not interfere with the formation of the motor-MT complex. The binding is not competitive with respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol is more active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates that monastrol inhibits ADP release by forming an Eg5-ADP-monastrol ternary complex. Monastrol reversibly inhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of the kinesin superfamily: MC5 (Drosophila melanogaster Ncd), HK379 (H. sapiens conventional kinesin), DKH392 (D. melanogaster conventional kinesin), BimC1-428 (Aspergillus nidulans BimC), Klp15 (Caenorhabditis elegans C-terminal motor), or Nkin460GST (Neurospora crassa conventional kinesin).
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PMID:Interaction of the mitotic inhibitor monastrol with human kinesin Eg5. 1252 61

To reveal the mechanism of mitosis, the development of M phase-specific inhibitors is an important strategy. We have been screening microbial products to find specific M phase inhibitors that do not directly target tubulins, and rediscovered terpendole E (TerE) as a novel Eg5 inhibitor. TerE did not affect microtubule integrity in interphase, but induced formation of a monoastral spindle in M phase. TerE inhibited both motor and microtubule-stimulated ATPase activities of human Eg5, but did not affect conventional kinesin from either Drosophila or bovine brain. Although terpendoles have been reported as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT), the Eg5 inhibitory activity of TerE was independent of ACAT inhibition. Taken together, we demonstrate that TerE is a novel Eg5 inhibitor isolated from a fungal strain.
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PMID:A novel action of terpendole E on the motor activity of mitotic Kinesin Eg5. 1261 85

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

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

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

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

Monastrol is a small, cell-permeable molecule that arrests cells in mitosis by specifically inhibiting Eg5, a member of the Kinesin-5 family. We have used steady-state and presteady-state kinetics as well as equilibrium binding approaches to define the mechanistic basis of S-monastrol inhibition of monomeric human Eg5/KSP. In the absence of microtubules (Mts), the basal ATPase activity is inhibited through slowed product release. In the presence of microtubules, the ATPase activity is also reduced with weakened binding of Eg5 to microtubules during steady-state ATP turnover. Monastrol-treated Eg5 also shows a decreased relative affinity for microtubules under equilibrium conditions. The Mt.Eg5 presteady-state kinetics of ATP binding and the subsequent ATP-dependent isomerization are unaffected during the first ATP turnover. However, monastrol appears to stabilize a conformation that allows for reversals at the ATP hydrolysis step. Monastrol promotes a dramatic decrease in the observed rate of Eg5 association with microtubules, and ADP release is slowed without trapping the Mt.Eg5.ADP intermediate. We propose that S-monastrol binding to Eg5 induces a stable conformational change in the motor domain that favors ATP re-synthesis after ATP hydrolysis. The aberrant interactions with the microtubule and the reversals at the ATP hydrolysis step alter the ability of Eg5 to generate force, thereby yielding a nonproductive Mt.Eg5 complex that cannot establish or maintain the bipolar spindle.
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PMID:Monastrol inhibition of the mitotic kinesin Eg5. 1566 80


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