nsc-83265 and monastrol

Cell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1-3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but-unlike mitosis-not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes.

Topics: Animals; Cell Division; Cysteine; Female; Kinesins; Kinetochores; M Phase Cell Cycle Checkpoints; Meiosis; Mice; Oocytes; Paclitaxel; Pyrimidines; Thiones; Tubulin Modulators

The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.

Topics: Allosteric Regulation; Animals; Cysteine; Drosophila melanogaster; Drosophila Proteins; Humans; Kinesins; Microtubule-Associated Proteins; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrimidines; Recombinant Fusion Proteins; Thiones

The kinesin Eg5 plays an essential role in bipolar spindle formation. A variety of structurally diverse inhibitors of the human kinesin Eg5, including monastrol and STLC, share the same binding pocket on Eg5, composed by helix alpha2/loop L5, and helix alpha3 of the Eg5 motor domain. Previous biochemical analysis in the inhibitor binding pocket of Eg5 identified key residues in the inhibitor binding pocket of Eg5 that in the presence of either monastrol or STLC exhibited ATPase activities similar to the untreated wild type Eg5. Here we evaluated the ability of full-length human Eg5 carrying point mutations in the drug binding pocket to confer resistance in HeLa and U2OS cells to either monastrol or STLC, as measured by the formation of bipolar spindles. Both transfected cells expressing wild type Eg5 and untransfected cells were equally sensitive to both inhibitors. Expression of Eg5 single point mutants R119A, D130A, L132A, I136A, L214A and E215A conferred significant resistance to monastrol. Certain mutations inducing monastrol resistance such as R119A, D130A and L214A also conferred significant resistance to STLC. For the first time at a cellular level, the propensity of selected Eg5 point mutants to confer drug resistance confirms the target specificity of monastrol and STLC for Eg5. These data also suggest a possible mechanism by which drug resistance may occur in tumors treated with agents targeting Eg5.

Topics: Antineoplastic Agents; Binding Sites; Cysteine; Drug Resistance; Humans; Inhibitory Concentration 50; Kinesins; Models, Molecular; Mutation; Protein Binding; Pyrimidines; Thiones

The inhibition of kinesin Eg5 by small molecules such as monastrol is currently evaluated as an approach to develop a novel class of antiproliferative drugs for the treatment of malignant tumours. Therefore, we studied the effects of the new monastrol analogues enastron, dimethylenastron and vasastrol VS-83 on the proliferation of human glioblastoma cells in the kinetic crystal violet assay. Compared to monastrol, the new cell cycle specific compounds showed an at least one order of magnitude higher anti proliferative activity against U-87 MG, U-118 MG, and U-373 MG glioblastoma cells. The compounds were neither inactivated by hydrolysis nor by binding to serum proteins. Moreover, we demonstrated the characteristic monoaster formation after incubation of cells with the new compounds by confocal laser scanning microscopy. We also showed that the arrangement of beta-actin and tubulin, vital components of the cyto-skeleton of mitotic and quiescent cells, were not affected by the new compounds. Due to the necessity of overcoming the blood-brain barrier in the treatment of brain tumours, we investigated if the new monastrol analogues are modulators or substrates of the p-glycoprotein (p-gp) 170 by a flow cytometric calcein-AM efflux assay. The tested compounds showed no modulating effects on the p-gp function. With respect to the treatment of primary and secondary CNS tumours, the results of our experiments suggest that the new monastrol analogues represent an interesting class of potential anticancer drugs, predicted to be less neurotoxic in comparison to classical tubulin inhibitors.

Topics: Acridines; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine; Dose-Response Relationship, Drug; Flow Cytometry; Fluoresceins; Glioblastoma; Humans; Insecticides; Kinesins; Molecular Structure; Paclitaxel; Pyrimidines; Quinazolines; Rotenone; Spindle Apparatus; Tetrahydroisoquinolines; Thiones; Time Factors; Tubulin; Tubulin Modulators; Vinblastine

HsEg5 (Eg5) is a kinesin required for proper execution of mitosis. Several compounds that specifically block Eg5 are in clinical development and have the potential to be used in the treatment of breast cancer. In this study, we investigated the interaction between Eg5 and estrogen receptor signaling. We observed decreased Eg5 expression after treatment of estrogen receptor-positive human breast cancer MCF-7 cells with the estrogen receptor downregulator fulvestrant. Downregulation of Eg5 expression in response to fulvestrant was also observed in another estrogen receptor-positive cell line ZR-75, but not in the estrogen receptor-negative breast cancer cell line MDA-231. Moreover, in MCF-7 cells previously arrested in the G0/G1 phase of the cell cycle by fulvestrant, addition of estrogen increased Eg5 expression. This upregulation correlated with progression through S-phase. Nevertheless, the effect of fulvestrant in Eg5 expression could not be explained solely by cell cycle arrest, because treatments that blocked cell cycle progression did not consistently decrease Eg5 expression. Pharmacological inhibition of Eg5 function, with either S-trityl-L-cysteine or monastrol, prevented growth of estrogen-treated MCF-7 cells with an IC50 of 0.46 and 29.71 micromol/l, respectively. Simultaneous inhibition of estrogen receptor function with fulvestrant increased the IC50 for S-trityl-L-cysteine to 2.30 micromol/l and for monastrol to 112.69 micromol/l. Our results suggest that pharmacological inhibition of Eg5 may be an effective treatment for estrogen receptor-positive breast cancer, even without concomitant hormonal therapy.

Topics: Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cysteine; Dose-Response Relationship, Drug; Down-Regulation; Estradiol; Estrogen Antagonists; Estrogens; Female; Flow Cytometry; Fulvestrant; Humans; Inhibitory Concentration 50; Kinesins; Mitosis; Pyrimidines; Receptors, Estrogen; Signal Transduction; Thiones; Up-Regulation

Fifteen KSP inhibitors were docked into the receptor and the binding mode was analyzed for the first time. It was considered that in addition to the main binding pocket all the inhibitors merged in, there exists a cooperative minor binding pocket, which could be explored for significantly increased binding affinity. In addition, a good linear relationship between the biological activities and the lowest binding free energies has also been found. This may help in predicting the binding affinity of newly designed KSP inhibitors.

Topics: Amino Acid Sequence; Amino Acids; Cysteine; Diterpenes; Hydrogen Bonding; Indoles; Kinesins; Models, Molecular; Phenols; Protein Binding; Pyrimidines; Spindle Apparatus; Thiones

The mitotic kinesin Eg5 plays an essential role in establishing the bipolar spindle. Recently, several antimitotic inhibitors have been shown to share a common binding region on Eg5. Considering the importance of Eg5 as a potential drug target for cancer chemotherapy it is essential to understand the molecular mechanism, by which these agents block Eg5 activity, and to determine the "key residues" crucial for inhibition. Eleven residues in the inhibitor binding pocket were mutated and the effects were monitored by kinetic analysis and mass spectrometry. Mutants R119A, D130A, P131A, I136A, V210A, Y211A and L214A abolish the inhibitory effect of monastrol. Results for W127A and R221A are less striking, but inhibitor constants are still considerably modified compared to wild-type Eg5. Only one residue, Leu214, was found to be essential for inhibition by STLC. W127A, D130A, V210A lead to increased K(i)(app) values, but binding of STLC is still tight. R119A, P131A, Y211A and R221A convert STLC into a classical rather than a tight-binding inhibitor with increased inhibitor constants. These results demonstrate that monastrol and STLC interact with different amino acids within the same binding region, suggesting that this site is highly flexible to accommodate different types of inhibitors. The drug specificity is due to multiple interactions not only with loop L5, but also with residues located in helices alpha2 and alpha3. These results suggest that tumour cells might develop resistance to Eg5 inhibitors, by expressing Eg5 point mutants that retain the enzyme activity, but prevent inhibition, a feature that is observed for certain tubulin inhibitors.

Topics: Amino Acid Sequence; Antimitotic Agents; Binding Sites; Cysteine; Drug Resistance; Kinesins; Kinetics; Mass Spectrometry; Mitosis; Models, Molecular; Molecular Sequence Data; Mutagenesis; Point Mutation; Protein Binding; Pyrimidines; Thiones

Monastrol, a chemical inhibitor specific to the Kinesin-5 family of motor proteins, was used to examine the functional roles of Kinesin-5 proteins during the first, asymmetric cell division cycle in the brown alga Silvetia compressa.. Monastrol treatment had no effect on developing zygotes prior to entry into mitosis. After mitosis entry, monastrol treatment led to formation of monasters and cell cycle arrest in a dose dependent fashion. These findings indicate that Kinesin-5 motors maintain spindle bipolarity, and are consistent with reports in animal cells. At low drug concentrations that permitted cell division, spindle position was highly displaced from normal, resulting in abnormal division planes. Strikingly, application of monastrol also led to formation of numerous cytasters throughout the cytoplasm and multipolar spindles, uncovering a novel effect of monastrol treatment not observed in animal cells.. We postulate that monastrol treatment causes spindle poles to break apart forming cytasters, some of which capture chromosomes and become supernumerary spindle poles. Thus, in addition to maintaining spindle bipolarity, Kinesin-5 members in S. compressa likely organize microtubules at spindle poles. To our knowledge, this is the first functional characterization of the Kinesin-5 family in stramenopiles.

Topics: Cell Polarity; Cysteine; Cytokinesis; Dinitrobenzenes; Kinesins; Microtubules; Mitosis; Molecular Motor Proteins; Paclitaxel; Phaeophyceae; Pyrimidines; Spindle Apparatus; Sulfanilamides; Thiones; Zygote

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.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Antineoplastic Agents; Cysteine; Deuterium Exchange Measurement; Genetic Vectors; Growth Inhibitors; Humans; Kinesins; Mitosis; Molecular Motor Proteins; Molecular Sequence Data; Neurospora crassa; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Pyrimidines; Spectrometry, Mass, Electrospray Ionization; Thiones