Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5

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.     

纺锤体驱动蛋白抑制剂*

对纺锤体驱动蛋白(kinesin spindle protein,KSP)进行抑制代表着一种新颖的抗肿瘤机制,能避免直接破坏微管的药物所具有的不可避免的神经毒性。自第一个选择性的小分子KSP抑制剂monastrol报道以来,已有多种类型的KSP抑制剂有了文献报道。本文介绍了近年来KSP抑制剂的结构和功能,以及作为一个新颖的靶点在抗肿瘤药物研究中的作用;讨论了该类抑制剂的构效关系,并对该类抑制剂的研究前景进行了展望。     

BP-M345, a New Diarylpentanoid with Promising Antimitotic Activity

Previously, we reported the in vitro growth inhibitory effect of diarylpentanoid BP-M345 on human cancer cells. Nevertheless, at that time, the cellular mechanism through which BP-M345 exerts its growth inhibitory effect remained to be explored. In the present work, we report its mechanism of action on cancer cells. The compound exhibits a potent tumor growth inhibitory activity with high selectivity index. Mechanistically, it induces perturbation of the spindles through microtubule instability. As a consequence, treated cells exhibit irreversible defects in chromosome congression during mitosis, which induce a prolonged spindle assembly checkpoint-dependent mitotic arrest, followed by massive apoptosis, as revealed by live cell imaging. Collectively, the results indicate that the diarylpentanoid BP-M345 exerts its antiproliferative activity by inhibiting mitosis through microtubule perturbation and causing cancer cell death, thereby highlighting its potential as antitumor agent.     

UA62784 Is a cytotoxic inhibitor of microtubules, not CENP-E

A recent screen for compounds that selectively targeted pancreatic cancer cells isolated UA62784. We found that UA62784 inhibits microtubule polymerization in?vitro. UA62784 interacts with tubulin dimers ten times more potently than colchicine, vinblastine, or nocodazole. Competition experiments revealed that UA62784 interacts with tubulin at or near the colchicine-binding site. Nanomolar doses of UA62784 promote the accumulation of mammalian cells in mitosis, due to aberrant mitotic spindles, as shown by immunofluorescence and live cell imaging. Treatment of cancerous cell lines with UA62784 is lethal, following activation of apoptosis signaling. By monitoring mitotic spindle perturbations and apoptosis, we found that the effects of UA62784 and of some known microtubule-depolymerizing drugs are additive. Finally, high content screening of H2B-GFP HeLa cells revealed that low doses of UA62784 and vinblastine potentiate each other to inhibit proliferation.     

The Design,Synthesis, and Biological Activities of Pyrrole-Based Carboxamides: The Novel Tubulin Inhibitors Targeting the Colchicine-Binding Site

Microtubule targeting agents (MTAs) that interfere with the dynamic state of the mitotic spindle are well-known and effective chemotherapeutic agents. These agents interrupt the microtubule network via polymerization or depolymerization, halting the cell cycle progression and leading to apoptosis. We report two novel pyrrole-based carboxamides (CAs) (CA-61 and -84) as the compounds exhibiting potent anti-cancer properties against a broad spectrum of epithelial cancer cell lines, including breast, lung, and prostate cancer. The anti-cancer activity of CAs is due to their ability to interfere with the microtubules network and inhibit tubulin polymerization. Molecular docking demonstrated an efficient binding between these ligands and the colchicine-binding site on the tubulin. CA-61 formed two hydrogen bond interactions with THR 179 (B) and THR 353 (B), whereas two hydrogen bonds with LYS 254 (B) and 1 with ASN 101 (A) were identified for CA-84. The binding energy for CA-84 and CA-61 was −9.910 kcal/mol and −9.390 kcal/mol. A tubulin polymerization assay revealed a strong inhibition of tubulin polymerization induced by CA-61 and -84. The immunofluorescence data revealed the disruption of the tubulin assembly in CA-treated cancer cells. As an outcome of the tubulin inhibition, these compounds halted the cell cycle progression in the G2/M phase, leading to the accumulation of the mitotic cells, and further induced apoptosis. Lastly, the in vivo study indicated that CAs significantly inhibited the HCC1806 breast cancer xenograft tumor growth in a nude mouse model. Collectively, we identified the novel CAs as potent MTAs, inhibiting tubulin polymerization via binding to the colchicine-binding site, disrupting the microtubule network, and exhibiting potent pro-apoptotic activities against the epithelial cancer cell lines both in vitro and in vivo.     

MITOTIC INHIBITION BY PHENYLPORPHINES AND TETRASULFONATED ALUMINIUM PHTHALOCYANINE IN COMBINATION WITH LIGHT

This work relates to studies on modes of phototoxicity by tetrasulfonated aluminium phthalocyanine (AlPcS4), tetrahydroxy- and monosulfonated meso-tetraphenylporphines (3-THPP and TPPS1) on culture cells. Toxicity at moderate light exposures appears to be related to inhibition of microtubule function. Treatment of human cervix carcinoma cells of the line NHIK 3025 incubated for 18 h with the sensitizers and exposed to light inhibits multiplication for the first hours after light exposure, a significant fraction of the cells accumulating in mitosis. For the first hours after treatment, the mitotic cells were always mainly found in metaphase; generally seen as c-metaphases and three-group metaphases. During this time, anaphase and telophase cells were absent or greatly reduced in number. Indirect immunofluorescence staining of beta-tubulin showed that the spindle apparatus of mitotic cells was perturbed in all cases. The accumulation in mitosis was more extensive after treatment with AlPcS4 and light than after treatment with 3-THPP or TPPS1 and light. This may be related to the great difference in the lipophilic properties of these sensitizers; i.e. AlPcS4 being highly water soluble while TPPS1 and 3-THPP are lipophilic sensitizers. The lipophilicity of several sensitizers has been measured by two different methods, the partition between an aqueous and a lipophilic phase (Triton X-114) and the binding strength to a reverse phase column. The results show that the measured relative lipophilicity of the sensitizers may be influenced by the method of analysis.     

JNK inhibitor SP600125 promotes the formation of polymerized tubulin, leading to G2/M phase arrest, endoreduplication, and delayed apoptosis

The JNK inhibitor SP600125 strongly inhibits cell proliferation in many human cancer cells by blocking cell-cycle progression and inducing apoptosis. Despite extensive study, the mechanism by which SP600125 inhibits mitosis-related effects in human leukemia cells remains unclear. We investigated the effects of SP600125 on the inhibition of cell proliferation and the cell cycle, and on microtubule dynamics in vivo and in vitro. Treatment of synchronized leukemia cells with varying concentrations of SP600125 results in significant G₂/M cell cycle arrest with elevated p21 levels, phosphorylation of histone H3 within 24 h, and endoreduplication with elevated Cdk2 protein levels after 48 h. SP600125 also induces significant abnormal microtubule dynamics in vivo. High concentrations of SP600125 (200 µM) were required to disorganize microtubule polymerization in vitro. Additionally, SP600125-induced delayed apoptosis and cell death was accompanied by significant poly ADP-ribose polymerase (PARP) cleavage and caspase-3 activity in the late phase (at 72 h). Endoreduplication showed a greater increase in ectopic Bcl-2-expressing U937 cells at 72 h than in wild-type U937 cells without delayed apoptosis. These results indicate that Bcl-2 suppresses apoptosis and SP600125-induced G₂/M arrest and endoreduplication. Therefore, we suggest that SP600125 induces mitotic arrest by inducing abnormal spindle microtubule dynamics.     

Induction of mitotic arrest and aneuploidy by organic arsenic compounds in human lymphocytes

Inorganic arsenic is methylated in the mammalian body to methylarsonic acid (MMA), dimethylarsinic acid (DMA) and trimethylarsine oxide (TMA). To achieve a more precise understanding of arsenic carcinogenicity, we examined the genotoxic effects of organic arsenic compounds on human lymphocytes by assessing induction of mitotic arrest, sister chromatid exchange (SCE) and aneuploidy. MMA, DMA and TMA arrested mitosis, DMA induced hyperdiploid cells, and DMA and TMA induced tetraploid cells. Of the three arsenic metabolites tested, DMA had the strongest effects on cell mitosis and aneuploidy induction. DMA arrested mitosis and induced c-mitosis significantly. These results suggest that DMA arrests mitosis and induces aneuploidy through spindle disruptions similar to those observed with known spindle poisons, such as colchicine or vinblastine. Since aneuploidy has been thought to be associated with tumor induction or neoplastic transformation, induction of aneuploidy by organic metabolites of arsenic may play a major role in arsenic carcinogenesis in humans. © 1997 John Wiley & Sons, Ltd.     

Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy

Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.     

Identification of a novel protein regulating microtubule stability through a chemical approach

To identify novel proteins regulating the microtubule cytoskeleton, we screened a library of purine derivatives using mitotic spindle assembly in Xenopus egg extracts as an assay. Out of a collection of 1561 compounds, we identified 15 that destabilized microtubules without targeting tubulin directly, resulting in small spindles. Affinity chromatography with one compound, named diminutol, revealed a potential target as NQO1, an NADP-dependent oxidoreductase. A role for NQO1 in influencing microtubule polymerization was confirmed through inhibition studies using known inhibitors and immunodepletion. Therefore, this chemical approach has identified a novel factor required for microtubule morphogenesis and cell division.     

Sec13 induces genomic instability in U2OS cells

Sec13p has been known as an endoplasmic reticulum-Golgi transport protein. Recently, it has also been shown to be required for the formation of septation in the fission yeast Schizosaccharomyces pombe. In the present study, we focused on the role of a human homolog of Saccharomyces cerevisiae SEC13, Sec13 protein during mitosis in U2OS cells. We found that the expression of Sec13 was constant throughout the cell cycle, and localized to the kinetochores at metaphase during mitosis. By using green fluorescent protein technology, we observed that Sec13 is required for evasion of mitotic arrest in response to spindle damage, leading to G1-like phase and apoptotic cell death. In addition, cells expressing exogenous Sec13 showed giant nuclei compared to endogenous ones in the absence of nocodazole. These results demonstrate that Sec13 is involved in the regulation of the metaphase/anaphase transition and may be functionally associated with mitotic machinery to maintain genomic stability during mitosis.     

The beta-carboline analog Mana-Hox causes mitotic aberration by interacting with DNA

Mana-Hox, an analog of beta-carbolines with anticancer activity, induces aberrant mitosis and delays mitotic exit. However, the cellular target is not known. In this study, we visualized the intracellular localization of Mana-Hox. Mana-Hox rapidly penetrated into cells (within 1 min) and concentrated on disorganized metaphase chromosomes after 13 hr of exposure. We demonstrated that Mana-Hox is a noncovalent DNA binder that can interact with DNA through intercalation and/or through minor groove binding. Furthermore, Mana-Hox also inhibits topoisomerase II relaxation activity in vitro, suggesting that Mana-Hox could perturb mitotic chromosome decatenation. Overall, Mana-Hox binding to DNA plays a critical role in the induction of aberrant mitosis and contributes to its anticancer activity.     

SUMOylation in Control of Accurate Chromosome Segregation during Mitosis

Posttranslational protein modification by small ubiquitin-related modifier (SUMO) has emerged as an important regulatory mechanism for chromosome segregation during mitosis. This review focuses on how SUMOylation regulates the centromere and kinetochore activities to achieve accurate chromosome segregation during mitosis. Kinetochores are assembled on the specialized chromatin domains called centromeres and serve as the sites for attaching spindle microtubule to segregate sister chromatids to daughter cells. Many proteins associated with mitotic centromeres and kinetochores have been recently found to be modified by SUMO. Although we are still at the early stage of elucidating how SUMOylation controls chromosome segregation during mitosis, a substantial progress has been achieved over the past decade. Furthermore, a major theme that has emerged from the recent studies of SUMOylation in mitosis is that both SUMO conjugation and deconjugation are critical for kinetochore assembly and disassembly. Lastly, we propose a model that SUMOylation coordinates multiple centromere and kinetochore activities to ensure accurate chromosome segregation.     

The β-Carboline Analog Mana-Hox Causes Mitotic Aberration by Interacting with DNA

Mana-Hox, an analog of beta-carbolines with anticancer activity, induces aberrant mitosis and delays mitotic exit. However, the cellular target is not known. In this study, we visualized the intracellular localization of Mana-Hox. Mana-Hox rapidly penetrated into cells (within 1 min) and concentrated on disorganized metaphase chromosomes after 13 hr of exposure. We demonstrated that Mana-Hox is a noncovalent DNA binder that can interact with DNA through intercalation and/or through minor groove binding. Furthermore, Mana-Hox also inhibits topoisomerase II relaxation activity in vitro, suggesting that Mana-Hox could perturb mitotic chromosome decatenation. Overall, Mana-Hox binding to DNA plays a critical role in the induction of aberrant mitosis and contributes to its anticancer activity.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

纺锤体驱动蛋白抑制剂的设计、合成与生物活性研究

纺锤体驱动蛋白(kinesin spindle protein, KSP/Eg5)作为潜在的肿瘤治疗靶点, 使发现KSP抑制剂成为热点. 设计并合成了4-氧基-β-四氢咔啉衍生物作为新型的KSP抑制剂, 并测定了其对KSP的抑制活性, 均优于阳性对照物. 其中化合物9c抑制KSP的IC50＝0.065 μmol•L－1, 优于阳性对照物Monastro l100多倍. 生物活性研究表明为抗肿瘤药物提供了新结构类型的候选化合物.     

Hierarchical dynamics in allostery following ATP hydrolysis monitored by single molecule FRET measurements and MD simulations

We report on a study that combines advanced fluorescence methods with molecular dynamics (MD) simulations to cover timescales from nanoseconds to milliseconds for a large protein. This allows us to delineate how ATP hydrolysis in a protein causes allosteric changes at a distant protein binding site, using the chaperone Hsp90 as test system. The allosteric process occurs via hierarchical dynamics involving timescales from nano- to milliseconds and length scales from Ångstroms to several nanometers. We find that hydrolysis of one ATP is coupled to a conformational change of Arg380, which in turn passes structural information via the large M-domain α-helix to the whole protein. The resulting structural asymmetry in Hsp90 leads to the collapse of a central folding substrate binding site, causing the formation of a novel collapsed state (closed state B) that we characterise structurally. We presume that similar hierarchical mechanisms are fundamental for information transfer induced by ATP hydrolysis through many other proteins.

We report on a study that combines advanced fluorescence methods with molecular dynamics simulations to cover timescales from nanoseconds to milliseconds for a large protein, the chaperone Hsp90.     

Design and synthesis of a new class of membrane-permeable triazaborolopyridinium fluorescent probes

A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.