Aluminum-induced kinesin inactivation as potential molecular cause of impairment of neuronal transport processes.

It is commonly accepted that aluminum ions may initiate the development of diverse diseases, including neurological disorders. So far, our knowledge of the molecular mechanisms of the interaction of aluminum with defined cellular structures has been still fragmentary. As functional key tasks of neuronal cells essentially depend on the activity of kinesin, we wanted to find out whether this motor protein represents a molecular target for aluminum. We demonstrate that aluminum ions inhibit (IC50 ∼50 µM) the ATPase of the neuron-specific kinesin KIF5A. The ATPase-active center itself, which is located in the kinesin motor domain, does not seem to be directly affected by aluminum. Our results suggest that inhibition is preferentially caused by aluminum binding to some sequence within the kinesin stalk leading to a conformational state of the kinesin molecule, similar to those described in cases of kinesin autoinhibition caused by motor domain-tail binding. Because of the relative high sequence conservation of mammalian kinesin-1 (to which KIF5A belongs), we assume that also in non-neuronal cells the intracellular transport can be affected by aluminum ions.

Elevated copper ion levels as potential cause of impaired kinesin-dependent transport processes.

Copper is a trace element required to maintain essential life processes. In healthy organisms, copper metabolism is well balanced. If this balance is destroyed, the cellular level of free copper might increase and cause toxic effects. So far, the molecular mechanisms of copper intoxication are understood only partly. The present study revealed that the kinesin-dependent transport system is strongly affected by copper(II) ions. Both the microtubules, along which kinesin moves, and the kinesin itself were found to be the target structures of copper ions: Microtubule formation was suppressed by copper ions (IC50 26-70 µM) apparently chiefly by inhibition of binding of microtubule-associated proteins to tubulin. This inhibition could be widely compensated by the microtubule-stabilising agent paclitaxel. In addition, copper ions strongly inhibited the ATPase activity of neuron-specific kinesin KIF5A. At final KIF5A concentration of 112 nM, an IC50 of 1.3 µM was determined. Correspondingly, the motility activity of KIF5A, measured as velocity of microtubules gliding across a kinesin-covered surface, was blocked. The effects of copper ions on microtubules and on KIF5A are suggested to contribute to impaired transport processes within brain and other organs in cases of copper ion surplus.

α-Synuclein oligomers impair neuronal microtubule-kinesin interplay.

Early α-synuclein (α-Syn)-induced alterations are neurite pathologies resulting in Lewy neurites. α-Syn oligomers are a toxic species in synucleinopathies and are suspected to cause neuritic pathology. To investigate how α-Syn oligomers may be linked to aberrant neurite pathology, we modeled different stages of α-Syn aggregation in vitro and investigated the interplay of α-Syn aggregates with proteins involved in axonal transport. The interaction of wild type α-Syn (WTS) and α-Syn variants (E57K, A30P, and aSyn(30-110)) with kinesin, tubulin, and the microtubule (MT)-associated proteins, MAP2 and Tau, is stronger for multimers than for monomers. WTS seeds but not α-Syn oligomers significantly and dose-dependently reduced Tau-promoted MT assembly in vitro. In contrast, MT gliding velocity across kinesin-coated surfaces was significantly decreased in the presence of α-Syn oligomers but not WTS seeds or fibrils (aSyn(30-110) multimers). In a human dopaminergic neuronal cell line, mild overexpression of the oligomerizing E57K α-Syn variant significantly impaired neurite network morphology without causing profound cell death. In accordance with these findings, MT stability, neuritic kinesin, and neuritic kinesin-dependent cargoes were significantly reduced by the presence of α-Syn oligomers. In summary, different α-Syn species act divergently on the axonal transport machinery. These findings provide new insights into α-Syn oligomer-driven neuritic pathology as one of the earliest events in synucleinopathies.

Phenylimino-10H-anthracen-9-ones as novel antimicrotubule agents-synthesis, antiproliferative activity and inhibition of tubulin polymerization.

A novel series of phenylimino-10H-anthracen-9-ones and 9-(phenylhydrazone)-9,10-anthracenediones were synthesized and evaluated for interaction with tubulin and for cytotoxicity against a panel of human tumor cell lines. The 10-(3-hydroxy-4-methoxy-phenylimino)-10H-anthracen-9-one 15h and its dichloro analog 16b were identified as potent inhibitors of tumor cell growth (16b, IC(50) K562 0.11 µM), including multidrug resistant phenotypes. Compound 15h had excellent activity as an inhibitor of tubulin polymerization. Concentration-dependent cell cycle analyzes by flow cytometry confirmed that KB/HeLa cells treated by 15h and 16b were arrested in the G2/M phases of the cell cycle. In competition experiments, 15h strongly displaced radiolabeled colchicine from its binding site on tubulin, showing IC(50) values similar to that of colchicine. The results obtained demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.

Kinesin passing permanent blockages along its protofilament track.

During movement along microtubules, kinesin usually follows a track parallel to the axis of a single protofilament. The question arises what happens when kinesin encounters blockages. The present study describes the movement of kinesin labeled by 20-nm gold beads along immobilized microtubules artificially decorated with blocking proteins. To guarantee that exactly the kinesin-binding sites were occupied and to avoid steric effects exerted by large molecules, the KIF5A motor domain was used for blocking. After binding, the blockages were irreversibly cross-linked to the microtubules to make them non-exchangeable. Under such conditions, kinesin movement became a non-continuous one. As a rule, after temporary stopping the kinesin moved on without being released from the microtubule. The results strongly suggest a bypassing mechanism based on the postulation that kinesin changes to and continues movement along a neighbouring protofilament. Bypassing is considered to ensure an efficient long-distance transport of cellular cargoes by kinesins.

Recovery of tubulin functions after freeze-drying in the presence of trehalose.

Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at -80 degrees C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assembly-competent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.

Synthesis of 2,6-aceanthrylenedione, a cyclic vinylog of anthraquinone.

The first synthesis of 2,6-aceanthrylenedione (6), a cyclic vinylog of anthraquinone and a useful starting material for the synthesis of 1-phenylaceanthrylene-2,6-diones such as 7, 8, and 9, is described. (10-Oxo-10H-anthracen-9-ylidene) acetyl chloride (5) cyclizes intramolecularly at room temperature in the presence of AlCl(3) to give 6. We found that 6 is a cytotoxic compound that inhibits tubulin polymerization.

Sulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone as highly active antimicrotubule agents. Synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

Benzenesulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone were prepared and found to inhibit microtubule formation by an in vitro tubulin polymerization assay. Several analogues showed potent cytotoxic activity in an assay based on K562 leukemia cells with IC50 values of <100 nM. The methylamino analogue 14i was the most active compound in this assay (14i, IC50 K562: 0.05 muM). Antiproliferative activities of selected compounds were additionally evaluated against a panel of 12 tumor cell lines, including multi-drug-resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with selected compounds were arrested in the G2/M phases of the cell cycle. In competition experiments, these compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values lower than that of colchicine. The results demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.

Toxic effects of zinc ions on kinesin - Potential molecular cause of impaired intracellular transport.

In healthy organisms the metabolism of the trace element zinc is well balanced. If this balance becomes destroyed the free zinc level might increase and cause toxic effects. The present study demonstrates that under definite conditions zinc ions are able to inhibit the ATPase activity of neuron-specific KIF5A (kinesin-1). Correspondingly, the motility activity of KIF5A also decreased. The inhibition rates have been found to depend on the magnesium ion concentration. Lowering the magnesium concentration weakens the inhibition. In addition, also decreases of temperature or increasing the ATP concentration result in reduced inhibition. Zinc ion-mediated inhibition of KIF5A activity might be one molecular cause contributing to impaired transport processes within brain and other organs in cases of zinc dyshomeostasis.

N-Heterocyclic (4-Phenylpiperazin-1-yl)methanones Derived from Phenoxazine and Phenothiazine as Highly Potent Inhibitors of Tubulin Polymerization.

We report here a series of 27 10-(4-phenylpiperazin-1-yl)methanones derived from tricyclic heterocycles which were screened for effects on tumor cell growth, inhibition of tubulin polymerization, and induction of cell cycle arrest. Several analogues, among them the 10-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-10H-phenoxazine-3-carbonitrile (16o), showed excellent antiproliferative properties, with low nanomolar GI50 values (16o, mean GI50 of 3.3 nM) against a large number (93) of cancer cell lines. Fifteen compounds potently inhibited tubulin polymerization. Analysis of cell cycle by flow cytometry revealed that inhibition of tumor cell growth was related to an induction of G2/M phase cell cycle blockade. Western blotting and molecular docking studies suggested that these compounds bind efficiently to ß-tubulin at the colchicine binding site. Our studies demonstrate the suitability of the phenoxazine and phenothiazine core and also of the phenylpiperazine moiety for the development of novel and potent tubulin polymerization inhibitors.

9-Benzylidene-naphtho[2,3-b]thiophen-4-ones as novel antimicrotubule agents-synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

A novel series of 9-benzylidene-naphtho[2,3-b]thiophen-4-ones and structurally related compounds were synthesized and evaluated for their ability to inhibit tubulin polymerization. The 4-hydroxy-3,5-dimethoxy-benzylidene analogue 15d was identified as a potent cytotoxic agent in an assay based on K562 leukemia cells. Antiproliferative activity of 15d and the 2,4-dimethoxy-3-hydroxy-benzylidene analogue 15e was additionally evaluated against a panel of 12 tumor cell lines, including multidrug resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that K562 cells as well as KB/HeLa cells treated by 15d were arrested in the G2/M phases of the cell cycle. Moreover, four compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds. In competition experiments, the most active compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values virtually 3- to 4-fold lower than that of colchicine.

High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc.

Genotoxicity of inorganic lead salts and disturbance of microtubule function.

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 microM lead chloride and 0.05 microM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 microM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 microM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 microM and reached half-maximal inhibition of motility at about 50 microM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II).

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 microM of complexed Hg(II), and for inhibition of motility it was 0.05 microM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 microM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

Kinesin-dependent motility generation as target mechanism of cadmium intoxication.

The anterograde vesicle transport within neurons critically depends on microtubules and on the activity of kinesin. The present study demonstrates that cadmium ions inhibit the in vitro assembly of microtubules from tubulin, whereby at high cadmium levels (∼500 µM) unstructured protein aggregates were formed. Cadmium ions also significantly lower both the ATPase and motility activity of neuron-specific kinesin KIF5A in concentration-dependent manner. For the inhibition of KIF5A ATPase activity, an IC50 value of 10.4±1.5 µM was determined. Inhibition could be widely compensated by addition of EGTA, but not by addition of thiols. The inhibitory effect of cadmium on KIF5A was considerably weakened by increasing ATP concentration. As nucleoside triphosphate binding is known to be accompanied by conformational changes within the kinesin motor domain, it might be suggested that these changes protect the motor domain against cadmium. The effects of cadmium ions on the kinesin-microtubule motility generating system are considered to contribute to the development of neuronal disorders caused by cadmium intoxication.

The Ca2+ sensor protein swiprosin-1/EFhd2 is present in neurites and involved in kinesin-mediated transport in neurons.

Swiprosin-1/EFhd2 (EFhd2) is a cytoskeletal Ca2+ sensor protein strongly expressed in the brain. It has been shown to interact with mutant tau, which can promote neurodegeneration, but nothing is known about the physiological function of EFhd2 in the nervous system. To elucidate this question, we analyzed EFhd2-/-/lacZ reporter mice and showed that lacZ was strongly expressed in the cortex, the dentate gyrus, the CA1 and CA2 regions of the hippocampus, the thalamus, and the olfactory bulb. Immunohistochemistry and western blotting confirmed this pattern and revealed expression of EFhd2 during neuronal maturation. In cortical neurons, EFhd2 was detected in neurites marked by MAP2 and co-localized with pre- and post-synaptic markers. Approximately one third of EFhd2 associated with a biochemically isolated synaptosome preparation. There, EFhd2 was mostly confined to the cytosolic and plasma membrane fractions. Both synaptic endocytosis and exocytosis in primary hippocampal EFhd2-/- neurons were unaltered but transport of synaptophysin-GFP containing vesicles was enhanced in EFhd2-/- primary hippocampal neurons, and notably, EFhd2 inhibited kinesin mediated microtubule gliding. Therefore, we found that EFhd2 is a neuronal protein that interferes with kinesin-mediated transport.

Characterization of a new class of androgen receptor antagonists with potential therapeutic application in advanced prostate cancer.

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the same mode of action on the androgen receptor. Although initially effective, resistance to these androgen receptor antagonists usually develops and the cancer quickly progresses to castration-resistant and metastatic states. Yet even in these late-stage patients, the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for novel chemical classes of androgen receptor antagonists that could help overcome the problem of resistance. In this study, we implemented and used the synergetic combination of virtual and experimental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound (VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft model. These results provide a foundation for the development of a new class of androgen receptor antagonists that can help address the problem of antiandrogen resistance in prostate cancer.

N-benzoylated phenoxazines and phenothiazines: synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

A total of 53 N-benzoylated phenoxazines and phenothiazines, including their S-oxidized analogues, were synthesized and evaluated for antiproliferative activity, interaction with tubulin, and cell cycle effects. Potent inhibitors of multiple cancer cell lines emerged with the 10-(4-methoxybenzoyl)-10H-phenoxazine-3-carbonitrile (33b, IC(50) values in the range of 2-15 nM) and the isovanillic analogue 33c. Seventeen compounds strongly inhibited tubulin polymerization with activities higher than or comparable to those of the reference compounds such as colchicine. Concentration-dependent flow cytometric studies revealed that inhibition of K562 cell growth was associated with an arrest in the G2/M phases of the cell cycle, indicative of mitotic blockade. Structure-activity relationship studies showed that best potencies were obtained with agents bearing a methoxy group placed para at the terminal phenyl ring and a 3-cyano group in the phenoxazine. A series of analogues highlight not only the phenoxazine but also the phenothiazine structural scaffold as valuable pharmacophores for potent tubulin polymerization inhibitors, worthy of further investigation.

Synthesis, antiproliferative activity and inhibition of tubulin polymerization by anthracenone-based oxime derivatives.

A series of anthracenone-based oxime ethers and -esters were synthesized in order to evaluate their antiproliferative activity. Several investigated compounds displayed strong antiproliferative activity against K562 leukemia cells and proved to be strong inhibitors of tubulin polymerization. In this context, anthracenone-based oxime ethers and -esters are considered to contribute to the development of novel antiproliferative drugs, based on tubulin interaction.

Synthesis, antiproliferative activity and inhibition of tubulin polymerization by 1,5- and 1,8-disubstituted 10H-anthracen-9-ones bearing a 10-benzylidene or 10-(2-oxo-2-phenylethylidene) moiety.

A novel series of 1,5- and 1,8-disubstituted 10-benzylidene-10H-anthracen-9-ones and 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones was synthesized to assess the substituent effects on biological activity. The 3-hydroxy-2,4-dimethoxy-benzylidene analogue 16 h displayed strong antiproliferative activity against several tumor cell lines, including multi-drug resistant phenotypes. Flow cytometric studies showed that KB/HeLa cells treated by elected compounds were arrested in the G2/M phases of the cell cycle. Among the compounds tested for inhibition of tubulin polymerization, 14 compounds proved to be exceptionally active with IC(50) values < 1 microM. In the 1,5-dichloro-derived series of benzylideneanthracenones, E/Z isomers were separated and biological effects were monitored. We found that the olefinic geometry had no significant effect on biological activity. Furthermore, the E isomeric 1,5-dichloro-substituted phenacylidenes entirely proved to be more potent inhibitors of tubulin polymerization than the recently described 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones. In conclusion, the present study improves understanding of the action of anthracenone-based tubulin polymerization inhibitors and contributes to the design of further potent anti-tubulin drugs.