Interaction Of Tubulin Research Articles

The Mechanism of Tubulin Assembly into Microtubules: Insights from Structural Studies.

SummaryMicrotubules are cytoskeletal components involved in pivotal eukaryotic functions such as cell division, ciliogenesis, and intracellular trafficking. They assemble from αβ-tubulin heterodimers and disassemble in a process called dynamic instability, which is driven by GTP hydrolysis. Structures of the microtubule and of soluble tubulin have been determined by cryo-EM and by X-ray crystallography, respectively. Altogether, these data define the mechanism of tubulin assembly-disassembly at atomic or near-atomic level. We review here the structural changes that occur during assembly, tubulin switching from a curved conformation in solution to a straight one in the microtubule core. We also present more subtle changes associated with GTP binding, leading to tubulin activation for assembly. Finally, we show how cryo-EM and X-ray crystallography are complementary methods to characterize the interaction of tubulin with proteins involved either in intracellular transport or in microtubule dynamics regulation.

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells.

αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1-100 μM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1-10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles.

A Review on Anticancer Activities of Thiophene and Its Analogs.

Cancer is the world's second-largest cause of mortality and one of the biggest global health concerns. The prevalence and mortality rates of cancer remain high despite significant progress in cancer therapy. The search for more effective, as well as less toxic treatment methods for cancer, is at the focus of current studies. Thiophene and its derivatives have surged as an influential scaffold, which, because of their appreciable diversity in biological activities, has drawn the concerned interest of the researchers in the field of medicinal chemistry. By the affluent introduction of its derivatives, which have antioxidant, anti-inflammatory, antimicrobial, and anticancer activities, the adaptability of the thiophene moiety has been displayed. The nature and positioning of the substitutions significantly impacted thiophene moiety activity. This decent array in the living response account about this moiety has picked plentiful researcher's consideration to inquire about it to its peculiar potential across certain activities. In the field of cancer therapy against different cancer cells, the structure-activity relationship for each of the derivatives showed an excellent understanding of thiophene moiety. Information from the various articles revealed the key role of thiophene moiety and its derivatives to develop the vital lead compound. The essential anticancer mechanisms identified include inhibition of the topoisomerase, inhibition of tyrosine kinase, tubulin interaction and apoptosis induction through the activation of reactive oxygen species. This review is an endeavor to promote the anticancer potential of the derivatives, whether having thiophene or condensed thiophene as a core moiety or as a substituent that can lead in the future to synthesize varieties of chemotherapeutic entities in the field of cancer treatment.

Novel colchicine conjugate with unusual effect on the microtubules of cancer cells

Abstract Colchicine derivative bearing substituted bispidine moiety, namely N-{7-(3,7-Di-(tert-butoxycarbonyl)-1,5-dimethyl-3,7-diazabicyclo[3.3.1]nonan-9-yl)-oxy-7-oxoheptanoyl}-N-deacetylcolchicine, was synthesized and tested for its effect on the net of microtubules (MT) in lung cancer cells A549. The compound induced not only MT depolymerization but stimulated the formation of small tubulin aggregates and long tubulin fibrils localized mainly around nuclei. The assemblies were morphologically different from tubulin clusters induced by structurally related anticancer agent tubuloclustin. The biotests data demonstrate that the depolymerization takes place for both pure tubulin and tubulin in cellulo, while fibrils are formed only in the cells. The research data of structure–activity relationship for several similar colchicine derivatives synthesized in the work give evidence for the proposition that the initial conjugate may interact not only with tubulin and MT in the cells, but also with MT-associated proteins, involved in the process of tubulin polymerization. The ability to affect simultaneously MAP – tubulin interactions opens attractive prospects in the design of novel anticancer agents.

Biophysical Characterization of Covalently Modified Protein Tau: Oligomers, Aggregation, and Tubulin Interactions

The molecular interactions that drive the onset and spread of Alzheimer's disease (AD) remain poorly understood. In AD, tau neurofibrillary tangles correlate with cognitive decline and have historically been associated with tau's toxicity. Recently, tau oligomers have been implicated as stable toxic subunits that contribute to the formation of paired helical filaments and eventual neurofibrillary tangles. Covalently modified tau is a major component of neurofibrillary tangles and plays a significant role in tau's ability to dissociate from microtubules, aggregate, and become toxic. Elucidating the effects of covalent modifications on oligomer formation could aide our understanding of the molecular mechanisms behind tau's toxicity. The complexity of tau, partially due to its intrinsically disordered nature, allows for phosphorylation, acetylation, and succinylation to occur at multiple sites and influence the physiological and pathological roles of tau. Specific residue modifications can play a major role in modulating tau function and aggregation. This has led to the use of mimics in cell, mouse, and in-vitro models of tau pathology. Site-specific mimics of phosphorylation, acetylation, and succinylation can aide our ability to biophysically characterize the effect of covalently modified tau on oligomer formation, aggregation propensity, and tubulin interactions; Ultimately aiding our understanding of tau function and structure. We apply nuclear magnetic resonance, fluorescence, and light scattering to show that covalently modified tau mimics of phosphorylation, acetylation, and succinylation perturb tau-T2R interactions (a model of tau-tubulin interactions), induce filament formation, and form stable toxic oligomers with different levels of efficiency. Our results reinforce phosphorylation as a contributor to filament formation, acetylation as a driver of oligomer formation, and introduces tau succinylation as a novel modifier of tau structure and function.

Design, Synthesis and Characterisation of Novel Phenothiazine‐Based Triazolopyridine Derivatives: Evaluation of Anti‐Breast Cancer Activity on Human Breast Carcinoma

AbstractA series of novel phenothiazine based [1,2,4]triazolo[4, 3‐a]pyridine scaffolds were designed and synthesized in good yields by the oxidative cyclisation of phenothiazine pyridylhydrazones. Biological responses of all compounds toward a panel of human breast cancer cells (MDA‐MB‐231, MDA‐MB‐468, MCF7, SKBR3 and T47D) and human non‐tumorigenic epithelial breast cells (MCF10 A) were evaluated. Structure‐activity relationship revealed that compound with pendant phenyl ring on phenothiazine exhibited significant cytotoxic activity and apoptotic induction effects against breast cancer cell line with IC50 value 10.2 to 17.6 μM. Notably, the cytotoxic effect was 3.5 fold higher on cancer than non‐cancer cells, indicating potential control of breast cancer with lower side effects. Molecular docking studies confirmed the presence of hydrophobic contacts between appended phenyl ring, triazolopyridine and phenothiazine moieties with adjacent residues within the binding pocket of tubulin. One of the nitrogen in the triazolo ring also showed hydrogen bonding with tubulin. These tubulin interactions were also found with the taxane ring of paclitaxel. Cell cycle analysis confirmed the G2/M arrest induced by this compound on human breast cancer cells. Therefore, the potential anti‐cancer, pro‐apoptotic, and cell cycle arrest warrant further development of this molecule as a new class of anticancer agent.

Abstract 105: MTH1 promotes mitotic progression to avoid oxidative DNA damage in cancer cells

Abstract MTH1 prevents incorporation of oxidized deoxynucleoside triphosphates (e.g., 8-oxodGTP) into DNA. Earlier we demonstrated that MTH1 inhibitors TH588 and TH1579 possesses anti-cancer activity in various tumour types, while other structurally distinct MTH1 inhibitors failed to kill cancer cells or elevate the levels of oxidized nucleotides in DNA. In our current study, we found a novel function of MTH1 in mitosis, as it plays an important role in correct spindle assembly in cancer cells and progression through mitosis, and furthermore, we demonstrate MTH1 binds tubulin in vivo. We also observed that structurally distinct MTH1 inhibitors display differential abilities to break the MTH1 tubulin interaction, and also demonstrate that TH588 and TH1579 can directly inhibit tubulin polymerisation in vitro. Consistent with our results, we establish that TH588 and TH1579 kill cancer cells by a mitosis-dependent mechanism and implicate reactive oxygen species (ROS) accumulation and incorporation of oxidised nucleotides into DNA in this process. Furthermore, we found that non-cytotoxic MTH1 inhibitors can become cytotoxic and increase incorporation of oxidised nucleotides into DNA when they are combined with sub-therapeutic concentrations of mitotic inhibitors. In conclusion, MTH1 inhibitors killing cancer cells have a unique and complex mechanism of action: (1) causing a mitotic arrest, induced by targeting MTH1 which is required for mitotic progression, but also through direct inhibition of microtubule polymerisation, in-turn leading to an increase in ROS and likely 8-oxodGTP and (2) MTH1-dependent 8-oxodGTPase inhibitory activity causing incorporation of damaged nucleotides into DNA to kill cancer cells. Citation Format: Kumar Sanjiv, Helge gad, Sean G. Rudd, Oliver Mortusewicz, Ailine Stolz, Nuno Amaral, Lars Brautigham, Linda Pudelko, Christina Kaldéren, Ann-Sofie Jemth, Ingrid Almlöf, Torkild Visnes, Niklas Schultz, Johan Boström, José Manuel Calderon Montano1, Anna Hagenkort, Petra Groth, Camilla Gokturk, Tobias Koolmeister, Prasad Wakchaure, Evert Homan, Mikael Altun, Cecilia Ström, Martin Scobie, Holger Bastians, Ulrika Warpman Berglund, Thomas Helleday. MTH1 promotes mitotic progression to avoid oxidative DNA damage in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 105.

Targeted Polypeptide–Microtubule Aggregation with Cucurbit[8]uril for Enhanced Cell Apoptosis

AbstractTunable protein assemblies not only hold a dominant position in vital biological events but are also a significant theme in supramolecular chemistry. Herein, we demonstrated that the intertubular aggregation of microtubules (MTs) could be efficiently regulated by a synergistic polypeptide–tubulin interaction and host–guest complexation. The benzylimidazolium‐modified antimitotic peptide (BP) could recognize the MTs and concurrently form stable inclusion complexes with avirulent cucurbit[7]uril (CB[7]) and cucurbit[8]uril (CB[8]) in different binding stoichiometries. The self‐assembling morphology of MTs was converted from fibrous to nanoparticulate aggregates via extensive BP⊂CB[8] cross‐linkage, leading to significant cell apoptosis and tumor ablation in vivo. The targeted (BP⊂CB[8])@MT ternary assembly provides a facile supramolecular method to enhance the protein–protein interactions, which may be developed as a therapy for degenerative diseases, such as cancer.

Synthesis and anti-breast cancer activity of novel indibulin related diarylpyrrole derivatives.

During recent years, a number of anti-tubulin agents were introduced for treatment of diverse types of cancer. Despite their potential in the treatment of cancer, drug resistance and adverse toxicity, such as peripheral neuropathy, are some of the negative effects of anti-tubulin agents. Among anti-tubulin agents, indibulin was found to cause minimal peripheral neuropathy. Thus far, however, indibulin has not entered clinical usage, caused in part by its poor aqueous solubility and other developmental problems in preclinical evaluation. With respect to need for finding potent and safe anticancer agents, in our current research work, we synthesized several indibulin-related diarylpyrrole derivatives and investigated their anti-cancer activity. Cell cultur studies were perfomred using the MTT cell viability assay on the breast cancer cell lines MCF-7, T47-D, and MDA-MB231 and also NIH-3T3 cells as representative of a normal cell line. The activity of some of the synthesized compounds for tubulin interaction was studied using colchicine binding and tubulin polymerization assays. The annexin V-FITC/PI method and flow cytometric analysis were used for studying apoptosis induction and cell cycle distribution. Two of the synthesized compounds, 4f and 4g, showed high activity on the MDA-MB231 cell line (IC50= 11.82 and 13.33μM, (respectively) and low toxicity on the normal fibroblast cells (IC50> 100μM). All of the tested compounds were more potent on T47-D cancer cells and less toxic on NIH-3T3 normal cells in comparison to reference compound, indibulin. The tubulin polymerization inhibition assay and [3H]colchicine binding assay showed that the main mechanism of cell death by the potent synthesized compounds was not related to an interaction with tubulin. In the annexin V/PI staining assay, the induction of apoptosis in the MCF-7 and MDA-MB231 cell lines was observed. Cell cycle analysis illustrated an increased percentage of sub-G-1 cells in the MDA-MB231 cell line as a further indication of cell death through induction of apoptosis. Graphical abstract Novel Indibulin analogous as anti-breast cancer agents.

Virtual screening identification of novel chemical inhibitors for aberrant interactions between pathogenic mutant SOD1 and tubulin

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease caused by selective motor neuron death. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) belong to one of the four major mutation clusters responsible for pathogenesis of ALS. Toxic gain-of-function (not loss-of-function) of SOD1 mutants causes motor neuron degeneration. Aberrant protein–protein interactions (PPI) between mutant SOD1 and other proteins are involved in this toxic gain-of-function. Therefore, PPI inhibitors of mutant SOD1 not only increase understanding of ALS pathogenesis, but can also be used as novel therapeutics for ALS. Although it is challenging to identify PPI inhibitors, prior knowledge of the binding site can increase success probability. We have previously reported that tubulin interacts with N-terminal residues 1–23 of mutant SOD1. In the present study, we performed virtual screening by docking simulation of 32,791 compounds using this N-terminal binding site as prior knowledge. An established assay system for interaction inhibition between mutant SOD1–tubulin was used as an in-house model system to identify mutant SOD1 PPI inhibitors, with the goal of developing novel therapeutics for ALS. Consequently, five of six assay-executable compounds among top-ranked compounds during docking simulation inhibited the mutant SOD1–tubulin interaction in vitro. Binding mode analysis predicted that some inhibitors might bind the tubulin binding site of G85R SOD1 by pi electron interaction with the aromatic ring of the Trp32 residue of G85R SOD1. Our screening methods may contribute to the identification of lead compounds for treating ALS.

Electromechanical vibration of microtubules and its application in biosensors.

An electric field (EF) has the potential to excite the vibration of polarized microtubules (MTs) and thus enable their use as a biosensor for the biophysical properties of MTs or cells. To facilitate the development, this paper aims to capture the EF-induced vibration modes and the associated frequency for MTs. The analyses were carried out based on a molecular structural mechanics model accounting for the structural details of MTs. Transverse vibration, radial breathing vibration and axial vibration were achieved for MTs subject to a transverse or an axial EF. The frequency shift and stiffness alteration of MTs were also examined due to the possible changes of the tubulin interactions in physiological or pathological processes. The strong correlation achieved between the tubulin interaction and MT vibration excited by EF provides a new avenue to a non-contacting technique for the structural or property changes in MTs, where frequency shift is used as a biomarker. This technique can be used for individual MTs and is possible for those in cells when the cytosol damping on MT vibrations is largely reduced by the unique features of MT-cytosol interface.

Structural Basis for the Lipid-Mediated Interaction of Tubulin with VDAC Revealed by Neutron Reflectometry

A preponderance of evidence suggests that the mitochondrial outer membrane (MOM) binds dimeric tubulin, which blocks the flux of metabolites through VDAC, the major MOM channel, by inserting its negatively charged, disordered C-terminal tail into the VDAC pore. The discovery of this novel regulatory mechanism of mitochondrial respiration has raised several fundamental questions, including how tubulin binds to the MOM surface, whether its membrane-bound conformation is consistent with the accessibility of its C-termini to the membrane surface, and what role mitochondrial lipids assume in mediating the tubulin-VDAC interaction. Here we report neutron reflectivity (NR) studies on tubulin-coated and VDAC-containing biomimetic membranes. In combination with molecular dynamics simulations, these reveal, first, that dimeric tubulin is anchored to the membrane surface by an amphipathic a-helix of the a-tubulin subunit in an orientation that presents the C-termini of both tubulin subunits to the membrane surface. The structural model is supported by electrophysiological evidence using recombinant tubulin constructs in which C-terminal tails bound to either subunit are individually observed in a VDAC nanopore. Second, we demonstrate using NR that surface-immobilized VDAC can be reconstituted into a complete lipid membrane, thus creating a “protein-tethered bilayer lipid membrane” (ptBLM) over a large sample area. The ptBLM architecture allows dynamic changes of the solution environment, while NR reveals the corresponding structural response of the protein and its lipid environment. This allows us to experimentally observe the effects of pH-induced changes in VDAC's structure on the local lipid environment. Molecular dynamics simulations are in good agreement with NR experiments. We speculate that local curvature or lipid packing stress induced by membrane-embedded VDAC and its structural dynamics serves to localize cytosolic regulators such as tubulin to the vicinity of the channel.

Characterization of Microtubule-Associated Proteins (MAPs) and Tubulin Interactions by Isothermal Titration Calorimetry (ITC).

Microtubules are highly dynamic structures which play a central role in many cellular processes such as cell division, intracellular transport, and migration. Their dynamics is tightly regulated by stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Many approaches have been developed to study interactions between tubulin and MAPs. However, isothermal titration calorimetry (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization of the interaction after a single titration experiment. We provide here the protocols to apply ITC to tubulin interaction with either stathmin or tau, which will help to avoid the common pitfalls in this very powerful and sensitive method.

Diverse balances of tubulin interactions and shape change drive and interrupt microtubule depolymerization.

Microtubules are stiff biopolymers that self-assemble via the addition of GTP-tubulin (αβ-dimer bound to GTP), but hydrolysis of GTP- to GDP-tubulin within the tubules destabilizes them toward catastrophically-fast depolymerization. The molecular mechanisms and features of the individual tubulin proteins that drive such behavior are still not well-understood. Using molecular dynamics simulations of whole microtubules built from a coarse-grained model of tubulin, we demonstrate how conformational shape changes (i.e., deformations) in subunits that frustrate tubulin-tubulin binding within microtubules drive depolymerization of stiff tubules via unpeeling "ram's horns" consistent with experiments. We calculate the sensitivity of these behaviors to the length scales and strengths of binding attractions and varying degrees of binding frustration driven by subunit shape change, and demonstrate that the dynamic instability and mechanical properties of microtubules can be produced based on either balanced or imbalanced strengths of lateral and vertical binding attractions. Finally, we show how catastrophic depolymerization can be interrupted by small regions of the microtubule containing undeformed dimers, corresponding to incomplete lattice hydrolysis. The results demonstrate a mechanism by which microtubule rescue can occur.

A magic bullet for targeting cancers with supernumerary centrosomes.

Centrosome amplification is frequently observed in aggressive human cancers, and it has been proposed as a promising target in cancer therapy. Work by Mariappan et al (2019) in this issue details the identification of a small‐molecule inhibitor that perturbs the CPAP–tubulin interaction, prevents efficient centrosome clustering during mitosis, and exhibits in vivo anti‐tumor activity. This study demonstrates the potential of developing tumor‐specific treatment by specifically targeting centrosome amplification.

Inhibition of CPAP–tubulin interaction prevents proliferation of centrosome‐amplified cancer cells

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP‐dependent peri‐centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP–tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP–tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de‐clustering, prolonged multipolar mitosis, and cell death. 3D‐organotypic invasion assays reveal that CCB02 has broad anti‐invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)‐resistant EGFR‐mutant non‐small‐cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug‐resistant cancers exhibiting high incidence of centrosome amplification.

Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α-actinin, linking innate immunity to the cytoskeleton.

Suppressor of IKKepsilon (SIKE) is associated with the type I interferon response of the innate immune system through TANK‐binding kinase 1 (TBK1). Originally characterized as an endogenous inhibitor of TBK1 when overexpressed in viral infection and pathological cardiac hypertrophic models, a mechanistic study revealed that SIKE acts as a high‐affinity substrate of TBK1, but its function remains unknown. In this work, we report that scratch assay analysis of parental and SIKE CRISPR/Cas9 knockout HAP1 cells showed an ~ 20% decrease in cell migration. Investigation of the SIKE interaction network through affinity purification/mass spectrometry showed that SIKE formed interactions with cytoskeletal proteins. In immunofluorescence assays, endogenous SIKE localized to cytosolic puncta in both epithelial and myeloid cells and to nuclear puncta in myeloid cells, while in epithelial cells additional staining occurred in stress fiber‐like structures and adjacent to the plasma membrane. Using cellular markers, co‐occurrence of SIKE fluorescence with actin, α‐actinin, and ezrin was detected. Reciprocal immunoprecipitation revealed a SIKE:tubulin interaction sensitive to the phosphorylation state of SIKE, but a SIKE:α‐actinin interaction was unchanged by SIKE phosphorylation. In vitro precipitation assays confirmed a direct SIKE interaction with tubulin and α‐actinin. These results indicate that SIKE may promote cell migration by directly associating with the cytoskeleton. In this role, SIKE may mediate cytoskeletal rearrangement necessary in innate immunity, but also link a key catalytic hub, TBK1, to the cytoskeleton.DatabaseThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD007262.

Elucidation of the Tubulin-targeted Mechanism of Action of 9-(3-pyridyl) Noscapine.

We have recently reported the synthesis and antiproliferative potential of a series of biaryl type α-noscapine congeners. Among them, 9-(3-pyridyl) noscapine 3f (9-PyNos, henceforth), which was synthesized by adding pyridine unit to the tetrahydroisoquinoline part of natural α-noscapine core, was found to be the most effective one to inhibit proliferation of a variety of cancer cell lines. However, details of its interactions with its cellular target, tubulin, remain poorly understood. In this report, we examined the nature of interactions of 9-PyNos with tubulin based on the methodologies of spectrofluorimetry, circular dichroism, and turbidimetry techniques. Far-UV circular dichroism spectra indicated perturbation of tubulin secondary structure in the presence of 9-PyNos, not amounting, however, to the perturbation induced by noscapine. The noscapinoid nevertheless altered the surface configuration of the protein considerably, as indicated by an anilinonaphthalene sulphonate binding assay, and promoted colchicine binding to tubulin, the latter indicating its adjacent binding site with colchicine. 9-PyNos however, did not alter microtubule assembly considerably. Investigating the possible reason behind this apparent lack of strong inhibition of microtubule assembly, we found that the binding interactions of tubulin with 9-PyNos do not involve modification of cysteine residues of tubulin. Taken together, our data suggest that the antiproliferative mechanism of action of 9-PyNos involves disruption of structural integrity of tubulin without strong inhibition of tubulin assembly.

A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase Activity

Kinesin-8 motors regulate the size of microtubule structures, using length-dependent accumulation at the plus end to preferentially disassemble long microtubules. Despite extensive study, the kinesin-8 depolymerase mechanism remains under debate. Here, we provide evidence for an alternative, tubulin curvature-sensing model of microtubule depolymerization by the budding yeast kinesin-8, Kip3. Kinesin-8/Kip3 uses ATP hydrolysis, like other kinesins, for stepping on the microtubule lattice, but at the plus end Kip3 undergoes a switch: its ATPase activity issuppressed when it binds tightly to the curved conformation of tubulin. This prolongs plus-end binding, stabilizes protofilament curvature, and ultimately promotes microtubule disassembly. The tubulin curvature-sensing model is supported by our identification of Kip3 structural elements necessary and sufficient for plus-end binding and depolymerase activity, as well as by the identification of an α-tubulin residue specifically required for the Kip3-curved tubulin interaction. Together, these findings elucidate a major regulatory mechanism controlling the size of cellular microtubule structures.

11-Substituted Benzo[c]phenanthridines: New Structures and Insight into Their Mode of Antiproliferative Action.

The synthesis of various new structures of a library of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines (BP) and 11-substituted 6-aminobenzo[c]phenanthridines (BP-D) is presented. These structures, further synthetic modifications, and the preparation of follow-up products which delivered about 40 new derivatives are described. Their potential as antiproliferative drug candidates was investigated by comparison of NCI 60 developmental therapeutics program (DTP) human tumor cell line screening data based on the results of in vitro tumor cell growth inhibition, including about 40 hitherto unpublished compound test results with up to 60 cancer cell lines. NCI-COMPARE studies helped to suggest the modes of action of the highly active antiproliferative drugs. These findings are supported by in vitro biological investigations showing either inhibition of tubulin polymerization and depolymerization or topoisomerase inhibition. Together with physicochemical parameters of the drug candidates, structure-activity relationships are critically discussed. Tubulin interaction or inhibition of topoisomerase I and IIα/β activity are two rationales that can explain the antiproliferative activity observed in the NCI 60 DTP human tumor cell line screen. However, it can also be reasonably assumed that these compounds address several targets, thus prohibiting the identification of simple structure-activity relationships. The new structures described herein are thought to act as so-called multitarget drugs, thus being of special interest in the area of multidrug resistance.