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Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, [gamma]-tubulin ring complex ([gamma]-TuRC) has been identified, precisely how [gamma]-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis [gamma]-TuRC. We reveal a high [gamma]-/[alpha][beta]-tubulin affinity, which facilitates assembly of a MT from [gamma]-TuRC. Whereas spontaneous nucleation requires assembly of 8 [alpha][beta]-tubulins, nucleation from [gamma]-TuRC occurs efficiently with a cooperativity of 4 [alpha][beta]-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated [alpha][beta]-tubulins drive [gamma]-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h [gamma]-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal [gamma]-/[alpha][beta]-tubulin interaction.

In this study, we aimed to develop a pharmacophore-based three-dimensional quantitative structure activity relationship (3D-QSAR) for a set including sixty-two cytotoxic quinolines (1-62) as anticancer agents with tubulin inhibitory activity. A total of 279 pharmacophore hypotheses were generated based on the survival score to build QSAR models. A six-point pharmacophore model (AAARRR.1061) was identified as the best model which consisted of three hydrogen bond acceptors (A) and three aromatic ring (R) features. The model showed a high correlation coefficient (R2=0.865), cross-validation coefficient (Q2=0.718), and F value (72.3). The best pharmacophore model was then validated by the Y-Randomization test and ROC-AUC analysis. The generated 3D contour maps were used to reveal the structure activity relationship of the compounds. The IBScreen database was screened against AAARRR.1061, and after calculating ADMET properties, 10 compounds were selected for further docking study. Molecular docking analysis showed that compound STOCK2S-23597 with the highest docking score (-10.948 kcal/mol) had hydrophobic interactions and can form four hydrogen bonds with active site residues.

Tubulin, an extensively studied self-assembling protein, forms filaments in eukaryotic cells that affect cell shape, among other functions. The model archaeon Haloferax volcanii uses two tubulin-like proteins (FtsZ1/FtsZ2) for cell division, similar to bacteria, but has an additional six related tubulins called CetZ. One of them, CetZ1, was shown to play a role in cell shape. Typically, discoid and rod shapes are observed in planktonic growth, but under biofilm formation conditions (i.e., attached to a substratum), H. volcanii can grow filamentously. Here, we show that the deletion mutants of all eight tubulin-like genes significantly impacted morphology when cells were allowed to form a biofilm. ΔftsZ1, ΔcetZ2, and ΔcetZ4-6 created longer, less round cells than the parental and a higher percentage of filaments. ΔcetZ1 and ΔcetZ3 were significantly rounder than the parental, and ΔftsZ2 generated larger, flat, amorphic cells. The results show all tubulin homologs affect morphology at most timepoints, which therefore suggests these genes indeed have a function.

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquidmalignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin–GB1 complex.

Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the ‘tubulin code’. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.The mechanical and dynamic properties of microtubules are determined by their complement of subunits, known as tubulin isotypes, and the post-translational modifications found on these isotypes. This concept is known as the ‘tubulin code’. The regulation of microtubules and microtubule-associated proteins by this code is critical for the correct function of a range of tissues. Consequently, recent studies have linked perturbation of the tubulin code to disease, including neurodegenerative diseases.

The auristatin class of microtubule destabilizers are highly potent cytotoxic agents against several cancer cell types when delivered as antibody drug conjugates. Here we describe the high resolution structures of tubulin in complex with both monomethyl auristatin E and F and unambiguously define the trans-configuration of both ligands at the Val-Dil amide bond in their tubulin bound state. Moreover, we illustrate how peptidic vinca-site agents carrying terminal carboxylate residues may exploit an observed extended hydrogen bond network with the M-loop Arg278 to greatly improve the affinity of the corresponding analogs and to maintain the M-loop in an incompatible conformation for productive lateral tubulin-tubulin contacts in microtubules. Our results highlight a potential, previously undescribed molecular mechanism by which peptidic vinca-site agents maintain unparalleled potency as microtubule-destabilizing agents.

Cancer accounts for numerous deaths each year, and it is one of the most common causes of death worldwide, despite many breakthroughs in the discovery of novel anticancer candidates. Each new year the FDA approves the use of new drugs for cancer treatments. In the last years, the biological targets of anticancer agents have started to be clearer and one of these main targets is tubulin protein; this protein plays an essential role in cell division, as well as in intracellular transportation. The inhibition of microtubule formation by targeting tubulin protein induces cell death by apoptosis. In the last years, numerous novel structures were designed and synthesized to target tubulin, and this can be achieved by inhibiting the polymerization or depolymerization of the microtubules. In this review article, recent novel compounds that have antiproliferation activities against a panel of cancer cell lines that target tubulin are explored in detail. This review article emphasizes the recent developments of tubulin inhibitors, with insights into their antiproliferative and anti-tubulin activities. A full literature review shows that tubulin inhibitors are associated with properties in the inhibition of cancer cell line viability, inducing apoptosis, and good binding interaction with the colchicine binding site of tubulin. Furthermore, some drugs, such as cabazitaxel and fosbretabulin, have been approved by FDA in the last three years as tubulin inhibitors. The design and development of efficient tubulin inhibitors is progressively becoming a credible solution in treating many species of cancers.

A series of N-acylhydrazones bearing a 1,4-dihydroindeno[1,2-b]pyrrole ring, along with benzene and thiophene rings substituted with chlorine or methyl groups, was synthesized and evaluated for their antiproliferative and cytotoxic activity against the melanoma A375 cell line and to measure the inhibition of tubulin polymerization. Four compounds elicited interesting activity: derivatives, 1g and 1h showed a 25% slowdown of tubulin polymerization, whereas compounds 2c and 2d caused a slowdown of 40% and 60%, respectively. Molecular modelling results have confirmed that the most active N-acylhydrazones (1g, 1h, 2c, and 2d) may act as tubulin polymerization inhibitors.

Combretastatins isolated from the Combretum caffrum tree belong to a group of closely related stilbenes. They are colchicine binding site inhibitors which disrupt the polymerization process of microtubules in tubulins, causing mitotic arrest. In vitro and in vivo studies have proven that some combretastatins exhibit antitumor properties, and among them, combretastatin A-4 is the most active mitotic inhibitor. In this study, a series of novel combretastatin A-4 analogs containing carboxylic acid, ester, and amide moieties were synthesized and their cytotoxic activity against six tumor cell lines was determined using sulforhodamine B assay. For the most cytotoxic compounds (8 and 20), further studies were performed. These compounds were shown to induce G0/G1 cell cycle arrest in MDA and A549 cells, in a concentration-dependent manner. Moreover, in vitro tubulin polymerization assays showed that both compounds are tubulin polymerization enhancers. Additionally, computational analysis of the binding modes and binding energies of the compounds with respect to the key human tubulin isotypes was performed. We have obtained a satisfactory correlation of the binding energies with the IC50 values when weighted averages of the binding energies accounting for the abundance of tubulin isotypes in specific cancer cell lines were computed.

A series of naphthalene-chalcone derivatives (3a-3t) were prepared and evaluated as tubulin polymerisation inhibitor for the treatment of breast cancer. All compounds were evaluated for their antiproliferative activity against MCF-7 cell line. The most of compounds displayed potent antiproliferative activity. Among them, compound 3a displayed the most potent antiproliferative activity with an IC 50 value of 1.42 ± 0.15 µM, as compared to cisplatin (IC 50  = 15.24 ± 1.27 µM). Additionally, the promising compound 3a demonstrated relatively lower cytotoxicity on normal cell line (HEK293) compared to tumour cell line. Furthermore, compound 3a was found to induce significant cell cycle arrest at the G 2 /M phase and cell apoptosis. Compound 3a displayed potent tubulin polymerisation inhibitory activity with an IC 50 value of 8.4 µM, which was slightly more active than the reference compound colchicine (IC 50  = 10.6 µM). Molecular docking analysis suggested that 3a interact and bind at the colchicine binding site of the tubulin.