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Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

Tubulin is an electrostatically negative protein that forms cylindrical polymers termed microtubules, which are crucial for a variety of intracellular roles. Exploiting the electrostatic behavior of tubulin and microtubules within functional microfluidic and optoelectronic devices is limited due to the lack of understanding of tubulin behavior as a function of solvent composition. This work displays the tunability of tubulin surface charge using dimethyl sulfoxide (DMSO) for the first time. Increasing the DMSO volume fractions leads to the lowering of tubulin's negative surface charge, eventually causing it to become positive in solutions >80% DMSO. As determined by electrophoretic mobility measurements, this change in surface charge is directionally reversible, i.e., permitting control between -1.5 and + 0.2 cm2 (V s)-1 . When usually negative microtubules are exposed to these conditions, the positively charged tubulin forms tubulin sheets and aggregates, as revealed by an electrophoretic transport assay. Fluorescence-based experiments also indicate that tubulin sheets and aggregates colocalize with negatively charged g-C3 N4 sheets while microtubules do not, further verifying the presence of a positive surface charge. This study illustrates that tubulin and its polymers, in addition to being mechanically robust, are also electrically tunable.

Background/Aims: Protein tyrosine phosphatases are crucial enzymes controlling numerous physiological and pathophysiological events and can be regulated by oxidation of the catalytic domain cysteine residue. Peracids are highly oxidizing compounds, and thus may induce inactivation of PTPs. The aim of the present study was to evaluate the inhibitory effect of peracids with different length of hydrocarbon chain on the activity of selected PTPs. Methods: The enzymatic activity of human CD45, PTP1B, LAR, bacterial YopH was assayed under the cell-free conditions, and activity of cellular CD45 in human Jurkat cell lysates. The molecular docking and molecular dynamics were performed to evaluate the peracids binding to the CD45 active site. Results: Here we demonstrate that peracids reduce enzymatic activity of recombinant CD45, PTP1B, LAR, YopH and cellular CD45. Our studies indicate that peracids are more potent inhibitors of CD45 than hydrogen peroxide (with an IC50 value equal to 25 nM for peroctanoic acid and 8 µM for hydrogen peroxide). The experimental data show that the inactivation caused by peracids is dependent on hydrocarbon chain length of peracids with maximum inhibitory effect of medium-chain peracids (C8-C12 acyl chain), which correlates with calculated binding affinities to the CD45 active site. Conclusion: Peracids are potent inhibitors of PTPs with the strongest inhibitory effect observed for medium-chain peracids.

In this paper we provide an overview of the status of various colchicine derivatives in pre-clinical development with special focus on their anti-cancer activity. We discuss several groups of compounds that have been designed to differentially bind with specific affinities for tubulin β isotypes, especially in regard to βIII, which is commonly over-expressed in cancer. Computational prediction, protein-based and cell-based assays are summarized as well as some animal tests conducted on these compounds. It is concluded that an untapped potential exists for exploiting the colchicine scaffold as a pharmacophore with the possibility of increasing its affinity for tubulin isotypes over-expressed in cancer and decreasing it for normal cells thereby widening the therapeutic window.

The binding free energies on human tubulin of selected colchicine and thiocolchicine compounds were determined. Two methods were used for the determination of binding free energies: one is based on theoretical prediction simulating the dissociation of the compound from tubulin using a series of molecular dynamics simulations, and the other method involves a series of experiments that measured the affinity of the compound on a synthetically expressed and purified tubulin protein using a spectrofluorometric technique.

Tubulin is the target for very widely used anti-tumor drugs, including Vinca alkaloids, taxanes, and epothilones, which are an important component of chemotherapy in breast cancer and other malignancies. Paclitaxel and other tubulin-targeting drugs bind to the β subunit of tubulin, which is a heterodimer of α and β subunits. β-Tubulin exists in the form of multiple isotypes, which are differentially expressed in normal and neoplastic cells and differ in their ability to bind to drugs. Among them, the βIII isotype is overexpressed in many aggressive and metastatic cancers and may serve as a prognostic marker in certain types of cancer. The underpinning mechanisms accounting for the overexpression of this isotype in cancer cells are unclear. To better understand the role of β-tubulin isotypes in cancer, we analyzed over 1000 clones from 90 breast cancer patients, sequencing their β-tubulin isotypes, in search of novel mutations. We have elucidated two putative emerging molecular subgroups of invasive breast cancer, each of which involve mutations in the βI-, βIIA-, or βIVB isotypes of tubulin that increase their structural, and possibly functional, resemblance to the βIII isotype. A unifying feature of the first of the two subgroups is the mutation of the highly reactive C239 residue of βI- or βIVB-tubulin to L239, R239, Y239, or P239, culminating in probable conversion of these isotypes from ROS-sensitive to ROS-resistant species. In the second subgroup, βI, βIIA, and βIVB have up to seven mutations to the corresponding residues in βIII-tubulin. Given that βIII-tubulin has emerged as a pro-survival factor, overexpression of this isotype may confer survival advantages to certain cancer cell types. In this mini-review, we bring attention to a novel mechanism by which cancer cells may undergo adaptive mutational changes involving alternate β-tubulin isotypes to make them acquire some of the pro-survival properties of βIII-tubulin. These "hybrid" tubulins, combining the sequences and/or properties of two wild-type tubulins (βIII and either βI, βIIA, or βIVB), are novel isotypes expressed solely in cancer cells and may contribute to the molecular understanding and stratification of invasive breast cancer and provide novel molecular targets for rational drug development.

Tubulin is the target for many small-molecule natural compounds, which alter microtubules dynamics, and lead to cell cycle arrest and apoptosis. One of these compounds is colchicine, a plant alkaloid produced by Colchicum autumnale. While C. autumnale produces a potent cytotoxin, colchicine, and expresses its target protein, it is immune to colchicine’s cytotoxic action and the mechanism of this resistance is hitherto unknown. In the present paper, the molecular mechanisms responsible for colchicine resistance in C. autumnale are investigated and compared to human tubulin. To this end, homology models for C. autumnale α-β tubulin heterodimer are created and molecular dynamics (MD) simulations together with molecular mechanics Poisson–Boltzmann calculations (MM/PBSA) are performed to determine colchicine’s binding affinity for tubulin. Using our molecular approach, it is shown that the colchicine-binding site in C. autumnale tubulin contains a small number of amino acid substitutions compared to human tubulin. However, these substitutions induce significant reduction in the binding affinity for tubulin, and subsequently fewer conformational changes in its structure result. It is suggested that such small conformational changes are insufficient to profoundly disrupt microtubule dynamics, which explains the high resistance to colchicine by C. autumnale.

We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron donating groups. These derivative structures were characterized by their spectral and analytical data. The newly synthesized hexahydropyrazole analogues were evaluated for their in vitro anticancer activity against breast and lung cancer cell lines using a cytotoxicity bioassay. To understand their mechanism of action, tubulin binding assays were performed which pointed to their binding to microtubules in a mode similar to but not identical to colchicine, as evidenced by their KD value evaluation. Computational docking studies also suggested binding near the colchicine binding site on tubulin. These results were further confirmed by colchicine-binding assays on the most active compounds, which indicated that they bound to tubulin near but not at the colchicine site. The moderate cytotoxic effects of these compounds may be due to the presence of electron donating groups on the para-position of the phenyl ring, along with the hexahydropyrazole core nucleus. The observed anti-cancer activity based on inhibition of microtubule formation may be helpful in designing more potent compounds with a hexahydropyrazole moiety.

Microtubules are the primary target for many anti-cancer drugs, the majority of which bind specifically to beta-tubulin. The existence of several beta-tubulin isotypes, coupled with their varied expression in normal and cancerous cells provides a platform upon which to construct selective chemotherapeutic agents. We have examined five prevalent human beta-tubulin isotypes and identified the colchicine-binding site as the most promising for drug design based on specificity. Using this binding site as a template, we have designed several colchicine derivatives and computationally probed them for affinity to the beta-tubulin isotypes. These compounds were synthesized and subjected to cytotoxicity assays to determine their effectiveness against several cancerous cell lines. We observed a correlation between computational-binding predictions and experimentally determined IC(50) values, demonstrating the utility of computational screening in the design of more effective colchicine derivatives. The most promising derivative exhibited an IC(50) approximately threefold lower than values previously reported for either colchicine or paclitaxel, demonstrating the utility of computational design and assessment of binding to tubulin.

Our previous work identified an intermediate binding site for taxanes in the microtubule nanopore. The goal of this study was to test derivatives of paclitaxel designed to bind to this intermediate site differentially depending on the isotype of β-tubulin. Since β-tubulin isotypes have tissue-dependent expression--specifically, the βIII isotype is very abundant in aggressive tumors and much less common in normal tissues--this is expected to lead to tubulin targeted drugs that are more efficacious and have less side effects. Seven derivatives of paclitaxel were designed and four of these were amenable for synthesis in sufficient purity and yield for further testing in breast cancer model cell lines. None of the derivatives studied were superior to currently used taxanes, however computer simulations provided insights into the activity of the derivatives. Our results suggest that neither binding to the intermediate binding site nor the final binding site is sufficient to explain the activities of the derivative taxanes studied. These findings highlight the need to iteratively improve on the design of taxanes based on their activity in model systems. Knowledge gained on the ability of the engineered drugs to bind to targets and bring about activity in a predictable manner is a step towards personalizing therapies.

Hydrogen peroxide induces oxidation and consequently inactivation of many protein tyrosine phosphatases. It was found that hydrogen peroxide, in the presence of carboxylic acids, was efficiently activated to form even more potent oxidant - peroxy acid. We have found that peroxytetradecanoic acid decreases the enzymatic activity of CD45 phosphatase significantly more than hydrogen peroxide. Our molecular docking computational analysis suggests that peroxytetradecanoic acid has a higher binding affinity to the catalytic center of CD45 than hydrogen peroxide.

To explore possible ways in which yew tree tubulin is naturally resistant to paclitaxel. While the yew produces a potent cytotoxin, paclitaxel, it is immune to paclitaxel's cytotoxic action.Tubulin sequence data for plant species were obtained from Alberta 1000 Plants Initiative. Sequences were assembled with Trinity de novo assembly program and tubulin identified. Homology modeling using MODELLER software was done to generate structures for yew tubulin. Molecular dynamics simulations and molecular mechanics Poisson-Boltzmann calculations were performed with the Amber package to determine binding affinity of paclitaxel to yew tubulin. ClustalW2 program and PHYLIP package were used to perform phylogenetic analysis on plant tubulin sequences.We specifically analyzed several important regions in tubulin structure: the high-affinity paclitaxel binding site, as well as the intermediate binding site and microtubule nanopores. Our analysis indicates that the high-affinity binding site contains several substitutions compared to human tubulin, all of which reduce the binding energy of paclitaxel.The yew has achieved a significant reduction of paclitaxel's affinity for its tubulin by utilizing several specific residue changes in the binding pocket for paclitaxel.

Background A maximum entropy approach is proposed to predict the cytotoxic effects of a panel of colchicine derivatives in several human cancer cell lines. Data was obtained from cytotoxicity assays performed with 21 drug molecules from the same family of colchicine compounds and correlate these results with independent tubulin isoform expression measurements for several cancer cell lines. The maximum entropy method is then used in conjunction with computed relative binding energy values for each of the drug molecules against tubulin isotypes to which these compounds bind with different affinities. Results We have found by using our analysis that αβ I and αβ III tubulin isoforms are the most important isoforms in establishing predictive response of cancer cell sensitivity to colchicine derivatives. However, since αβ I tubulin is widely distributed in the human body, targeting it would lead to severe adverse side effects. Consequently, we have identified tubulin isotype αβ III as the most important molecular target for inhibition of microtubule polymerization and hence cancer cell cytotoxicity. Tubulin isotypes αβ I and αβ II are concluded to be secondary targets. Conclusions The benefit of being able to correlate expression levels of specific tubulin isotypes and the resultant cell death effect is that it will enable us to better understand the origin of drug resistance and hence design optimal structures for the elimination of cancer cells. The conclusion of the study described herein identifies tubulin isotype αβ III as a target for optimized chemotherapy drug design.