Microtubule Assembly Research Articles

Novel chalcone-derived pyrazoles as potential therapeutic agents for the treatment of non-small cell lung cancer

Lung cancer is considered to account for approximately one-fifth of all malignant tumor-related deaths worldwide and is therefore one of the most lethal malignancies. Pyrazole scaffold possesses a wide range of biological and pharmacological activities, which play important roles in medicinal chemistry. The present study reports the synthesis and in vitro biological characterization of nine pyrazoles derived from chalcones as potential anticancer agents for non-small cell lung cancer A-549, H226, and H460 cell lines. Most of the compounds efficiently inhibited the growth of all the tested cancer cell lines at micromolar concentrations. One of the most active compounds (PCH-1) was further evaluated for its effect on cell cycle distribution, apoptosis, migration, epithelial–mesenchymal transition, and oxidative stress. Furthermore, studies on the mechanism of action revealed that PCH-1 disrupts microtubule assembly, leading to cancer cell death. Molecular modeling studies confirmed the potent interaction of PCH-1 with the vinblastine binding site on tubulin. Overall, this study provides novel opportunities to identify anticancer agents in the pyrazole series.

Re-evaluation of the Fijianolide/Laulimalide Chemotype Suggests an Alternate Mechanism of Action for C-15/C-20 Analogs.

Herein, we report on naturally derived microtubule stabilizers with activity against triple negative breast cancer (TNBC) cell lines, including paclitaxel, fijianolide B/laulimalide (3), fijianolide B di-acetate (4), and two new semisynthetic analogs of 3, which include fijianolide J (5) and fijianolide L (6). Similar to paclitaxel, compound 3 demonstrated classic microtubule stabilizing activity with potent (GI50 = 0.7–17 nM) antiproliferative efficacy among the five molecularly distinct TNBC cell lines. Alternatively, compounds 5 or 6, generated from oxidation of C-20 or C-15 and C-20 respectively, resulted in a unique profile with reduced potency (GI50 = 4–9 μM), but improved efficacy in some lines, suggesting a distinct mechanism of action. The C-15, C-20 di-acetate, and dioxo modifications on 4 and 6 resulted in compounds devoid of classic microtubule stabilizing activity in biochemical assays. While 4 also had no detectable effect on cellular microtubules, 6 promoted a reorganization of the cytoskeleton resulting in an accumulation of microtubules at the cell periphery. Compound 5, with a single C-20 oxo substitution, displayed a mixed phenotype, sharing properties of 3 and 6. These results demonstrate the importance of the C-15/C-20 chiral centers, which appear to be required for the potent microtubule stabilizing activity of this chemotype and that oxidation of these sites promotes unanticipated cytoskeletal alterations that are distinct from classic microtubule stabilization, likely through a distinct mechanism of action.

The role of non-enzymatic glycation on Tau-DNA interactions: Kinetic and mechanistic approaches

Despite the regulatory role of Tau protein in the stabilization and assembly of microtubules, this protein has an important function in the protection and stabilizing of DNA molecules in the cell nucleus. In the present study, it has been indicated that glycation of lysine residues (Lys-267, Lys-274, and Lys-280) in the microtubule-binding domain (MBD) can considerably decrease its binding affinity to DNA molecules. The structural analysis also confirmed that the decreased glycated tau-DNA complex's stability was due to structural modification of this protein after the glycation process. The study of hippocampal cells under hyperglycemic conditions showed that near to 70% of Tau proteins glycated in these cells, although the expression of Tau remained unaffected. The assessment of H3K9me2, as a marker for binding of Tau to pericentromeric heterochromatin (PCH), indicated that localization of Tau protein on PCH was remarkably decreased at high glucose conditions relative to the controls. It is suggested that increasing the structural stability of Tau protein limits the ability of this protein for DNA binding, while the molecular and physical barrier of glycated Lys residues should not be neglected.

Ashwagandha-polyphenols-functionalized gold nanoparticles facilitate apoptosis by perturbing microtubule assembly dynamics in breast cancer cells

Ashwagandha (Withania somnifera) is a medicinal plant used in several traditional medical practices for treating a variety of ailments. With its multitude of polyphenols, ashwagandha possesses strong anticancer potential. In an effort to improve the anticancer efficacy and target specificity of ashwagandha, we coated gold nanoparticles with ashwagandha polyphenols, characterized the particles, evaluated their antiproliferative efficacy, and elucidated their mechanism of action in the cells. The Withania somnifera-coated gold nanoparticles (“Ws-GNPs”) were characterized using UV–visible spectroscopy, energy-dispersive X-ray spectroscopy, transmission electron microscopy, dynamic light scattering, zeta potential analysis, and Fourier-transform infrared spectroscopy. The coating of ashwagandha polyphenols on the nanoparticles was confirmed by 1H nuclear magnetic resonance spectroscopy. The Ws-GNPs were then subjected to cellular-level analysesusing the triple-negative breast cancer cell line, MDA-MB-231, as the cell model. Ws-GNPs inhibited proliferation of these malignant breast cancer cells with an IC50 of 90 ± 10 μg/mL, which was half the IC50 of the ashwagandha-extract (Ws-EX)-treated cells (180 ± 23 μg/mL). The nanoparticles strongly inhibited the clonogenicity and migration of the cells, as evidenced from a clonogenic assay and wound-healing assay, respectively. Ws-GNPs showed concentration-dependent binding to purified tubulin as confirmed by an in vitro, spectrofluorometry-based tryptophan-quenching assay and an anilinonaphthalene sulfonate-binding assay. The binding perturbed the secondary structure of the protein, as evidenced by far-UV circular dichroism spectroscopy. In cells, the interactions between tubulin and the nanoparticles resulted in the suppressed dynamicity of the microtubules, the subsequent halting of the cell cycle progression through mitosis, and finally, apoptosis.

Exploring the interaction of clonazepam and diazepam with tau protein: Multispectral and molecular docking studies

Tau, as a neuronal protein, has a crucial role in microtubule assembly and stabilization. In the current study, fluorescence spectroscopy, surface plasmon resonance (SPR), as well as molecular docking analysis were used to explore the influence of benzodiazepines, clonazepam (CZP), and diazepam (DZP), on tau protein structure. SPR thermodynamic parameters of drugs-protein complexes were calculated and the results showed that both complexes have negative ∆H and ∆S and positive ∆G values. According to these findings, the roles of hydrogen bonds and van der Waals forces are extremely substantial in the formation of a complex between DZP/CZP and tau protein. It was observed that the KSV values declined following increasing temperature, therefore the fluorescence quenching mechanism of tau-CZP/DZP was static quenching. Molecular docking studies represented that hydrogen bonds and hydrophobic interactions are important in the formation of complexes between CZP/DZP and tau protein. This study provides general and significant information about the structural behavior of tau protein following the binds to both benzodiazepine derivatives CZP and DZP. Also, this investigation led us to the idea that CZP and DZP could be effective compounds to prevent the aggregation of tau protein and the treatment of tauopathy.

WDR72 regulates vesicle trafficking in ameloblasts

As the hardest tissue in the human body, tooth enamel formation is a highly regulated process involving several stages of differentiation and key regulatory genes. One such gene, tryptophan‐aspartate repeat domain 72 (WDR72), has been found to cause a tooth enamel defect when deleted or mutated, resulting in a condition called amelogenesis imperfecta. Unlike the canonical genes regulating tooth development, WDR72 remains intracellularly and is not secreted to the enamel matrix space to regulate mineralization, and is found in other major organs of the body, namely the kidney, brain, liver, and heart. To date, a link between intracellular vesicle transport and enamel mineralization has been suggested, however identification of the mechanistic regulators has yet to be elucidated, in part due to the limitations associated with studying highly differentiated ameloblast cells. Here we show compelling evidence that WDR72 regulates endocytosis of proteins, both in vivo and in a novel in vitro ameloblast cell line. We elucidate WDR72’s function to be independent of intracellular vesicle acidification while still leading to defective enamel matrix pH extracellularly. We identify a vesicle function associated with microtubule assembly and propose that WDR72 directs microtubule assembly necessary for membrane mobilization and subsequent vesicle transport. Understanding WDR72 function provides a mechanistic basis for determining physiologic and pathologic tissue mineralization.

Integrated multiple analytes and semi-mechanistic population pharmacokinetic model of tusamitamab ravtansine, a DM4 anti-CEACAM5 antibody-drug conjugate

Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC), combining a humanized monoclonal antibody (IgG1) targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and a potent cytotoxic maytansinoid derivative, DM4, inhibiting microtubule assembly. SAR408701 is currently in clinical development for the treatment of advanced solid tumors expressing CEACAM5. It is administered intravenously as a conjugated antibody with an average Drug Antibody Ratio (DAR) of 3.8. During SAR408701 clinical development, four entities were measured in plasma: conjugated antibody (SAR408701), naked antibody (NAB), DM4 and its methylated metabolite (MeDM4), both being active. Average DAR and proportions of individual DAR species were also assessed in a subset of patients. An integrated and semi-mechanistic population pharmacokinetic model describing the time-course of all entities in plasma and DAR measurements has been developed. All DAR moieties were assumed to share the same drug disposition parameters, excepted for clearance which differed for DAR0 (i.e. NAB entity). The conversion of higher DAR to lower DAR resulted in a DAR-dependent ADC deconjugation and was represented as an irreversible first-order process. Each conjugated antibody was assumed to contribute to DM4 formation. All data were fitted simultaneously and the model developed was successful in describing the pharmacokinetic profile of each entity. Such a structural model could be translated to other ADCs and gives insight of mechanistic processes governing ADC disposition. This framework will further be expanded to evaluate covariates impact on SAR408701 pharmacokinetics and its derivatives, and thus can help identifying sources of pharmacokinetic variability and potential efficacy and safety pharmacokinetic drivers.

Lab-on-chip microscope platform for electro-manipulation of a dense microtubules network

Pulsed electric field (PEF) technology is promising for the manipulation of biomolecular components and has potential applications in biomedicine and bionanotechnology. Microtubules, nanoscopic tubular structures self-assembled from protein tubulin, serve as important components in basic cellular processes as well as in engineered biomolecular nanosystems. Recent studies in cell-based models have demonstrated that PEF affects the cytoskeleton, including microtubules. However, the direct effects of PEF on microtubules are not clear. In this work, we developed a lab-on-a-chip platform integrated with a total internal reflection fluorescence microscope system to elucidate the PEF effects on a microtubules network mimicking the cell-like density of microtubules. The designed platform enables the delivery of short (microsecond-scale), high-field-strength (le 25 kV/cm) electric pulses far from the electrode/electrolyte interface. We showed that microsecond PEF is capable of overcoming the non-covalent microtubule bonding force to the substrate and translocating the microtubules. This microsecond PEF effect combined with macromolecular crowding led to aggregation of microtubules. Our results expand the toolbox of bioelectronics technologies and electromagnetic tools for the manipulation of biomolecular nanoscopic systems and contribute to the understanding of microsecond PEF effects on a microtubule cytoskeleton.

Cdk5rap2 dimers activate microtubule nucleation by opposing inhibitory pathways

Microtubule assembly is a critical cellular process that is tightly regulated in space and time via the universal microtubule nucleator, the gamma-tubulin ring complex (γTuRC). Despite being present throughout the cytoplasm, γTuRC's activity is restricted to specific sites including the centrosome and Golgi. To better understand how γTuRC is activated, we focused on a well-conserved γTuRC nucleation activator (γTuNA) domain within the centrosomal scaffold protein, Cdk5rap2, which has previously been reported to increase the number of microtubules generated by γTuRC.

Protein conformations in Tau condensates probed by smFRET and FLIM-FRET

Tau-rich neurofibrillary tangles are the defining neuropathologic feature of Alzheimer’s disease. Liquid-liquid phase separation play critical roles in Tau function and dysfunction by promoting microtubule assembly and acting as an intermediate step towards protein fibrillation and disease pathology. To track Tau conformational and morphological evolution during LLPS-mediated microtubule assembly and aggregation, we used FRET as a conformational reporter both at the single-molecule resolution (smFRET) and the ensemble regime (FLIM-FRET).

Magneto-mechanical manipulation of full-lenght human Tau40 in live-cell neuron cultures

The microtubule-associated protein Tau is well known to aid in stabilizing the axonal cytoskeleton in brain cells. The longest of six human Tau isoforms, Tau40, contains four microtubule-binding domains, facilitates rapid assembly of axonal microtubules, and is the most common isoform in the adult human brain. Previous studies have shown that mechanical forces administered to the intracellular space of rodent cortical neurons impacted the distribution of Tau proteins. It remains, however, unclear what direct underlying mechanism is at play between Tau protein distribution and nanoparticle-mediated force manipulation.

Isolation of a tau binding peptide by phage display

The microtubule-associated protein tau is mainly expressed in the central nervous system and promotes the microtubule assembly. While tau is highly stable under normal conditions, several factors, including mutation, post-translational modification, and interaction with polyanions or proteins, have been found to induce tau aggregation. Formation of tau aggregates in neuron is a hallmark of a series of neurodegenerative diseases known as tauopathies, such as Alzheimer’s disease, Pick’s disease, chronic traumatic encephalopathy, and corticobasal degeneration.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.

The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.

Cytotoxic mechanism of tioconazole involves cell cycle arrest at mitosis through inhibition of microtubule assembly.

The online version contains supplementary material available at 10.1007/s10616-021-00516-w.

Therapeutic potential of colchicine in cardiovascular medicine: a pharmacological review.

Colchicine is an ancient herbal drug derived from Colchicum autumnale. It was first used to treat familial Mediterranean fever and gout. Based on its unique efficacy as an anti-inflammatory agent, colchicine has been used in the therapy of cardiovascular diseases including coronary artery disease, atherosclerosis, recurrent pericarditis, vascular restenosis, heart failure, and myocardial infarction. More recently, colchicine has also shown therapeutic efficacy in alleviating cardiovascular complications of COVID-19. COLCOT and LoDoCo2 are two milestone clinical trials that confirm the curative effect of long-term administration of colchicine in reducing the incidence of cardiovascular events in patients with coronary artery disease. There is growing interest in studying the anti-inflammatory mechanisms of colchicine. The anti-inflammatory action of colchicine is mediated mainly through inhibiting the assembly of microtubules. At the cellular level, colchicine inhibits the following: (1) endothelial cell dysfunction and inflammation; (2) smooth muscle cell proliferation and migration; (3) macrophage chemotaxis, migration, and adhesion; (4) platelet activation. At the molecular level, colchicine reduces proinflammatory cytokine release and inhibits NF-κB signaling and NLRP3 inflammasome activation. In this review, we summarize the current clinical trials with proven curative effect of colchicine in treating cardiovascular diseases. We also systematically discuss the mechanisms of colchicine action in cardiovascular therapeutics. Altogether, colchicine, a bioactive constituent from an ancient medicinal herb, exerts unique anti-inflammatory effects and prominent cardiovascular actions, and will charter a new page in cardiovascular medicine.

Spatial and Temporal Scaling of Microtubules and Mitotic Spindles.

During cell division, the mitotic spindle, a macromolecular structure primarily comprised of microtubules, drives chromosome alignment and partitioning between daughter cells. Mitotic spindles can sense cellular dimensions in order to adapt their length and mass to cell size. This scaling capacity is particularly remarkable during early embryo cleavage when cells divide rapidly in the absence of cell growth, thus leading to a reduction of cell volume at each division. Although mitotic spindle size scaling can occur over an order of magnitude in early embryos, in many species the duration of mitosis is relatively short, constant throughout early development and independent of cell size. Therefore, a key challenge for cells during embryo cleavage is not only to assemble a spindle of proper size, but also to do it in an appropriate time window which is compatible with embryo development. How spatial and temporal scaling of the mitotic spindle is achieved and coordinated with the duration of mitosis remains elusive. In this review, we will focus on the mechanisms that support mitotic spindle spatial and temporal scaling over a wide range of cell sizes and cellular contexts. We will present current models and propose alternative mechanisms allowing cells to spatially and temporally coordinate microtubule and mitotic spindle assembly.

Changing in the production of anticancer drugs (vinblastine and vincristine) in Catharanthus roseus (L.) G. Don by potassium and ascorbic acid treatments

Catharanthus roseus seedling was treated with different concentrations (1.5, 3.16, 15, and 30 mmol) and forms (K<sub>2</sub>SO<sub>4</sub> and KNO<sub>3</sub>) of potassium (K<sup>+</sup>) via Hoagland’s nutrient solution. Ascorbic acid (AsA) was sprayed twice (plant days 68 and 78) with different concentrations (750 and 1 500 mg/L) on the leaves. Vinblastine, vincristine, tryptophan contents, D4H and DAT genes expression, peroxidase activity, and H<sub>2</sub>O<sub>2</sub> content of leaves were measured. Potassium in KNO<sub>3</sub> form increased vinblastine (60%) and vincristine (50%), compared to 30% and 20% using K<sub>2</sub>SO<sub>4</sub>. Vinblastine and vincristine inhibit microtubule assembly and ultimately metaphase-arrested caused by the polymerisation. The genes expression was higher 3 times in KNO<sub>3</sub> and 2.5 times in K<sub>2</sub>SO<sub>4</sub> in excess of K<sup>+</sup>. Foliar application of 750 mg/L AsA led to an increase in vinblastine (20%) and vincristine (16%). Both concentrations of AsA had the same additional effect on the expression of D4H and DAT about 30% and 60%, respectively, compared to the control plant. Tryptophan decreased 2.5 times in excess of K<sup>+</sup> and 35% due to the exterior of AsA. H<sub>2</sub>O<sub>2</sub> decreased while peroxidase activity increased along with AsA treatment. A positive interaction existed between the K<sup>+</sup> and AsA on the amount of vinblastine, vincristine, tryptophan, and gene expression.

Altered succinylation of mitochondrial proteins, APP and tau in Alzheimer\u2019s disease

Abnormalities in brain glucose metabolism and accumulation of abnormal protein deposits called plaques and tangles are neuropathological hallmarks of Alzheimer’s disease (AD), but their relationship to disease pathogenesis and to each other remains unclear. Here we show that succinylation, a metabolism-associated post-translational protein modification (PTM), provides a potential link between abnormal metabolism and AD pathology. We quantified the lysine succinylomes and proteomes from brains of individuals with AD, and healthy controls. In AD, succinylation of multiple mitochondrial proteins declined, and succinylation of small number of cytosolic proteins increased. The largest increases occurred at critical sites of amyloid precursor protein (APP) and microtubule-associated tau. We show that in vitro, succinylation of APP disrupted its normal proteolytic processing thereby promoting Aβ accumulation and plaque formation and that succinylation of tau promoted its aggregation to tangles and impaired microtubule assembly. In transgenic mouse models of AD, elevated succinylation associated with soluble and insoluble APP derivatives and tau. These findings indicate that a metabolism-linked PTM may be associated with AD.

Functional midbody assembly in the absence of a central spindle.

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F-like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2-dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle-independent midbodies appeared to form via contractile ring constriction-driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.