Tubulin Dynamics Research Articles

AlphaFold2-guided engineering of split-GFP technology enables labeling of endogenous tubulins across species while preserving function.

Dynamic properties are essential for microtubule (MT) physiology. Current techniques for in vivo imaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of the AlphaFold2 pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis in Caenorhabditis elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies.

Promising anti-proliferative indolic benzenesulfonamides alter mechanisms with sulfonamide nitrogen substituents

Agents that cause apoptotic cell death by interfering with tubulin dynamics, such as vinblastine and paclitaxel, are an important class of chemotherapeutics. Unfortunately, these compounds are substrates for multidrug resistance (MDR) pumps, allowing cancer cells to gain resistance to these chemotherapeutics. The indolesulfonamide family of tubulin inhibitors are not excluded by MDR pumps and have a promising activity profile, although their high lipophilicity is a pharmacokinetic limitation for their clinical use. Here we present a new family of N-indolyl-3,4,5-trimethoxybenzenesulfonamide derivatives with modifications on the indole system at positions 1 and 3 and on the sulfonamide nitrogen. We synthesized and screened against HeLa cells 34 novel indolic benzenesulfonamides. The most potent derivatives (1.7–109 nM) were tested against a broad panel of cancer cell lines, which revealed that substituted benzenesulfonamides analogs had highest potency. Importantly, these compounds were only moderately toxic to non-tumorigenic cells, suggesting the presence of a therapeutic index. Consistent with known clinical anti-tubulin agents, these compounds arrested the cell cycle at G2/M phase. Mechanistically, they induced apoptosis via caspase 3/7 activation, which occurred during M arrest. The substituents on the sulfonamide nitrogen appeared to determine different mechanistic results and cell fates. These results suggest that the compounds act differently depending on the bridge substituents, thus making them very interesting as mechanistic probes as well as potential drugs for further development.

Click estradiol dimers with novel aromatic bridging units: synthesis and anticancer evaluation

Estradiol dimers (EDs) possess significant anticancer activity by targeting tubulin dynamics. In this study, we synthesised 12 EDs variants via copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction, focusing on structural modifications within the aromatic bridge connecting two estradiol moieties. In vitro testing of these EDs revealed a marked improvement in selectivity towards cancerous cells, particularly for ED1–8. The most active compounds, ED3 (IC50 = 0.38 μM in CCRF-CEM) and ED5 (IC50 = 0.71 μM in CCRF-CEM) demonstrated cytotoxic effects superior to 2-methoxyestradiol (IC50 = 1.61 μM in CCRF-CEM) and exhibited anti-angiogenic properties in an endothelial cell tube-formation model. Cell-based experiments and in vitro assays revealed that EDs interfere with mitotic spindle assembly. Additionally, we proposed an in silico model illustrating the probable binding modes of ED3 and ED5, suggesting that dimers with a simple linker and a single substituent on the aromatic central ring possess enhanced characteristics compared to more complex dimers.

Discovery and biological profile of pyridachlometyl

AbstractPyridachlometyl is a novel tubulin dynamics modulator fungicide developed by Sumitomo as a new agent designed to tackle fungicide resistance. Pyridachlometyl is being developed as a first‐in‐class molecule with an anti‐tubulin mode of action, the chemical structure of which is characterized by a unique tetrasubstituted pyridazine ring. The first commercial product ‘Fuseki flowable’ received initial registration in 2023 in Japan. The concepts of the discovery project, optimization of chemical structures, and biological profiles are reviewed herein. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Antiproliferative and Pro-Apoptotic Activity and Tubulin Dynamics Modulation of 1H-Benzimidazol-2-yl Hydrazones in Human Breast Cancer Cell Line MDA-MB-231.

(1) Background: The aim of the work is the evaluation of in vitro antiproliferative and pro-apoptotic activity of four benzimidazole derivatives containing colchicine-like and catechol-like moieties with methyl group substitution in the benzimidazole ring against highly invasive breast cancer cell line MDA-MB-231 and their related impairment of tubulin dynamics. (2) Methods: The antiproliferative activity was assessed with the MTT assay. Alterations in tubulin polymerization were evaluated with an in vitro tubulin polymerization assay and a docking analysis. (3) Results: All derivatives showed time-dependent cytotoxicity with IC50 varying from 40 to 60 μM after 48 h and between 13 and 20 μM after 72 h. Immunofluorescent and DAPI staining revealed the pro-apoptotic potential of benzimidazole derivatives and their effect on tubulin dynamics in living cells. Compound 5d prevented tubulin aggregation and blocked mitosis, highlighting the importance of the methyl group and the colchicine-like fragment. (4) Conclusions: The benzimidazole derivatives demonstrated moderate cytotoxicity towards MDA-MB-231 by retarding the initial phase of tubulin polymerization. The derivative 5d containing a colchicine-like moiety and methyl group substitution in the benzimidazole ring showed potential as an antiproliferative agent and microtubule destabilizer by facilitating faster microtubule aggregation and disrupting cellular and nuclear integrity.

Reversible Influence of Hemipiperazine Photochromism on the Early Development of Zebrafish Embryo.

This study explores the potential of controlling organismal development with light by using reversible photomodulation of activity in bioactive compounds. Specifically, our research focuses on plinabulin 1, an inhibitor of tubulin dynamics that contains a photochromic motif called hemipiperazine. The two isomeric forms, Z-1 and E-1, can partially interconvert with light, yet show remarkable thermal stability in darkness. The Z-isomer exhibits higher cytotoxicity due to stronger binding to α-tubulin's colchicine site. The less toxic E-1 form, considered a "pro-drug", can be isolated in vitro and stored. Upon activation by blue or cyan light, it predominantly generates the more toxic Z-1 form. Here we demonstrate that 1 can effectively photomodulate epiboly, a critical microtubule-dependent cell movement during gastrulation in zebrafish embryos. This research highlights the potential of photomodulation for precise and reversible control of cellular activities and organismal development.

3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines inhibit tubulin polymerisation and act as anticancer agents

A series of cis-restricted 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines as novel tubulin polymerisation inhibitors was designed based on molecular docking. Compound 9p, exhibited potent antiproliferative activity against HeLa, MCF-7, and A549 cell lines. Mechanism studies indicated that 9p potently inhibited tubulin polymerisation and disrupted the microtubule dynamics of tubulin in HeLa cells. Moreover, 9p could cause G2/M phase cell cycle arrest and apoptosis in HeLa cells. In addition, the prediction of physicochemical properties disclosed that 9p conformed well to the Lipinski’s rule of five. The initial results suggest that the 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines could serve as a promising scaffold for the development of novel anticancer drugs.

Mechanical communication within the microtubule through network-based analysis of tubulin dynamics

The identification of the mechanisms underlying the transfer of mechanical vibrations in protein complexes is crucial to understand how these super-assemblies are stabilized to perform specific functions within the cell. In this context, the study of the structural communication and the propagation of mechanical stimuli within the microtubule (MT) is important given the pivotal role of the latter in cell viability. In this study, we employed molecular modelling and the dynamical network analysis approaches to analyse the MT. The results highlight that β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta$$\\end{document}-tubulin drives the transfer of mechanical information between protofilaments (PFs), which is altered at the seam due to a different interaction pattern. Moreover, while the key residues involved in the structural communication along the PF are generally conserved, a higher diversity was observed for amino acids mediating the lateral communication. Taken together, these results might explain why MTs with different PF numbers are formed in different organisms or with different β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta$$\\end{document}-tubulin isotypes.

Determinants of cytoplasmic microtubule depolymerization during ciliogenesis in Chlamydomonas.

At the core of cilia are microtubules which establish length and assist ciliary assembly and disassembly; however, microtubules outside of the cilium can regulate ciliogenesis. The microtubule cytoskeleton polymerizes and depolymerizes rapidly. These processes have been studied across various organisms with chemical and genetic perturbations. However, these have generated conflicting data in terms of the role of cytoplasmic microtubules (CytoMTs) and free tubulin dynamics during ciliogenesis. Here, we look at the relationship between ciliogenesis and CytoMT dynamics in Chlamydomonas reinhardtii using chemical and mechanical perturbations. We find that not only can stabilized CytoMTs allow for normal ciliary assembly, but high calcium concentrations and low pH-induced deciliation cause CytoMTs to depolymerize separately from ciliary shedding. In addition, ciliary shedding through mechanical shearing allows cilia to regenerate earlier despite intact CytoMTs. Our data suggest that CytoMTs are not a sink for a limiting pool of cytoplasmic tubulin in Chlamydomonas, depolymerization after deciliation is a consequence rather than a requirement for ciliogenesis, and intact tubulin in the cytoplasm and proximal cilium support more efficient ciliary assembly.

Design, synthesis and anticancer activity of N-aryl indolylsulfoximines: Identification of potent and selective anticancer agents

A facile and efficient approach utilizing copper-mediated cross-coupling reaction of N-boc-3-indolylsulfoximines with aryl iodides was developed to synthesize a diverse range of N-arylated indolylsulfoximines 11a-m in excellent yields (up to 91%). The key precursors, free NH sulfoximines 9 were readily prepared by the treatment of N-boc-3-methylthioindoles 8 with a combination of IBD and ammonium carbamate. Under similar conditions NH-free indolylsulfoximine 9a was successfully prepared in gram-scale quantities. The reaction is highly chemoselective and tolerant of a wide range of functional groups. The process is environmentally friendly and is amenable to scale-up. Among the prepared N-arylated indolylsulfoximines 11a-m, compounds 11i-j (2.68–2.76 μM), 11f-g (1.9–3.7 μM) and 11k (1.28 μM) showed potent and selective cytotoxicity against 22Rv1, C4-2 and MCF7 cells, respectively. Indolylsulfoximine derivative 11l displayed a broad spectrum of activity (1.7–8.2 μM) against the tested cancer cell lines. These compounds were found to be non-cytotoxic to normal HEK293 cells, indicating their potential selectivity for cancer cells. We analysed the impact of 11l on various cellular assays to uncover its mechanism of action. Cellular assay shows that 11l increases the endogenous level of ROS, leading to the increased level of p-53 and c-jun inducing apoptosis. 11l also induced mitochondrial dysfunction, further promoting apoptotic pathways. Besides, 11l also restricts cell invasiveness, indicating that it could serve as an effective anti-metastatic agent. As oxidative stress severe F actin causing tubulin depolymerization, we examined the impact of 11l on tubulin dynamics. Accordingly, 11l treatment decreased the levels of polymerized tubulin in 22Rv1 and C4-2 cells. Although future studies are needed to determine their exact molecular target(s), our data shows that N-aryl indolylsulfoximines could serve as effective anti-cancer agents.

Design, synthesis, and biological evaluation of 3, 5-disubsituted-1H-pyrazolo[3,4-b]pyridines as multiacting inhibitors against microtubule and kinases

Combination therapy of kinases inhibitors and chemotherapeutics targeting tubulin dynamics is an important strategy to improve therapeutic efficacy and overcome the resistance to single-target drug therapies. Inspired by this, we report herein the rational design of 3,5-disubsituted-1H-pyrazolo[3,4-b]pyridines as multiacting molecules that are capable of inhibiting tubulin and kinases simultaneously. Among them, 8g showed excellent antiproliferative activities toward a panel of cancer cell lines. 8g strongly inhibited tubulin assembly and demonstrated a potent inhibition toward FLT3 and Abl1 in both enzymatic and cellular assays. 8g caused a cell cycle arrest at G2/M phase, and significantly disrupted HUVEC tube formation. In vivo efficacy studies showed that 8g significantly inhibited tumor growth on the K562 leukemia xenograft model at 10 mg/kg. Collectively our studies suggest that the excellent antiproliferative potency of 8g may be attributed to its potent inhibitory activity against both microtubule and kinases.

FKBP25 regulates myoblast viability and migration and is differentially expressed in in vivo models of muscle adaptation.

FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role ofFKBP25 in non-transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline-inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.

Tetrabromobisphenol A-bis(2,3-dibromopropyl ether) impairs Postnatal Testis Development in Mice: The Microtubule Cytoskeleton as a Sensitive Target.

Tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE), a widely used flame retardant, has been frequently detected in various environmental compartments, but its health hazard remains largely unknown. Here, we investigated the adverse effects of TBBPA-BDBPE (50 and 1000 μg/kg/day) on postnatal testis development in CD-1 mice and the underlying mechanism. Following the first week of maternal exposure, neonatal mice in the high-dose group exhibited reduced seminiferous tubule area, fewer Sertoli cells and germ cells, and damaged microtubules in Sertoli cells; even microtubule damage was also observed in the low-dose group. When exposure extended to adulthood, male offspring in the high-dose group presented more remarkable alterations in reproductive parameters, including reduced sperm count; in the low-dose group, microtubule damage was also observable, along with blood-testis barrier impairment. Further molecular docking analysis and tubulin polymerization assay indicated that TBBPA-BDBPE could interact with tubulin and disrupt its polymerization. Moreover, we observed attenuated microtubules in mouse Sertoli cells in vitro (TM4) following TBBPA-BDBPE treatment, suggesting that TBBPA-BDBPE impaired testis development possibly by interfering with tubulin dynamics. This study not only highlights the male reproductive hazard of TBBPA-BDBPE but also greatly improved the understanding of the molecular mechanism for male reproductive toxicity of chemicals.

Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole

Although IFN-γ depletes tryptophan (Trp) as a defense against intracellular Chlamydia trachomatis (Ct) infected to hypoxic vagina, the presence of indole, a precursor of Trp, enables Ct to infect IFN-γ-exposed culture cells. Meanwhile, Trp-derived indole derivatives interact the aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor involved in the cellular homeostasis with tubulin dynamics. Here, the amounts of IFN-γ and indole in cervical swabs with known Ct infection status were measured, and Ct growth in the presence of indole was determined from the perspective of the AhR axis under hypoxia. A positive correlation between the amounts of IFN-γ and indole was found, and both of these amounts were lower in Ct-positive swabs than in Ct-negative ones. Indole as well as other AhR ligands inhibited Ct growth, especially under normoxia. Ct prompted the expression of detyrosinated tubulin (dTTub), but indole inhibited it. Indole did not stimulate the translocation of AhR to nucleus, and it blocked AhR activation in AhR-reporter cells. Ct growth was reduced more effectively under normoxia in AhR-knockdown cells, an effect that was enhanced by indole, which in turn diminished dTTub. Thus, Ct growth relies on the scavenger role of cytosolic AhR responsible for promoting dTTub expression.

Structural Perspective of Benzophenones Targeting Tubulin as Anticancer Agents.

Cancer is the leading cause of death and the most significant determinant of life expectancy in almost every country in this twenty-first century. According to the World Health Organization (WHO), cancer is responsible for the leading cause of death globally. Benzophenone derivatives are found in a variety of naturally occurring compounds which are known to be pharmacologically efficacious against a variety of diseases, including cancer. Microtubules are thought to be a good target for cancer chemotherapies. Microtubule polymerization and depolymerization are induced by a variety of natural, synthetic, and semisynthetic chemicals having a benzophenone nucleus, affecting tubulin dynamics. Several medications that affect microtubule dynamics are in various stages of clinical trials, including Combretastatins (phase II), Vincristine (clinically approved), Paclitaxel (in clinical usage), and epothilone (phase III), and only a few have been patented. Benzophenone derivatives target the colchicine binding site of microtubules, damage them and cause cell cycle arrest in the G2-M phase. Belonging to this class of molecules, phenstatin, a potent inhibitor of tubulin polymerization, has shown strongly inhibit cancer cell growth and arrest the G2/M phase of the cell cycle by targeting the colchicine binding site of microtubules. In the present manuscript, we described the benzophenone as tubulin polymerization inhibitors, their Structure-Activity Relationships (SARs) and molecular docking studies that reveal its binding affinity with the colchicine binding site.

Maytansinol Functionalization: Towards Useful Probes for Studying Microtubule Dynamics.

Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode. These encouraging results open new avenues for the design of new maytansine-based probes.

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

Per1/Per2 Disruption Reduces Testosterone Synthesis and Impairs Fertility in Elderly Male Mice.

Circadian rhythm disorders caused by genetic or environmental factors lead to decreased male fertility but the mechanisms are poorly understood. The current study reports that the mechanism of Per1/Per2 Double knockout (DKO) reduced the reproductive capacity of elderly male mice. The sperm motility and spermatogenic capacity of male DKO mice were weak. Hormone-targeted metabolomics showed reduced plasma levels of free testosterone in DKO male mice compared with WT male mice. Transcriptomic analysis of testicular tissue showed the down-regulation of testosterone synthesis-related enzymes (Cyp11a1, Cyp17a1, Hsd17b3, Hsd3b1, and Star) in the steroid hormone synthesis pathway. Spermatogenesis genes, Tubd1 and Pafah1b were down-regulated, influencing tubulin dynamics and leading to impaired motility. Seleno-compound metabolic loci, Scly and Sephs2, were up-regulated and Slc7a11 and Selenop were down-regulated. Western-blotting showed that steroid acute regulatory protein (StAR) and p-CREB, PKA and AC1 were reduced in testicular tissue of DKO mice compared to WT. Therefore, Per1/Per2 disruption reduced testosterone synthesis and sperm motility by affecting the PKA-StAR pathway, leading to decreased fertility.

Cover Picture: Optochemical Control of Therapeutic Agents through Photocatalyzed Isomerization (Angew. Chem. Int. Ed. 28/2022)

The photocatalyzed uphill isomerization of drugs using Ru(bpy3)2 is reported by Nicolas Winssinger et al. in their Research Article (DOI: 10.1002/anie.202203390). This chemistry was shown to locally convert inactive drugs into active ones that alter the tubulin dynamics in cells (green structures in the background of the picture).

Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution.

Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5′-triphosphate (GTP)–bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the “+” end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.