Tubulin Binding Research Articles

Potential of medicinal plant compounds to targeting Tau protein in the therapy of Alzheimer’s disease– A review

Alzheimer’s disease (AD) is a devastative neurodegenerative disorder with complex etiology. AD is characterized by blood-brain barrier disruption, oxidative stress, mitochondrial impairment, neuro- inflammation, hypo-metabolism; it decreases in acetylcholine levels and a reduction of cerebral blood flow. It is also not solely the end-product of aberrantly processed, misfolded, and aggregated oligomeric amyloid- beta peptides but hyper phosphorylated Tau (tubulin binding protein) which formed senile plaque and intracellular neurofibrillary tangles respectively. However, despite the long-term and worldwide effort for a more effective therapy, the only available treatment is a symptomatic use of acetylcholinesterase inhibitors and memantine. Then, many researchers focused their attention to modulate amyloid-beta peptides. These therapeutic approaches as well as those based on cholinergic or amyloid theory have not brought the desired benefits yet. Thus, the main features related with the Tau pathology found in AD are Tau phosphorylation and aggregation. Based on the biochemically diverse range of pathological Tau protein, a number of approaches have been proposed to develop new potential therapeutics like inhibition of Tau phosphorylation, proteolysis and aggregation; promotion of intra- and extracellular Tau clearance and stabilization of microtubules (MTs). Medicinal plants have been used in different systems of medicine and exhibited their powerful roles in the management and cure of memory disorders. This review paper discusses the potential of medicinal plant molecules to targeting Tau protein in Alzheimer’s disease therapy.
 

Comment on: an orally antitumor chalcone hybrid inhibited HepG2 cells growth and migration as the tubulin binding agent.

Recently we read a paper in Investigational New Drugs "An orally antitumor chalcone hybrid inhibited HepG2 cells growth and migration as the tubulin binding agent". Chalcone hybrid 9, a novel chalcone derivative, may be a promising agent for the treatment of hepatocellular carcinoma. However, there are some problems in this paper that are worthy of comment. Human hepatocellular carcinoma HepG2 cells from Shanghai Research Science Limited Company could generate xenograft in nude mice and chalcone hybrid 9 suppressed the growth of HepG2 tumor. However, according to the description of cell bank of Chinese Academy of Science and ATCC, HepG2 cells are no tumorigenic. Similarly, our lab also confirmed that HepG2 cells cannot demonstrate tumorigenic ability in nude mice. Therefore, this discrepancy raised our concern about HepG2 xenograft in the paper.

Mitochondrial bioenergetic dysfunction in the D2.mdx model of Duchenne muscular dystrophy is associated with microtubule disorganization in skeletal muscle

In Duchenne muscular dystrophy, a lack of dystrophin leads to extensive muscle weakness and atrophy that is linked to cellular metabolic dysfunction and oxidative stress. This dystrophinopathy results in a loss of tethering between microtubules and the sarcolemma. Microtubules are also believed to regulate mitochondrial bioenergetics potentially by binding the outer mitochondrial membrane voltage dependent anion channel (VDAC) and influencing permeability to ADP/ATP cycling. The objective of this investigation was to determine if a lack of dystrophin causes microtubule disorganization concurrent with mitochondrial dysfunction in skeletal muscle, and whether this relationship is linked to altered binding of tubulin to VDAC. In extensor digitorum longus (EDL) muscle from 4-week old D2.mdx mice, microtubule disorganization was observed when probing for α-tubulin. This cytoskeletal disorder was associated with a reduced ability of ADP to stimulate respiration and attenuate H2O2 emission relative to wildtype controls. However, this was not associated with altered α-tubulin-VDAC2 interactions. These findings reveal that microtubule disorganization in dystrophin-deficient EDL is associated with impaired ADP control of mitochondrial bioenergetics, and suggests that mechanisms alternative to α-tubulin’s regulation of VDAC2 should be examined to understand how cytoskeletal disruption in the absence of dystrophin may cause metabolic dysfunctions in skeletal muscle.

Concise Synthesis of 1,1-Diarylvinyl Sulfones and Investigations on their Antiproliferative Activity via Tubulin Inhibition.

Discovery of small molecules that inhibit tubulin polymerization is an attractive strategy for the development of new and improved anti-proliferative agents. A series of novel 2-sulfonyl-1,1-diarylethenes were designed towards this end keeping in view the favorable chemical and pharmacological virtues of unsaturated sulfones. Rapid, convenient and efficient two-step assembly of the designed molecules was achieved by the vicinal iodo-sulfonylation-Suzuki coupling sequence. As hypothesized, these compounds showed good anti-proliferative activity against different tissuespecific cancer cell lines: MCF-7, DU-145, A-549, HepG2, and HeLa. The most active compound, pnitrophenyl ring-bearing analog, exhibited an IC50 value of 0.90μM against A-549 cells. Flow cytometry studies on this derivative revealed that it arrests the cell cycle of A-549 cells at the G2/M phase. This compound exhibited molecular binding to tubulin as well as tubulin polymerization inhibition comparable to that of colchicine. A new class of potent, tubulin binding anticancer agents based on 1,1,-diarylvinyl sulfone scaffold has been designed and synthesized.

A cryptic tubulin-binding domain links MEKK1 to curved tubulin protomers

The MEKK1 protein is a pivotal kinase activator of responses to cellular stress. Activation of MEKK1 can trigger various responses, including mitogen-activated protein (MAP) kinases, NF-κB signaling, or cell migration. Notably, MEKK1 activity is triggered by microtubule-targeting chemotherapies, among other stressors. Here we show that MEKK1 contains a previously unidentified tumor overexpressed gene (TOG) domain. The MEKK1 TOG domain binds to tubulin heterodimers-a canonical function of TOG domains-but is unusual in that it appears alone rather than as part of a multi-TOG array, and has structural features distinct from previously characterized TOG domains. MEKK1 TOG demonstrates a clear preference for binding curved tubulin heterodimers, which exist in soluble tubulin and at sites of microtubule polymerization and depolymerization. Mutations disrupting tubulin binding decrease microtubule density at the leading edge of polarized cells, suggesting that tubulin binding may play a role in MEKK1 activity at the cellular periphery. We also show that MEKK1 mutations at the tubulin-binding interface of the TOG domain recur in patient-derived tumor sequences, suggesting selective enrichment of tumor cells with disrupted MEKK1-microtubule association. Together, these findings provide a direct link between the MEKK1 protein and tubulin, which is likely to be relevant to cancer cell migration and response to microtubule-modulating therapies.

Discovery of noscapine derivatives as potential β-tubulin inhibitors

Twenty novel 1,2,3-triazole noscapine derivatives were synthesized starting from noscapine by consecutive N-demethylation, reduction of lactone ring, N-propargylation and Huisgen 1,3-dipolar cycloaddition reaction. In order to select the most promising molecules to subject to further biophysical and biological evaluation, a molecular docking analysis round was performed using noscapine as reference compound. The molecules featuring docking predicted binding affinity better than that of noscapine were then subjected to MTT assay against MCF7 cell line. The obtained results disclosed that all the selected triazole derivatives exhibited a remarkably lower cell viability compared to noscapine in the range of 20 μM in 48 h. In an attempt to correlate the biological activity with the ability to bind tubulin, the surface plasmon resonance (SPR) assay was employed. Compounds 8a, 8h, 9c, 9f and 9j were able to bind tubulin with affinity constant values in the nanomolar range and higher if compared to noscapine. Integrating computational predictions and experimental evaluation, two promising compounds (8h and 9c) were identified, whose relevant cytotoxicity was supposed to be correlated with tubulin binding affinity. These findings shed lights onto structural modifications of noscapine toward the identification of more potent cytotoxic agents targeting tubulin.

An Antibody-Drug Conjugate Targeting MUC1-Associated Carbohydrate CA6 Shows Promising Antitumor Activities.

Glycosylation is a complex multienzyme-related process that is frequently deregulated in cancer. Aberrant glycosylation can lead to the generation of novel tumor surface-specific glycotopes that can be targeted by antibodies. Murine DS6 mAb (muDS6) was generated from serous ovary adenocarcinoma immunization. It recognizes CA6, a Mucin-1 (MUC1)-associated sialoglycotope that is highly detected in breast, ovarian, lung, and bladder carcinomas. SAR566658 antibody-drug conjugate (ADC) is a humanized DS6 (huDS6) antibody conjugated through a cleavable linker to the cytotoxic maytansinoid derivative drug, DM4. SAR566658 binds to tumor cells with subnanomolar affinity, allowing good ADC internalization and intracellular delivery of DM4, resulting in tumor cell death (IC50 from 1 to 7.3 nmol/L). SAR566658 showed in vivo antitumor efficacy against CA6-positive human pancreas, cervix, bladder, and ovary tumor xenografts and against three breast patient-derived xenografts. Tumor regression was observed in all tumor models with minimal effective dose correlating with CA6 expression. SAR566658 displayed better efficacy than standard-of-care nontargeted tubulin binders. These data support the development of SAR566658 in patients with CA6-expressing tumors.

Artificial polyploidy induction for improvement of ornamental and medicinal plants

Green plants provide food, fuel, materials, and medicine for humans. Amongst these, medicinal plants contain unique molecules with therapeutic applications. However, our understanding of these organisms is in its infancy. Landscaping plants also have a great impact on the human soul and emotional perception. These two groups of plants are part of natural wealth and have great economic value. One of the remarkable breeding strategies to improve the valuable properties of plants is artificial polyploidy induction. Medicinal and ornamental plants with duplicated whole sets of chromosomes, although not always, have more distinctive characteristics such as modified phytochemical profile, higher content of desired pharma molecules, plant form, flower color, size and style, fragrance, vase life, and prolonged flowering period. Therefore, artificial chromosome doubling (ACD) of ornamental and medicinal plants could have significant economic consequences. In medicinal plants with an ornamental flower/inflorescence, and/or in ornamental plants bearing essential oils, ACD is a dual beneficial breeding strategy. Working with both in vitro and in vivo chromosome doubling pathways, researchers are able to produce custom-designed plants with higher marketability. Some critical factors must be considered to establish a successful ACD protocol. The plant genotype and explant type are important parameters in this procedure. Type, dosage and duration of application (exposure time) of antimitotic agent (AMA) should also be considered as the main factors. Antimitotic agents can be classified according to their efficacy and toxicity. Although colchicine is the most applied and well-known AMA, there are other alternative mitotic spindle inhibitors with higher specificity for tubulin binding sites in plant materials and possessing less toxicity for animal tubulin. Testing the interaction effects of plant and AMA parameters is necessary for establishing an ACD breeding program and promoting the economic values of medicinal and ornamental plants. This paper reviews significant developments in polyploidization using different antimitotic agents over the last decade in the field of ornamental and medicinal plants. Artificial polyploidy induction is one of the applicable breeding methods in ornamental and medicinal plants. Increasing the efficiency of chromosome doubling and fast-reliable screening of desired phenotypes are prerequisites for production of custom-designed medicinal and ornamental plants through artificial polyploidy induction.

Structural Basis of Noscapine Activation for Tubulin Binding.

Noscapine is a natural alkaloid that is used as an antitussive medicine. However, it also acts as a weak anticancer agent in certain in vivo models through a mechanism that is largely unknown. Here, we performed structural studies and show that the cytotoxic agent 7A-O-demethoxy-amino-noscapine (7A-aminonoscapine) binds to the colchicine site of tubulin. We suggest that the 7A-methoxy group of noscapine prevents binding to tubulin due to a steric clash of the compound with the T5-loop of α-tubulin. We further propose that the anticancer activity of noscapine arises from a bioactive metabolite that binds to the colchicine site of tubulin to induce mitotic arrest through a microtubule cytoskeleton-based mechanism.

Tubulin targeted antimitotic agents based on adamantane lead compound: synthesis, SAR and molecular modeling

5-Hydroxymethyl-2-methoxyphenyl adamantane-1-acetate inhibits cell proliferation and stimulates depolymerization of microtubules of cancer cells to free tubulin. Its analogues were synthesized via the Steglich or Mitsunobu reactions to determine the role of structural subunits of the molecule in tubulin binding. Based on the structure–activity relationship studies, metabolically stable 1-[2-(5-hydroxymethyl-2-methoxyphenyl)ethyl]adamantane was invented, which exhibits a dual-target profile and retains in vitro activity observed for the lead compound.

Rational design of 9-vinyl-phenyl noscapine as potent tubulin binding anticancer agent and evaluation of the effects of its combination on Docetaxel

Docetaxel (DOX) based combination therapy is a novel therapeutic strategy that attracts great interest in breast cancer treatment but its clinical utility got limited due to side effects. In contrast, noscapine, an antitussive drug showed antitumor activity against many cancers without any side effects that targets microtubules and attenuates its dynamic instability. In the quest for an increase in the anticancer activity of noscapine, we strategically designed a novel derivative, 9-vinyl phenyl noscapine (VPN), based on our in silico molecular docking and molecular dynamics simulation effort. Molecular docking of VPN and DOX onto microtubule revealed a docking score of −4.82 kcal/mol and −6.67 kcal/mol respectively, while the docking score of VPN was changed to −3.23 kcal/mol when it was docked onto the co-complex of tubulin-DOX. Further, the binding free energy (ΔGbind,PBSA) of VPN and DOX with tubulin showed −24.04 and −18.65 kcal/mol respectively, while the binding free energy of DOX was increased further in combination with VPN (ΔGbind, PBSA was reduced to −21.41 kcal/mol), denoting combination effect of both ligands. The IC50 value amounted to 30.17 µM and 19.92 µM for VPN and 0.621 µM and 0.193 µM for DOX, respectively for 48 h and 72 h. The dose dependent cytotoxicity of DOX has been reduced considerably with the combination dose regimen of VPN. Further, the combine effect of both the agents improved the apoptotic cell death 28.5% compared to single agent treatment 5.71% and 10.5% for VPN and DOX, respectively. Both agents bind effectively to tubulin in single and in combination to interfere with cell cycle progression in G2/M transition. This study provides novel concept of combination treatment of DOX and VPN to amend efficiency in breast cancer treatment. Communicated by Ramaswamy H. Sarma

Targeting Tau Hyperphosphorylation via Kinase Inhibition: Strategy to Address Alzheimer's Disease.

Microtubule-associated protein tau is involved in the tubulin binding leading to microtubule stabilization in neuronal cells which is essential for stabilization of neuron cytoskeleton. The regulation of tau activity is accommodated by several kinases which phosphorylate tau protein on specific sites. In pathological conditions, abnormal activity of tau kinases such as glycogen synthase kinase-3 β (GSK3β), cyclin-dependent kinase 5 (CDK5), c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinase 1 and 2 (ERK1/2) and microtubule affinity regulating kinase (MARK) lead to tau hyperphosphorylation. Hyperphosphorylation of tau protein leads to aggregation of tau into paired helical filaments like structures which are major constituents of neurofibrillary tangles, a hallmark of Alzheimer's disease. In this review, we discuss various tau protein kinases and their association with tau hyperphosphorylation. We also discuss various strategies and the advancements made in the area of Alzheimer's disease drug development by designing effective and specific inhibitors for such kinases using traditional in vitro/in vivo methods and state of the art in silico techniques.

Structure-activity relationships of tubulysin analogues

The tubulysins are an emerging antibody-drug conjugate (ADC) payload that maintain potent anti-proliferative activity against cells that exhibit the multi-drug resistant (MDR) phenotype. These drugs possess a C-11 acetate known to be hydrolytically unstable in plasma, and loss of the acetate significantly attenuates cytotoxicity. Structure-activity relationship studies were undertaken to identify stable C-11 tubulysin analogues that maintain affinity for tubulin and potent cytotoxicity. After identifying several C-11 alkoxy analogues that possess comparable biological activity to tubulysin M with significantly improved plasma stability, additional analogues of both the Ile residue and N-terminal position were synthesized. These studies revealed that minor changes within the tubulin binding site of tubulysin can profoundly alter the activity of this chemotype, particularly against MDR-positive cell types.

Tubulin Resists Degradation by Cereblon-Recruiting PROTACs.

Dysregulation of microtubules and tubulin homeostasis has been linked to developmental disorders, neurodegenerative diseases, and cancer. In general, both microtubule-stabilizing and destabilizing agents have been powerful tools for studies of microtubule cytoskeleton and as clinical agents in oncology. However, many cancers develop resistance to these agents, limiting their utility. We sought to address this by developing a different kind of agent: tubulin-targeted small molecule degraders. Degraders (also known as proteolysis-targeting chimeras (PROTACs)) are compounds that recruit endogenous E3 ligases to a target of interest, resulting in the target’s degradation. We developed and examined several series of α- and β-tubulin degraders, based on microtubule-destabilizing agents. Our results indicate, that although previously reported covalent tubulin binders led to tubulin degradation, in our hands, cereblon-recruiting PROTACs were not efficient. In summary, while we consider tubulin degraders to be valuable tools for studying the biology of tubulin homeostasis, it remains to be seen whether the PROTAC strategy can be applied to this target of high clinical relevance.

Temporospatial distribution and transcriptional profile of retinal microglia in the oxygen-induced retinopathy mouse model.

Myeloid cells such as resident retinal microglia (MG) or infiltrating blood-derived macrophages (Mϕ) accumulate in areas of retinal ischemia and neovascularization (RNV) and modulate neovascular eye disease. Their temporospatial distribution and biological function in this process, however, remain unclarified. Using state-of-the-art methods, including cell-specific reporter mice and high-throughput RNA sequencing (RNA Seq), this study determined the extent of MG proliferation and Mϕ infiltration in areas with retinal ischemia and RNV in Cx3cr1CreERT2 :Rosa26-tdTomato mice and examined the transcriptional profile of MG in the mouse model of oxygen-induced retinopathy (OIR). For RNA Seq, tdTomato-positive retinal MG were sorted by flow cytometry followed by Gene ontology (GO) cluster analysis. Furthermore, intraperitoneal injections of the cell proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) were performed from postnatal day (p) 12 to p16. We found that MG is the predominant myeloid cell population while Mϕ rarely appears in areas of RNV. Thirty percent of retinal MG in areas of RNV were EdU-positive indicating a considerable local MG cell expansion. GO cluster analysis revealed an enrichment of clusters related to cell division, tubulin binding, ATPase activity, protein kinase regulatory activity, and chemokine receptor binding in MG in the OIR model compared to untreated controls. In conclusion, activated retinal MG alter their transcriptional profile, exhibit considerable proliferative ability and are by far the most frequent myeloid cell population in areas of ischemia and RNV in the OIR model thus presenting a potential target for future therapeutic approaches.

Measurement and Modeling of Microtubule Tip Dynamics

The ability of microtubules to undergo dynamic instability is a fundamental process in all cells and a target of antimitotic drugs. Traditionally, microtubule growth and shrinkage phases in vitro have been assessed through kymograph analysis, which provides limited insight into the precise dynamics at growing tips. Here, we used interference reflection microscopy (IRM) to image dynamic microtubules at 10 frames/s over 10s of minutes, and used a half-gaussian function to track the precise position of the growing plus- and minus-end positions. To study polymerization dynamics occurring during phases of growth and shrinkage, time-dependent fluctuations of tip position were fit to an equation for drift plus diffusion. We found that the plus-tip diffusivity and velocity both increase with increasing tubulin concentration. To understand tubulin on-and off-kinetics, we measured tip diffusivity, growth rate, shrinkage rate, and microtubule lifetimes, and incorporated them into a biochemical model. Using our measured parameters for both the plus- and minus-ends, we were able to put constraints on the on-rate, the longitudinal bond energy, and the lateral bond energy parameters. Current work is focused on comparing the growth of microtubules in GTP versus the non-hydrolyzable analog GMPCPP. Collectively, our measurements will be utilized to understand the impact of hydrolysis on reversible tubulin binding at the plus- and minus-ends.

Enhanced antitumor potential induced by chloroacetate-loaded benzophenones acting as fused tubulin-pyruvate dehydrogenase kinase 1 (PDHK1) ligands.

The majority of cancers detected every year are treated with anti-cancer compounds. Unfortunately, many tumors become resistant to antineoplastic drugs. One option is to use cocktails of compounds acting on different targets to try to overcome the resistant cells. This type of approach can produce good results, but is often accompanied by a sharp increase of associated side effects. The strategy presented herein focuses on the use of a single compound acting on two different biological targets enhancing potency and lowering the toxicity of the chemotherapy. In this light, the approach presented in the current study involves the dual inhibition of human pyruvate dehydrogenase kinase-1 (PDHK1) and tubulin polymerization using mono-, di- and tri-chloroacetate-loaded benzophenones and benzothiophenones. Synthesized molecules were evaluated in vitro on tubulin polymerization and on pyruvate dehydrogenase kinase 1. The cell cycle distribution after treatment of DA1-3b leukemic cells with active compounds was tested. Twenty-two benzo(thio)phenones have been selected by the National Cancer Institute (USA) for evaluation of their anti-proliferative potential against NCI-60 cancer cell lines including multidrug-resistant tumor cell lines. Seventeen molecules proved to be very effective in combating the growth of tumor cells exhibiting inhibitory activities up to nanomolar range. The molecular docking of best antitumor molecules in the study was realized with GOLD in the tubulin and PDHK1 binding sites, and allowed to understand the positioning of active molecules. Chloroacetate-loaded benzo(thio)phenones are dual targeted tubulin- and pyruvate dehydrogenase kinase 1 (PDHK1)-binding antitumor agents and exhibited superior antitumor activity compared to non-chlorinated congeners particularly on leukemia, colon, melanoma and breast cancer cell lines.

A Comprehension into Target Binding and Spatial Fingerprints of Noscapinoid Analogues as Inhibitors of Tubulin.

Owing to its potential to interfere in microtubule dynamics in the mitotic phase of cell cycle and selectively induce apoptosis in cancer cells without affecting normal cells, noscapine and its synthetic analogues have been investigated by other research groups in different cell lines for their capability to be used as anti-cancer agents. The present study is focused on the investigation of the mode of binding of noscapinoids with tubulin, prediction of target binding affinities and mapping of their spatial fingerprints (shape and electrostatic). Molecular docking assisted alignment based 3D-QSAR was used on a dataset (43 molecules) having an inhibitory activity (IC50 = 1.2-250 μM) against human lymphoblast (CEM) cell line. Key amino acid residues of target tubulin were mapped for the binding of most potent noscapine analogue (Compound 11) and were compared with noscapine. Spatial fingerprints of noscapinoids for favorable tubulin inhibitory activity were generated and are proposed herewith for further pharmacophoric amendments of noscapine analogues to design and develop novel potent noscapine based anti-cancer agents that may enter into drug development pipeline.

Pictorial Imaging-Histopathology Correlation in a Rabbit with Hepatic VX2 Tumor Treated by Transarterial Vascular Disrupting Agent Administration.

Cancer vasculature is immature, disorganized and hyperpermeable and can serve as a target for anti-cancer therapies. Vascular disrupting agents (VDAs) are tubulin protein binding and depolymerizing agents that induce rapid tumoral vascular shutdown and subsequent cancer necrosis. However, two clinical problems exist with all VDAs, i.e. 1) incomplete anticancer effect and 2) dose-dependent toxicity.To tackle these problems, in our ongoing research, a novel VDA C118P is applied by transarterial administration of half the intravenous dose in rabbits with implanted VX2 liver tumor to assess its therapeutic efficacy.Nearly complete tumor necrosis was achieved by only a single arterial dose of C118P at 5 mg/kg, which was documented in a representative case by in vivo digital subtraction arteriogram (DSA) and magnetic resonance imaging (MRI), and further confirmed by ex vivo microangiogram and histopathology.This convincing and promising preliminary outcome would warrant further comprehensive studies to explore the potentials of VDAs by transarterial administration either in mono-drug or in combination for management of solid cancers.

Testing the role of intraflagellar transport in flagellar length control using length-altering mutants of Chlamydomonas.

Cilia and flagella are ideal model organelles in which to study the general question of organelle size control. Flagellar microtubules are steady-state structures whose size is set by the balance of assembly and disassembly. Assembly requires intraflagellar transport (IFT), and measurements of IFT have shown that the rate of entry of IFT particles into the flagellum is a decreasing function of length. It has been proposed that this length dependence of IFT may be the basis for flagellar length control. Here, we test this idea by showing that three different long-flagella mutations in Chlamydomonas all cause increased IFT injection, thus confirming that IFT can influence length control. However, quantitative comparisons with mathematical models suggest that the increase in injection is not sufficient to explain the full increase in length seen in these mutants; hence, some other mechanism may be at work. One alternative mechanism that has been proposed is length-regulated binding of tubulin to the IFT particles. However, we find that the apparent length dependence of tubulin loading that has previously been reported may actually reflect length-dependent organization of IFT trains.This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.