A Force-Induced Directional Switch of a Molecular Motor Enables Parallel Microtubule Bundle Formation.

Microtubule-organizing centers (MTOCs) nucleate microtubules that can grow autonomously in any direction. To generate bundles of parallel microtubules originating from a single MTOC, the growth of multiple microtubules needs to coordinated, but the underlying mechanism is unknown. Here, we show that a conserved two-component system consisting of the plus-end tracker EB1 and the minus-end-directed molecular motor Kinesin-14 is sufficient to promote parallel microtubule growth. The underlying mechanism relies on the ability of Kinesin-14 to guide growing plus ends along existing microtubules. The generality of this finding is supported by yeast, Drosophila, and human EB1/Kinesin-14 pairs. We demonstrate that plus-end guiding involves a directional switch of the motor due to a force applied via a growing microtubule end. The described mechanism can account for the generation of parallel microtubule networks required for a broad range of cellular functions such as spindle assembly or cell polarization.

ORP8 inhibits renal cell carcinoma progression by accelerating Stathmin1 degradation and microtubule polymerization.

ORP8 has been reported to suppress tumor progression in various malignancies. However, the functions and underlying mechanisms of ORP8 are still unknown in renal cell carcinoma (RCC). Here, decreased expression of ORP8 was detected in RCC tissues and cell lines. Functional assays verified that ORP8 suppressed RCC cell growth, migration, invasion, and metastasis. Mechanistically, ORP8 attenuated Stathmin1 expression by accelerating ubiquitin-mediated proteasomal degradation and led to an increase in microtubule polymerization. Lastly, ORP8 knockdown partly rescued microtubule polymerization, as well as aggressive cell phenotypes induced by paclitaxel. Our findings elucidated that ORP8 suppressed the malignant progression of RCC by increasing Stathmin1 degradation and microtubule polymerization, thus suggesting that ORP8 might be a novel target for the treatment of RCC.

Cypin binds to tubulin heterodimers and microtubule protofilaments and regulates microtubule spacing in developing hippocampal neurons.

Cytosolic PSD-95 interactor (cypin) is a multifunctional, guanine deaminase that plays a major role in shaping the morphology of the dendritic arbor of hippocampal and cortical neurons. Cypin catalyzes the Zn2+-dependent deamination of guanine to xanthine, which is then metabolized to uric acid by xanthine oxidase. Cypin binds to tubulin heterodimers via its carboxyl terminal region (amino acids (aa) 350-454), which contains a collapsin response mediator protein (CRMP) homology domain (aa 350-403). Moreover, this region alone is not sufficient to facilitate microtubule polymerization; therefore, additional cypin regions must be involved in this process. Here, we asked whether cypin binds to fully formed microtubules and how overexpression of cypin regulates the microtubule cytoskeleton in dendrites of cultured hippocampal neurons. Protein-protein docking strategies confirm that the cypin homodimer binds to tubulin heterodimers via amino acids within aa 350-454. Biochemical pull-down data suggest that aa 1-220 are necessary for cypin binding to soluble tubulin heterodimers and to taxol-stabilized microtubules. Molecular docking of the cypin homodimer to soluble tubulin heterodimers reveals a consistently observed docking pose using aa 47-71, 113-118, 174-178, and 411-418, which is consistent with our biochemical data. Additionally, overexpression of cypin in hippocampal neurons results in decreased spacing between microtubules. Our results suggest that several protein domains facilitate cypin-mediated polymerization of tubulin heterodimers into microtubules, possibly through a mechanism whereby cypin dimers bind to multiple tubulin heterodimers.

Exceptional Repositioning of Dog Dewormer: Fenbendazole Fever.

Fenbendazole (FZ) is a benzimidazole carbamate drug with broad-spectrum antiparasitic activity in humans and animals. The mechanism of action of FZ is associated with microtubular polymerization inhibition and glucose uptake blockade resulting in reduced glycogen stores and decreased ATP formation in the adult stages of susceptible parasites. A completely cured case of lung cancer became known globally and greatly influenced the cancer community in South Korea. Desperate Korean patients with cancer began self-administering FZ without their physician's knowledge, which interfered with the outcome of the cancer treatment planned by their oncologists. On the basis of presented evidence, this review provides valuable information from PubMed, Naver, Google Scholar, and Social Network Services (SNS) on the effects of FZ in a broad range of preclinical studies on cancer. In addition, we suggest investigating the self-administration of products, including supplements, herbs, or bioactive compounds, by patients to circumvent waiting for long and costly FZ clinical trials.

Development of SKI-349, a dual-targeted inhibitor of sphingosine kinase and microtubule polymerization.

Our sphingosine kinase inhibitor (SKI) optimization studies originated with the optimization of the SKI-I chemotype by replacement of the substituted benzyl rings with substituted phenyl rings giving rise to the discovery of SKI-178. We have recently reported that SKI-178 is a dual-targeted inhibitor of both sphingosine kinase isoforms (SphK1/2) and a microtubule disrupting agent (MDA). In mechanism-of-action studies, we have shown that these two separate actions synergize to induce cancer cell death in acute myeloid leukemia (AML) cell and animal models. Owning to the effectiveness of SKI-178, we sought to further refine the chemotype while maintaining "on-target" SKI and MDA activities. Herein, we modified the "linker region" between the substituted phenyl rings of SKI-178 through a structure guided approach. These studies have yielded the discovery of an SKI-178 congener, SKI-349, with log-fold enhancements in both SphK inhibition and cytotoxic potency. Importantly, SKI-349 also demonstrates log-fold improvements in therapeutic efficacy in a retro-viral transduction model of MLL-AF9 AML as compared to previous studies with SKI-178. Together, our results strengthen the hypothesis that simultaneous targeting of the sphingosine kinases (SphK1/2) and the induction of mitotic spindle assembly checkpoint arrest, via microtubule disruption, might be an effective therapeutic strategy for hematological malignancies including AML.

PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment.

PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation.

Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization.

BACKGROUND: The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. METHODS: Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata) on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC) cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol's inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol's efficacy in vivo. RESULTS: Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. CONCLUSION: These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment.

Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell.

BACKGROUND/AIMS: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl--induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX) is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. METHODS: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. RESULTS: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. CONCLUSIONS: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

HSP70 regulates the function of mitotic centrosomes.

To establish a functional bipolar mitotic spindle, the centrosome expands and matures, acquiring enhanced activities for microtubule (MT) nucleation and assembly at the onset of mitosis. However, the regulatory mechanisms of centrosome maturation and MT assembly from the matured centrosome are largely unknown. In this study, we showed that heat shock protein (HSP) 70 considerably accumulates at the mitotic centrosome during prometaphase to metaphase and is required for bipolar spindle assembly. Inhibition or depletion of HSP70 impaired the function of mitotic centrosome and disrupted MT nucleation and polymerization from the spindle pole, and may thus result in formation of abnormal mitotic spindles. In addition, HSP70 may associate with NEDD1 and γ-tubulin, two pericentriolar material (PCM) components essential for centrosome maturation and MT nucleation. Loss of HSP70 function disrupted the interaction between NEDD1 and γ-tubulin, and reduced their accumulation at the mitotic centrosome. Our results thus demonstrate a role for HSP70 in regulating centrosome integrity during mitosis, and indicate that HSP70 is required for the maintenance of a functional mitotic centrosome that supports the assembly of a bipolar mitotic spindle.

Novel dual action PARP and microtubule polymerization inhibitor AMXI-5001 powerfully inhibits growth of esophageal carcinoma both alone and in combination with radiotherapy.

Esophageal cancer is one of the leading causes of cancer deaths globally with an incidence that is concentrated in specific hot spots in Eastern Asia, the Middle East, Eastern Africa, and South America. 10-year overall survival for patients treated with standard of care chemoradiation followed by surgical resection is below 40% highlighting the need for novel therapeutics to treat this disease. We assessed the effect of AMXI-5001, a novel small molecule poly ADP-Ribose polymerase (PARP) inhibitor and microtubule polymerization inhibitor on tumor growth inhibition in both in-vitro and in-vivo murine models. We found that AMXI-5001 was the most potent growth inhibitor of 8 out of 9 different esophageal carcinoma cell lines compared to other clinically available PARP inhibitors, Olaparib, Niraparib, Rucaparib, and Talazoparib. We then confirmed the previously described mechanism of action of AMXI-5001 as a PARP-inhibitor and microtubule polymerization inhibitor using both a PARP trapping assay and immunofluorescence. To further assess AMXI-5001's potential as a therapeutic for esophageal carcinoma we evaluated the effect of AMXI-5001 in combination with standard chemotherapy agents, Cisplatin and 5 Fluorouracil. We showed that AMXI-5001 synergistically inhibits growth in KYSE-70, a squamous esophageal cell line in combination with these drugs. In addition, we found that AMXI-5001 was an effective radiosensitizer, and squamous esophageal carcinoma cell lines treated 24 hours prior to external beam radiation showed significantly more growth inhibition compared to controls. Finally, we assessed the effect of AMXI-5001 monotherapy and in combination with radiotherapy in a xenograft mouse model implanted with subcutaneous KYSE-70 cells. Compared to vehicle control, and those treated with either AMXI-5001 alone or radiation alone, mice treated with both AMXI-5001 and radiation had significant tumor response. In conclusion, AMXI-5001 is an orally bioavailable dual-action PARP and microtubule polymerization inhibitor that holds promise in the treatment of esophageal carcinoma.

Chemical studies on Taxus canadensis.

A series of new taxanes, 1-93, have been isolated, together with 37 known taxoids including Taxol(®) (paclitaxel) and cephalomannine, from the Canadian yew, Taxus canadensis (Taxaceae) in the past 30 years. These new taxoids possess various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, and 6/12 ring systems and six new taxanes with four novel skeletons, i.e., a taxane with a 6/6/8/6 ring system, a taxane with a [3.3.3] propellane skeleton, three taxanes with [3.3.3] [3.4.5] dipropellane sytems, as well as a novel taxane with a unique 5/5/4/6/6/6 hexacyclic skeleton, containing a unique [3.3.2] propellane, were isolated for the first time from natural sources. It should be emphasized that 13-acetyl-9-dihydrobaccatin III, a very useful starting material for the semisynthesis of Taxol(®) and Taxotere(®) , represents the most abundant taxane in the needles of this yew tree. These findings establish the above mentioned yew tree as significantly different from the remaining species. On the other hand, some chemical modifications on the taxanes isolated from this plant were carried out.

A model for the catalytic activity of microtubule polymerases.

A XMAP215/Stu2/Alp14 polymerase can catalyze processively the tubulin addition to the microtubule (MT) plus end. In this work, a model is proposed for the underlying molecular mechanism of the polymerase activity, where the polymerase can not only catalyze processively the tubulin addition to but also promote the tubulin removal from the MT plus end. Based on the model the dynamics of both the wild-type and mutant polymerases is studied theoretically, explaining consistently and well various available experimental data. To further test the model, predicted results are provided.

A Novel Dual-Payload ADC for the Treatment of HER2+ Breast and Colon Cancer.

Antibody-drug conjugates (ADCs) have demonstrated a great therapeutic potential against cancer due to their target specificity and cytotoxicity. To exert a maximum therapeutic effect on cancerous cells, we have conjugated two different payloads to different amino acids, cysteines (cys) and lysines (lys), on trastuzumab, which is a humanised anti-HER2 monoclonal antibody. First, trastuzumab was conjugated with monomethyl auristatin E (MMAE), an antimitotic agent, through a cleavable linker (Val-Cit) to prepare ADC (Tmab-VcMMAE). Then, the ADC (Tmab-VcMMAE) was conjugated with a second antimitotic agent, Mertansine (DM1), via a non-cleavable linker Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) to form a dual conjugate (Tmab-VcMMAE-SMCC-DM1). Our results indicated that the dual-payload conjugate, Tmab-VcMMAE-SMCC-DM1, had a synergistic and superior cytotoxic effect compared to trastuzumab alone. Ultimately employing a dual conjugation approach has the potential to overcome treatment-resistance and tumour recurrences and could pave the way to employ other payloads to construct dual (or multiple) payload complexes.

PICT1 is critical for regulating the Rps27a-Mdm2-p53 pathway by microtubule polymerization inhibitor against cervical cancer.

In our previous study, it showed that P-3F, a podophyllotoxin derivative, causes the increased level of p53 expression by enhancing p53 stability, resulting from blockage of the Mdm2-p53 feedback loop via nucleolus-to-nucleoplasm translocation of Rps27a in human cervical cancer HeLa cell line. However, the mechanism of regulating Rps27a localization remains to be studied. In the current study, it has been demonstrated that the level of protein interacting with carboxyl terminus 1 (PICT1), originally identified as a tumor suppressor, was decreased in a concentration-dependent manner in response to P-3F, leading to inhibition of human cervical cancer cell lines proliferation. Also remarkably, reduction of serine phosphorylation of STMN1 at position 16 induced by P-3F was required in the downregulation of PICT1, in which p53 activity was likely to be directly involved. Note as well that, PICT1 also played an important role in p53 stability enhancement by inhibiting Mdm2-mediated p53 ubiquitination due to Rps27a translocation from the nucleolus to the nucleoplasm to interact with Mdm2 following treatment with P-3F. Collectively, these findings indicated that P-3F, a microtubule polymerization inhibitor, promotes the decreased level of PICT1 expression, which is critical for regulating the Rps27a-Mdm2-p53 pathway against cervical cancer.

Ligand- and structural-based discovery of potential small molecules that target the colchicine site of tubulin for cancer treatment.

Small molecules targeting the colchicine site of tubulin represent an attractive cancer treatment strategy. In this study, a total of 468 models derived from 1076 diverse inhibitors binding to the tubulin colchicine site were constructed based on fingerprints using three machine learning approaches: 1) naive Bayesian (NB); 2) single tree (ST); and 3) random forest (RF). The overall predictive accuracy of the best models exceeded 85.12% for both the training and test sets. We designed an integrated virtual screening (VS) strategy for identifying new tubulin inhibitors by combining established models, molecular docking, and similarity-based analog searching. Through two rounds of VS, compound 23g was identified as a novel potent anticancer agent exhibiting activity against MDA-MB-231, HeLa, A549, HepG2, CNE2, and HCT116 tumor cell lines with IC50 values of 5.45, 8.61, 7.47, 2.29, 2.91, and 4.10 µM, respectively. Compared with taxol, colchicine, and adriamycin, 23g also displayed potent cytotoxicity against the drug-resistant tumor cell lines, HepG2/ADR, A549/CDDP, and A549/TAX cells, with IC50 values of 4.12, 6.58, and 6.38 µM, respectively. Further mechanistic studies revealed that 23g inhibited microtubule polymerization by binding to the colchicine site of tubulin, arrested the cell cycle at the G2/M phase, induced cell apoptosis, and exhibited potent in vitro anti-metastasis activity. Finally, molecular docking, molecular dynamics, and free energy analyses were employed to explore the detailed binding interaction between 23g and tubulin. Collectively, these findings indicated that 23g should be further investigated as a potential novel potent antitumor agent targeting tubulin.

SYTL4 downregulates microtubule stability and confers paclitaxel resistance in triple-negative breast cancer.

Background: Taxanes are frontline chemotherapeutic drugs for patients with triple-negative breast cancer (TNBC); however, chemoresistance reduces their effectiveness. We hypothesized that the molecular profiling of tumor samples before and after neoadjuvant chemotherapy (NAC) would help identify genes associated with drug resistance. Methods: We sequenced 10 samples by RNA-seq from 8 NAC patients with TNBC: 3 patients with a pathologic complete response (pCR) and the other 5 with non-pCR. Differentially expressed genes that predicted chemotherapy response were selected for in vitro functional screening via a small-scale siRNAs pool. The clinical and functional significance of the gene of interest in TNBC was further investigated in vitro and in vivo, and biochemical assays and imaging analysis were applied to study the mechanisms. Results: Synaptotagmin-like 4 (SYTL4), a Rab effector in vesicle transport, was identified as a leading functional candidate. High SYTL4 expression indicated a poor prognosis in multiple TNBC cohorts, specifically in taxane-treated TNBCs. SYTL4 was identified as a novel chemoresistant gene as validated in TNBC cells, a mouse model and patient-derived organoids. Mechanistically, downregulating SYTL4 stabilized the microtubule network and slowed down microtubule growth rate. Furthermore, SYTL4 colocalized with microtubules and interacted with microtubules through its middle region containing the linker and C2A domain. Finally, we found that SYTL4 was able to bind microtubules and inhibit the in vitro microtubule polymerization. Conclusion: SYTL4 is a novel chemoresistant gene in TNBC and its upregulation indicates poor prognosis in taxane-treated TNBC. Further, SYTL4 directly binds microtubules and decreases microtubule stability.

Towards further Understanding the Structural Requirements of Combretastatin- like Chalcones as Inhibitors of Microtubule Polymerization.

BACKGROUND: Microtubules are dynamic filamentous cytoskeletal structures which play several key roles in cell proliferation and trafficking. They are supposed to contribute in the development of important therapeutic targeting tumor cells. Chalcones are important group of natural compounds abundantly found in fruits & vegetables that are known to possess anticancer activity. We have used QSAR and docking studies to understand the structural requirement of chalcones for understanding the mechanism of microtubule polymerization inhibition. METHODS: Three dimensional (3D) QSAR (CoMFA and CoMSIA), pharmacophore mapping and molecular docking studies were performed for the generation of structure activity relationship of combretastatin-like chalcones through statistical models and contour maps. RESULTS: Structure activity relationship revealed that substitution of electrostatic, steric and donor groups may enhance the biological activity of compounds as inhibitors of microtubule polymerization. From the docking study, it was clear that compounds bind at the active site of tubulin protein. CONCLUSION: The given strategies of modelling could be an encouraging way for designing more potent compounds as well as for the elucidation of protein-ligand interaction.

P-3F, a microtubule polymerization inhibitor enhances P53 stability through the change in localization of RPS27a.

Previously, we demonstrated that P-3F, a podophyllum derivative, exhibits a 297-fold enhancement in antitumor activity than VP-16, used as anticancer agent in clinical. The purpose of our present study was to investigate the precise antitumor mechanism action of P-3F. It showed that P-3F inhibited microtubule polymerization in a concentration-dependent manner. The results were in overall agreement with modeling and docking studies performed on P-3F and tubulin. In addition, P-3F increased the levels of P53, this in turn prolonged P53 half-life. Note as well that levels of P21 protein were increased along with P53 in a concentration dependent change. It suggested that enhancement in stabilization of P53 induced by P-3F may be critical for P53/P21 signaling pathway, resulting in cell cycle arrest at G2/M. Furthermore, release of RPS27a from the nucleolus into the nucleoplasm led to decrease phosphorylation of Mdm2 at serine residue 166 and inhibit Mdm2-mediated ubiquitination of P53 in (P-3F)-treated HeLa cells. Together, these data suggest that P-3F, a microtubule polymerization inhibitor, causes P53 accumulation via P53 stability enhancement, due to blockage of the P53-Mdm2 network through the change in localization of RPS27a.

α-Cyperone of Cyperus rotundus is an effective candidate for reduction of inflammation by destabilization of microtubule fibers in brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (Cyperaceae), commonly known as purple nutsedge or nut grass is one of the most invasive and endemic weeds in tropical, subtropical and temperate regions. This plant has been extensively used in traditional medicine for anti-arthritic, antidiarrheal and antiplatelet properties as well as treatment for several CNS disorders such as epilepsy, depression and inflammatory disorders. Inflammation is evidently occurring in pathologically susceptible regions of the Alzheimer's disease (AD) brain as well as other disorders. Many cellular processes are responsible in chronic inflammation. Microtubule-based inflammatory cell chemotaxis is a well-recognized process that influences production of cytokines and phagocytosis. The effect of α-Cyperone, one of main ingredients of Cyperus rotundus on microtubule assembly and dynamics has not been examined and is the purpose of this investigation. MATERIALS AND METHODS: Microtubules and tubulin were extracted in order to explore their interaction with α-Cyperone by utilization of turbidimetric examinations, intrinsic fluorescence and circular dichroism spectroscopy (CD) studies. The molecular docking analysis was executed in order to facilitate a more detail and stronger evidence of this interaction. The BINding ANAlyzer (BINANA) algorithm was used to evaluate and further substantiate the binding site of α-Cyperone. RESULTS: It was demonstrated that α-Cyperone had a pronounced influence on the tubulin structure, decreased polymerization rate and reduced concentration of polymerized tubulin in vitro. The CD deconvolution analysis concluded that significant conformational changes occurred, demonstrated by a drastic increase in content of ß-strands upon binding of α-Cyperone. The fluorescence spectroscopy revealed that a static type of quenching mechanism is responsible for binding of α-Cyperone to tubulin. Upon characterization of various biophysical parameters, it was further deduced that ligand binding was spontaneous and a single site of binding was confirmed. Transmission electron microscopy revealed that upon binding of α-Cyperone to microtubule the number and complexity of fibers were noticeably decreased. The computational analysis of docking suggested that α-Cyperone binds preferably to ß-tubulin at a distinct location with close proximity to the GTP binding and hydrolysis site. The ligand interaction with ß-tubulin is mostly hydrophobic and occurs at amino acid residues that are exclusively on random coil. The BINANA 1.2.0 algorithm which counts and tallies close molecular interaction by performing defined set of simulations revealed that amino acid residues Arg 48 and Val 62 have registered the highest scores and are possibly crucial in ligand-protein interaction. CONCLUSION: α-Cyperone binds and interacts with tubulin and is capable of distinctly destabilizing microtubule polymerization. The effect of this interaction could result in reduction of inflammation which would be highly beneficial for treatment of inflammatory diseases such as AD.

Establishing a High-content Analysis Method for Tubulin Polymerization to Evaluate Both the Stabilizing and Destabilizing Activities of Compounds.

Microtubules are important components of the cellular cytoskeleton that play roles in various cellular processes such as vesicular transport and spindle formation during mitosis. They are formed by an ordered organization of α-tubulin and ß-tubulin hetero-polymers. Altering microtubule polymerization has been known to be the mechanism of action for a number of therapeutically important drugs including taxanes and epothilones. Traditional cell-based assays for tubulin-interacting compounds rely on their indirect effects on cell cycle and/or cell proliferation. Direct monitoring of compound effects on microtubules is required to dissect detailed mechanisms of action in a cellular setting. Here we report a high-content assay platform to monitor tubulin polymerization status by directly measuring the acute effects of drug candidates on the cellular tubulin network with the capability to dissect the mechanisms of action. This high-content analysis distinguishes in a quantitative manner between compounds that act as tubulin stabilizers versus those that are tubulin destabilizers. In addition, using a multiplex approach, we expanded this analysis to simultaneously monitor physiological cellular responses and associated cellular phenotypes.