Disruption Of Microtubule Network Research Articles

Potent and Selective Benzothiazole-Based Antimitotics with Improved Water Solubility: Design, Synthesis, and Evaluation as Novel Anticancer Agents.

The design of colchicine site ligands on tubulin has proven to be a successful strategy to develop potent antiproliferative drugs against cancer cells. However, the structural requirements of the binding site endow the ligands with low aqueous solubility. In this work, the benzothiazole scaffold is used to design, synthesize, and evaluate a new family of colchicine site ligands exhibiting high water solubility. The compounds exerted antiproliferative activity against several human cancer cell lines, due to tubulin polymerization inhibition, showing high selectivity toward cancer cells in comparison with non-tumoral HEK-293 cells, as evidenced by MTT and LDH assays. The most potent derivatives, containing a pyridine moiety and ethylurea or formamide functionalities, displayed IC50 values in the nanomolar range even in the difficult-to-treat glioblastoma cells. Flow cytometry experiments on HeLa, MCF7, and U87MG cells showed that they arrest the cell cycle at the G2/M phases at an early time point (24 h), followed by apoptotic cell death 72 h after the treatment. Tubulin binding was confirmed by microtubule network disruption observed via confocal microscopy. Docking studies support favorable interaction of the synthesized ligands at the colchicine binding site. These results validate the proposed strategy to develop potent anticancer colchicine ligands with improved water solubility.

PO-01-170 PHARMACOLOGICAL AND GENE-THERAPY BASED INHIBITION OF NOX2-GENERATED ROS ATTENUATES DISRUPTION OF MICROTUBULE NETWORK AND EMERGENCE OF TRIGGERED CA2+WAVES IN ATRIAL MYOCYTES AND IN CHRONIC AF CANINE MODEL

Recent research indicate an important role for derailed proteostasis in atrial fibrillation (AF). Major pathways of derailed proteostasis include disruption of microtubule network (MTN). Experimental and clinical studies have documented a significant contribution of histone deactylase 6 (HDAC6) mediated disruption of MTN. Another major causative factor for AF is excessive oxidative stress (OS). However, a clear connection of disruption of MTN with OS is not known in atrial myocytes and chronic model of AF. In particular, it remains elusive whether disruption of MTN by NOX2-generated reactive oxygen species (ROS) contributes to the emergence of triggered Ca2+ waves (TCWs), which can cause membrane depolarization and promote early-afterdepolarization (EAD). To establish the connection between OS and disruption of MTN in isolated canine atrial myocytes and in atrial myocardium of a canine model of chronic AF Normal atrial myocytes were isolated from 4 dogs and pre-incubated in Colchicine (a microtubule disruptor) and H2O2. While myocytes, loaded with Ca2+ sensitive dye, were paced at different cycle lengths (1000ms to 200ms), line-scan confocal Ca2+ imaging were performed in the presence of gp91-tat (a NOX2 inhibitor) and TSA (a HDAC6 inhibitor). In the same cells, MTN was examined by Immunofluorescence. To determine the MTN organization in chronic AF, immunohischemistry was performed in atrial tissue sections from 3 normal animals, 4 control rapid atrial pacing (RAP) animals, and 4 NOX2-shRNA injected RAP animals. In Colchicine treated- isolated atrial myocytes, acute application of gp91-tat and TSA attenuated the disruption of MTN (a) and the generation of TCWs (b). Similarly, gp91-tat and TSA attenuated the disruption of MTN in H2O2 pretreated atrial myocytes (data not shown). Notably, while distinctive MTN was abolished in RAP induced chronic AF atrial sections, there was an attenuation of expected MTN decrease in atrial sections from NOX2-shRNA injected RAP dogs (c). Inhibition of NOX2 by either NOX2 shRNA injection in the chronic AF model or acute application of NOX2 inhibitor as well as HDAC6 inhibitor in atrial myocytes is associated with attenuation of the disruption of MTN and generation of TCWs. Identifying the connection between OS and derailed proteostasis not only sheds light on the molecular mechanisms underlying AF, but also may allow to develop novel therapeutic approach for AF.

Abstract 4943: SP-1-39 as a novel tubulin inhibitor overcomes taxane resistance in castration-resistant prostate cancer model

Abstract Colchicine binding site inhibitors have proven to have potent anti-cancer efficacy in many cancer types. Inhibition of tubulin polymerization, a popular mechanism of action for anti-cancer activities, will be activated once the inhibitor interacts with the colchicine binding site. We have previously developed and reported a potent tubulin inhibitor (SB-216). One of its derivatives, the compound SP-1-39, a pyrimidine dihydroquinoxalinone derivative, has been recently designed to further improve the potency and therapeutic properties. Here, we examined the impact of SP-1-39 on preclinical prostate cancer models in vitro and in vivo. EBI competition assay confirmed that SP-1-39 binds to colchicine-binding site and inhibits the formation of EBI:β-tubulin adduct. The anti-proliferation abilities of SP-1-39 was tested against melanoma, breast cancer, pancreatic cancer, prostate cancer, and paclitaxel-resistant prostate cancer cell lines by MTS assay, and defined IC50 values of 0.2-4 nmol/L. Other in vitro results showed SP-1-39 disrupted microtubule network, induced cell cycle arrest at G2/M phase, and caused apoptosis in paclitaxel-resistant human prostate cancer cells (PC-3/TxR). For in vivo study, 2.5mg/kg SP-1-39 significantly attenuated tumor growth in PC-3/TxR xenograft mouse model, inhibited angiogenesis, and overcame taxane resistance. Our findings demonstrate the preclinical therapeutic efficacy and safety of SP-1-39, and provide support for further exploration of novel tubulin inhibitors as anti-cancer agent for cancer therapy. Citation Format: Rui Wang, Kelli L. Hartman, Satyanarayana Pochampally, Duane D. Miller, Wei Li. SP-1-39 as a novel tubulin inhibitor overcomes taxane resistance in castration-resistant prostate cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4943.

Colchicine attenuates the electrical remodeling of post-operative atrial fibrillation through inhibited expression of immune-related hub genes and stabilization of microtubules.

Rationale: Acute inflammation is a major risk factor for post-operative atrial fibrillation (POAF), and epicardial adipose tissue (EAT) is considered as a source of inflammatory mediators. However, underlying mechanisms and pharmacological targets of POAF are poorly understood. Methods: Integrative analysis of array data from EAT and right atrial appendage (RAA) samples was conducted to identify potential hub genes. Lipopolysaccharide (LPS)-stimulated inflammatory models in mice and in induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) were used to examine the exact mechanism underlying POAF. Electrophysiological analysis, multi-electrode array, and Ca2+ imaging was employed to explore the alterations of electrophysiology and Ca2+ homeostasis under inflammation. Flow cytometry analysis, histology and immunochemistry were performed to investigate immunological alterations. Results: We observed electrical remodeling, enhanced atrial fibrillation (AF) susceptibility, immune cell activation, inflammatory infiltration, and fibrosis in LPS-stimulated mice. LPS-stimulated iPSC-aCMs showed arrhythmias, abnormal Ca2+ signaling, reduced cell viability, disrupted microtubule network and increased α-tubulin degradation. VEGFA, EGFR, MMP9 and CCL2 were identified as hub genes simultaneously targeted in the EAT and RAA of POAF patients. Notably, treatment of colchicine in LPS-stimulated mice resulted in a U-shape dose-response curve, where greatly improved survival rates were observed only at doses between 0.10-0.40 mg/kg. At this therapeutic dose level, colchicine inhibited the expression of all the identified hub genes and effectively rescued the pathogenic phenotypes observed in LPS-stimulated mice and iPSC-aCM models. Conclusions: Acute inflammation promotes α-tubulin degradation, induces electrical remodeling, and both recruits and facilitates the infiltration of circulating myeloid cells. A certain dose of colchicine attenuates electrical remodeling and decreases the recurrence of AF.

Disruption of Sarcoplasmic Reticulum-Mitochondrial Contacts Underlies Contractile Dysfunction in Experimental and Human Atrial Fibrillation: A Key Role of Mitofusin 2.

Background Atrial fibrillation (AF) is the most common and progressive tachyarrhythmia. Diabetes is a common risk factor for AF. Recent research findings revealed that microtubule network disruption underlies AF. The microtubule network mediates the contact between sarcoplasmic reticulum and mitochondria, 2 essential organelles for normal cardiomyocyte function. Therefore, disruption of the microtubule network may impair sarcoplasmic reticulum and mitochondrial contacts (SRMCs) and subsequently cardiomyocyte function. The current study aims to determine whether microtubule-mediated SRMCs disruption underlies diabetes-associated AF. Methods and Results Tachypacing (mimicking AF) and high glucose (mimicking diabetes) significantly impaired contractile function in HL-1 cardiomyocytes (loss of calcium transient) and Drosophila (reduced heart rate and increased arrhythmia), both of which were prevented by microtubule stabilizers. Furthermore, both tachypacing and high glucose significantly reduced SRMCs and the key SRMC tether protein mitofusin 2 (MFN2) and resulted in consequent mitochondrial dysfunction, all of which were prevented by microtubule stabilizers. In line with pharmacological interventions with microtubule stabilizers, cardiac-specific knockdown of MFN2 induced arrhythmia in Drosophila and overexpression of MFN2 prevented tachypacing- and high glucose-induced contractile dysfunction in HL-1 cardiomyocytes and/or Drosophila. Consistently, SRMCs/MFN2 levels were significantly reduced in right atrial appendages of patients with persistent AF compared with control patients, which was aggravated in patients with diabetes. Conclusions SRMCs may play a critical role in clinical AF, especially diabetes-related AF. Furthermore, SRMCs can be regulated by microtubules and MFN2, which represent novel potential therapeutic targets for AF.

Design, synthesis and biological evaluation of colchicine glycoconjugates as tubulin polymerization inhibitors

A series of new colchicine glycoconjugates as tubulin polymerization inhibitors were designed by targeting strategy based on Warburg effect. All of the colchicine glycoconjugates were synthesized and then evaluated for their antiproliferative activities against three human cancer lines HT-29, MCF-7 and Hep-3B. Among them, 1e exhibited greater than 10 times selectivity between GLUT1 highly expressed cells (HT-29 and MCF-7) and GLUT1 lowly expressed cells (Hep-3B), and also showed lower cytotoxicity against HUVECs compared with colchicine. Moreover, 1e significantly inhibited tubulin polymerization and disrupted microtubule networks. GLUT1 inhibitor-dependent cytotoxicity assay demonstrated that the uptake of 1e was regulated via GLUT1. Molecular docking studies showed that 1e could be a substrate of GLUT1 and bind to the colchicine site of tubulin.

IMB5476, a novel microtubule inhibitor, induces mitotic catastrophe and overcomes multidrug resistance in tumors

IMB5046 is a nitrobenzoate microtubule inhibitor we reported previously. During screening of its structural analogues, we identified a novel compound IMB5476 with increased aqueous solubility. Here, its antitumor activity and the underlying mechanism were investigated. IMB5476 disrupted microtubule networks in cells and arrested cell cycle at G2/M phase. It inhibited purified tubulin polymerization in vitro. Competition assay indicated that it bound to tubulin at the colchicine pocket. Further experiments proved that it induced cell death by mitotic catastrophe and apoptosis. Notably, it was a poor substrate of P-glycoprotein and exhibited potent cytotoxicity against drug-resistant tumor cells. In addition, IMB5476 could inhibit angiogenesis in vitro. IMB5476 also inhibited the growth of drug-resistant KBV200 xenografts in mice. Conclusively, our data reveal a novel nitrobenzoate microtubule inhibitor with improved aqueous solubility and can overcome multidrug resistance.

2-Methoxydiol derivatives as new tubulin and HDAC dual-targeting inhibitors, displaying antitumor and antiangiogenic response

Multi-target drugs design has become an active research field because of their advantages in cancer treatment. In present study, HDAC inhibitors pharmacophore and 2-methoxyestradiol(2ME2) were combined into a new hybrid molecule for the first time. Forty-seven 2ME2 derivatives were synthesized and evaluated for antiproliferative activity. In particular, compound 4s exhibited a dual inhibition of tubulin polymerization and HDAC (IC50 = 0.06 µM toward HDAC2) activity, as well as the most potent cytotoxicity IC50 values of 0.37–4.84 µM against six cancer cell lines. Compound 4s remarkably disrupted microtubule networks, arrested cell cycle at G2/M phase, induced mitochondrial membrane potential collapse and eventually apoptosis in A549 cells. Notably, 4s was discovered to potently imped the tube-formation of HUVECs and prohibited the proliferation, migration, and invasion of HUVECs, as well as A549 cells. In addition, the anti-angiogenic and anti-metastasis activities were demonstrated via a zebrafish model test. All these beneficial anticancer activities together with its high selectivity toward noncancer cells, suggested 4s may deserves consideration for cancer therapy.

Disassembly of microtubules by intense terahertz pulses.

The biological effects of terahertz (THz) radiation have been observed across multiple levels of biological organization, however the sub-cellular mechanisms underlying the phenotypic changes remain to be elucidated. Filamentous protein complexes such as microtubules are essential cytoskeletal structures that regulate diverse biological functions, and these may be an important target for THz interactions underlying THz-induced effects observed at the cellular or tissue level. Here, we show disassembly of microtubules within minutes of exposure to extended trains of intense, picosecond-duration THz pulses. Further, the rate of disassembly depends on THz intensity and spectral content. As inhibition of microtubule dynamics is a mechanism of clinically-utilized anti-cancer agents, disruption of microtubule networks may indicate a potential therapeutic mechanism of intense THz pulses.

Design, synthesis and bioevaluation of 2,7-diaryl-pyrazolo[1,5-a]pyrimidines as tubulin polymerization inhibitors

Two series of 2,7-diaryl-pyrazolo[1,5-a]pyrimidines as tubulin polymerization inhibitors were designed to restrict bioactive configuration of (E,Z)-vinylogous CA-4. All of the target compounds were synthesized and then evaluated for their in vitro antiproliferative activities against three cancer cell lines (MCF-7, SGC-7901 and A549). Among them, 6d exhibited the most potent antiproliferative activity against the MCF-7 with IC50 value of 0.047 μM. Moreover, 6d significantly inhibited tubulin polymerization, disrupted microtubule networks, arrested cell cycle at G2/M phase, induced apoptosis and hindered cancer cell migration. Colchicine competition assay and molecular docking studies suggested that 6d could interact with tubulin by binding to the colchicine site.

Discovery of Novel Diarylamide N-Containing Heterocyclic Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer Activity.

Tubulin has been regarded as an attractive and successful molecular target in cancer therapy and drug discovery. Vicinal diaryl is a simple scaffold found in many colchicine site tubulin inhibitors, which is also an important pharmacophoric point of tubulin binding and anti-cancer activity. As the continuation of our research work on colchicine binding site tubulin inhibitors, we designed and synthesized a series of diarylamide N-containing heterocyclic derivatives by the combination of vicinal diaryl core and N-containing heterocyclic skeletons into one hybrid though proper linkers. Among of these compounds, compound 15b containing a 5-methoxyindole group exhibited the most potent inhibitory activity against the tested three human cancer cell lines (MGC-803, PC-3 and EC-109) with IC50 values of 1.56 μM, 3.56 μM and 14.5 μM, respectively. Besides, the SARs of these compounds were preliminarily studied and summarized. The most active compound 15b produced the inhibition of tubulin polymerization in a dose-dependent manner and caused microtubule network disruption in MGC-803 cells. Therefore, compound 15b was identified as a novel tubulin polymerization inhibitor targeting the colchicine binding site. In addition, the results of molecular docking also suggested compound 15b could tightly bind into the colchicine binding site of β-tubulin.

Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruption

The proper organization of the microtubule-based spindle during cell division requires the collective activity of many different proteins. These include non-motor microtubule-associated proteins (MAPs), whose functions include crosslinking microtubules to regulate filament sliding rates and assemble microtubule arrays. One such protein is PRC1, an essential MAP that has been shown to preferentially crosslink overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps remains unclear. Here, we employ a modified microtubule gliding assay to rupture PRC1-mediated microtubule pairs using surface-bound kinesins. We discovered that PRC1 crosslinks always reduce bundled filament sliding velocities relative to single-microtubule gliding rates and do so via two distinct emergent modes of mechanical resistance to motor-driven sliding. We term these behaviors braking and coasting, where braking events exhibit substantially slowed microtubule sliding compared to coasting events. Strikingly, braking behavior requires the formation of two distinct high-density clusters of PRC1 molecules near microtubule tips. Our results suggest a cooperative mechanism for PRC1 accumulation when under mechanical load that leads to a unique state of enhanced resistance to filament sliding and provides insight into collective protein ensemble behavior in regulating the mechanics of spindle assembly.

Novel Semi-Synthetic Cu (II)-Cardamonin Complex Exerts Potent Anticancer Activity against Triple-Negative Breast and Pancreatic Cancer Cells via Inhibition of the Akt Signaling Pathway.

Cardamonin is a polyphenolic natural product that has been shown to possess cytotoxic activity against a variety of cancer cell lines. We previously reported the semi-synthesis of a novel Cu (II)–cardamonin complex (19) that demonstrated potent antitumour activity. In this study, we further investigated the bioactivity of 19 against MDA-MB-468 and PANC-1 cancer cells in an attempt to discover an effective treatment for triple-negative breast cancer (TNBC) and pancreatic cancer, respectively. Results revealed that 19 abolished the formation of MDA-MB-468 and PANC-1 colonies, exerted growth-inhibitory activity, and inhibited cancer cell migration. Further mechanistic studies showed that 19 induced DNA damage resulting in gap 2 (G2)/mitosis (M) phase arrest and microtubule network disruption. Moreover, 19 generated reactive oxygen species (ROS) that may contribute to induction of apoptosis, corroborated by activation of caspase-3/7, PARP cleavage, and downregulation of Mcl-1. Complex 19 also decreased the expression levels of p-Akt and p-4EBP1, which indicates that the compound exerts its activity, at least in part, via inhibition of Akt signalling. Furthermore, 19 decreased the expression of c-Myc in PANC-1 cells only, which suggests that it may exert its bioactivity via multiple mechanisms of action. These results demonstrate the potential of 19 as a therapeutic agent for TNBC and pancreatic cancer.

Two Modes of PRC1-Mediated Mechanical Resistance to Kinesin-Driven Microtubule Network Disruption

The proper structural organization of the microtubule-based spindle during cell division requires the activity of non-motor microtubule-associated proteins, which crosslink microtubules to regulate filament sliding rates and assemble microtubule arrays. PRC1 is an essential MAP that preferentially crosslinks overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps has been lacking.

Preservation of the microtubule network to beat atrial fibrillation: role of SR-mitochondrial contacts

Abstract Background Atrial fibrillation (AF), the most common tachyarrhythmia, is a progressive disease characterized by electrophathology, which is defined as the structural remodeling of atrial cardiomyocytes that underlies electrophysiological remodeling and contractile dysfunction and consequently AF progression. We recently discovered that disruption of microtubule network, by HDAC6 activation, is a key factor underlying structural remodeling and AF promotion in experimental models for AF and patients with AF. However, the molecular mechanism how microtubule disruption induces contractile dysfunction in AF is unclear. Sarcoplasmic reticulum (SR) and mitochondria are the central organelles for normal cardiomyocyte contraction by controlling the Ca2+ and energy (ATP) homeostasis. The crosstalk between SR and mitochondria via contacts, termed SR-mitochondria contacts (SMCs), is essential for normal mitochondrial and cardiac function. Interestingly, functional SMCs are highly dependent on intact microtubule network. This study aims to whether preservation of microtubule protects against AF via SMC pathway. Methods and results Tachypacing of HL-1 cardiomyocytes significantly impaired calcium transient (CaT) amplitude compared to normal paced cardiomyocytes. Pretreatment with microtubule stalilizer taxol and acetyl-CoA inducer β-hydroxybutyrate (βOHB) significantly protected against tachypacing-induced CaT loss. Moreover, by using immunoflurecent staining, mitochondrial associated membrane isolation and seahorse, we showed that tubacin, Taxol and βOHB also significantly inhibited the SMC reduction, attenuated tachypacing-induced mitochondrial dysfunction. Consistently, these microtubule stabilizer drugs also prevented tachypacing-induced contractile dysfunction in the Drosophila model for AF. Conclusions Preservation of microtubule network prevented the reduction of SMCs and the consequent mitochondrial and contractile dysfunction in HL-1 cardiomyocytes and Drosophila models for AF. Therefore, the microtubule-SMC pathway is a novel central therapeutic target for AF. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Dutch Heart Foundation

Triphala polyphenols-functionalized gold nanoparticles impair cancer cell survival through induction of tubulin dysfunction

Green-nanotechnology has enabled the development of several potential nanomedicines against diseases such as cancer. Triphala is an ayurvedic formulation with anticancer activities. The aim of the study was to synthesize, characterize, and biologically evaluate Trl-polyphenol-coated gold nanoparticles (Trl-GNPs) and to elucidate the mechanism of action of this formulation. Trl-GNPs were synthesized using the lyophilized powder of Trl extract and gold chloride trihydrate, and characterized by an assortment of spectroscopy techniques (UV–visible spectroscopy, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, size- and zeta potential-analysis, and one-dimensional proton nuclear magnetic resonance spectroscopy) and transmission electron microscopy. Cell viability analyses of the triple-negative breast cancer cell line, MDA-MB-231, were carried out using (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. The nature of interactions of the Trl-GNPs with the purified tubulin was studied using spectrofluorimetry and circular dichroism. Microtubule architectural defects were investigated using immunofluorescence microscopy. Trl-GNPs were found to be inhibitory to the proliferative potential of the cells. The antiproliferative mechanism of action of Trl-GNPs involved perturbation of the structural the integrity of the mitotic-spindle-building protein, tubulin, at the secondary and tertiary levels. These disruptions of tubulin manifested as disrupted microtubule network in cells facilitating cell cycle arrest. Our data suggest a potential method for enhancing the delivery of Trl polyphenols to cancer cells, and elucidate the antiproliferative mechanism of action of these particles.

Synthesis, and biological evaluation of 3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine analogues as new potent tubulin polymerization inhibitors

On the basis of our previous work, twenty-nine novel [1,2,4]triazolo[4,3-a]pyridines possessing 3,4,5-trimethoxylphenyl groups were designed, synthesized, and evaluated as tubulin polymerization inhibitors. The bioassay results revealed that some of the compounds displayed excellent antiproliferative efficacies in the nanomolar range against HeLa cells, and the most promising derivative 7i demonstrated almost comparable activity to that of the reference CA-4 and sixty-two fold more potent than the parent compound 6 with an IC50 value of 12 nM. Importantly, 7i exhibited high selectivity over the normal human embryonic kidney HEK-293 cells (IC50 > 100 μM). Further mechanism studies revealed that 7i significantly arrested cell cycle at G2/M phase, induced apoptosis with a dose-dependent manner, and disrupted microtubule networks. Additionally, the most active compound 7i effectively inhibited tubulin polymerization with a value similar to that of CA-4 (3.4 and 4.2 μM, respectively). Furthermore, molecular docking analysis suggested that 7i well occupied the colchicine binding pocket of tubulin. The present study highlights that compound 7i is a novel potential tubulin polymerization inhibitor and deserves further investigation for the treatment of cancers.

Exploring Diverse-Ring Analogues on Combretastatin A4 (CA-4) Olefin as Microtubule-Targeting Agents.

Combretastatin-4 (CA-4) as a tubulin polymerization inhibitor draws extensive attentions. However, due to its weak stability of cis-olefin and poor metabolic stability, structure modifications on cis-configuration are being performed. In this work, we constructed a series of novel CA-4 analogues with linkers on olefin containing diphenylethanone, cis-locked dihydrofuran, α-substituted diphenylethanone, cyclobutane and cyclohexane on its cis-olefin. Cytotoxic activity of all analogues was measured by an SRB assay. Among them, compound 6b, a by-product in the preparation of diphenylethanone analogues, was found to be the most potent cytotoxic agents against HepG2 cells with IC50 values of less than 0.5 μM. The two isomers of 6b induced cellular apoptosis tested by Annexin V-FITC and propidium iodide (PI) double staining, arrested cells in the G2/M phase by PI staining analysis, and disrupted microtubule network by immunohistochemistry study in HepG2 cells. Moreover, 6b-(E) displayed a dose-dependent inhibition effect for tubulin assembly in in vitro tubulin polymerization assay. In addition, molecular docking studies showed that two isomers of 6b could bind efficiently at colchicine binding site of tubulin similar to CA-4.

BF12, a Novel Benzofuran, Exhibits Antitumor Activity by Inhibiting Microtubules and the PI3K/Akt/mTOR Signaling Pathway in Human Cervical Cancer Cells.

BF12 [(2E)-3-[6-Methoxy-2-(3,4,5-trimethoxybenzoyl)-1-benzofuran-5-yl]prop-2-enoic acid], a novel derivative of combretastatin A-4 (CA-4), was previously found to inhibit tumor cell lines, with a particularly strong inhibitory effect on cervical cancer cells. In this study, we investigated the microtubule polymerization effects and apoptosis signaling mechanism of BF12. BF12 showed a potent efficiency against cervical cancer cells, SiHa and HeLa, with IC50 values of 1.10 and 1.06 μm, respectively. The cellular mechanism studies revealed that BF12 induced G2/M phase arrest and apoptosis in SiHa and HeLa cells, which were associated with alterations in the expression of the cell G2/M cycle checkpoint-related proteins (cyclin B1 and cdc2) and alterations in the levels of apoptosis-related proteins (P53, caspase-3, Bcl-2, and Bax) of these cells, respectively. Western blot analysis showed that BF12 inhibited the PI3 K/Akt/mTOR signaling pathway and induced apoptosis in human cervical cancer cells. BF12 was identified as a tubulin polymerization inhibitor, evidenced by the effective inhibition of tubulin polymerization and heavily disrupted microtubule networks in living SiHa and HeLa cells. By inhibiting the PI3 K/Akt/mTOR signaling pathway and inducing apoptosis in human cervical cancer cells, BF12 shows promise for use as a microtubule inhibitor.

Synthesis, molecular properties prediction and biological evaluation of indole-vinyl sulfone derivatives as novel tubulin polymerization inhibitors targeting the colchicine binding site

Twenty-two novel indole-vinyl sulfone derivatives were designed, synthesized and evaluated as tubulin polymerization inhibitors. The physicochemical and drug-likeness properties of all target compounds were predicted by Osiris calculations. All compounds were evaluated for their antiproliferative activities, among them, compound 7f exhibited the most potent activity against a panel of cancer cell lines, which was 2–7 folds more potent than our previously reported compound 4. Especially, 7f displayed about 8-fold improvement of selective index as compared with compound 4, indicating that 7f might have lower toxicity. Besides, 7f inhibited the microtubule polymerization by binding to the colchicine site of tubulin. Further investigations showed that compound 7f effectively disrupted microtubule network, caused cell cycle arrest at G2/M phase and induced cell apoptosis in K562 cells. Moreover, 7f reduced the cell migration and disrupted capillary-like tube formation in HUVEC cells. Importantly, the in vivo anti-tumor activity of 7f was validated in H22 liver cancer xenograft mouse model without apparent toxicity, suggesting that 7f is a promising anti-tubulin agent for cancer therapy.