Microtubules and Cell Division: Potential Pharmacological Targets in Cancer Therapy.

Microtubules are a well-known target in cancer chemotherapy because of their critical role in cell division. Chromosome segregation during mitosis depends on the establishment of the mitotic spindle apparatus through microtubule dynamics. The disruption of microtubule dynamics through the stabilization or destabilization of microtubules results in the mitotic arrest of the cells. Microtubule-targeted drugs, which interfere with microtubule dynamics, inhibit the growth of cells at the mitotic phase and induce apoptotic cell death. The principle of microtubule-targeted drugs is to arrest the cells at mitosis and reduce their growth because cancer is a disease of unchecked cell proliferation. Many anti-microtubule agents produce significant inhibition of cancer cell growth and are widely used as chemotherapeutic drugs for the treatment of cancer. The drugs that interact with microtubules generally bind at one of the three sites vinblastine site, taxol site, or colchicine site. Colchicine binds to the interface of tubulin heterodimer and induces the depolymerization of microtubules. The colchicine binding site on microtubules is a much sought-after target in the history of anti-microtubule drug discovery. Many colchicine-binding site inhibitors have been discovered, but their use in the treatment of cancer is limited due to their dose-limiting toxicity and resistance in humans. Combination therapy can be a new treatment strategy to overcome these drawbacks of currently available microtubule-targeted anticancer drugs. This review discusses the significance of microtubules as a potential pharmacological target for cancer and stresses the necessity of finding new microtubule inhibitors to fight the disease.

MD simulation-based screening approach identified tolvaptan as a potential inhibitor of Eg5.

We discovered tolvaptan as a new Eg5 inhibitor using molecular dynamics simulation-based virtual screening. The Eg5-monastrol, Eg5-ispinesib, and Eg5-STLC complexes with "closed" L5 conformation obtained in MD simulation were used to generate a combined pharmacophore model, and this model was used during the process of virtual screening. Further, the MD simulation for 1 µs showed that the binding of tolvaptan to Eg5 was stable due to the closure of the α2/L5/α3 pocket. Tolvaptan belongs to the class of drugs called vaptans which are non-peptide vasopressin receptor antagonists. Since our virtual search for mitotic inhibitors identified tolvaptan as a potential candidate, we were interested in unraveling its antimitotic mechanism. Tolvaptan bound to purified Eg5-437H with a dissociation constant of 27 ± 3.8 µM. Tolvaptan inhibited the growth of HeLa cells through the mitotic block, and around 70% of these mitotic cells exhibited a characteristic monopolar spindle. Tolvaptan bound to goat brain tubulin with a dissociation constant of 103 ± 13 µM. The binding location of tolvaptan on tubulin overlapped with that of colchicine, according to molecular docking analysis. The combination of tolvaptan with STLC augmented mitotic bock with monopolar cells, whereas its combination with vinblastine increased mitotic block with bipolar cells. Since tolvaptan is found to have a significant cytotoxic effect on HeLa cells, it can be developed as a prospective anticancer agent either alone or in combination with other antimitotic drugs. Tolvaptan was identified as an inhibitor of Eg5 in a MD simulation-based virtual screening using a combined pharmacophore model.

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

Tioconazole is one of the drugs used to treat topical mycotic infections. It exhibited severe toxicity during systemic administration; however, the molecular mechanism behind the cytotoxic effect was not well established. We employed HeLa cells as a model to investigate the molecular mechanism of its toxicity and discovered that tioconazole inhibited HeLa cell growth through mitotic block (37%). At the half-maximal inhibitory concentration (≈ 15 µM) tioconazole apparently depolymerized microtubules and caused defects in chromosomal congression at the metaphase plate. Tioconazole induced apoptosis and significantly hindered the migration of HeLa cells. Tioconazole bound to goat brain tubulin (K d, 28.3 ± 0.5 µM) and inhibited the assembly of microtubules in the in vitro assays. We report for the first time that tioconazole binds near to the colchicine site, based on the evidence from in vitro tubulin competition experiment and computational analysis. The conformation of tubulin dimer was found to be "curved" upon binding with tioconazole in the MD simulation. Tioconazole in combination with vinblastine synergistically inhibited the growth of HeLa cells and augmented the percentage of mitotic block by synergistically inhibiting the assembly of microtubules. Our study indicates that the systemic adverse effects of tioconazole are partly due to its effects on microtubules and cell cycle arrest. Since tioconazole is well tolerated at the topical level, it could be developed as a topical anticancer agent in combination with other systemic anticancer drugs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00516-w.

Sertaconazole induced toxicity in HeLa cells through mitotic arrest and inhibition of microtubule assembly.

Econazole, miconazole, and sertaconazole, the structurally related azoles with imidazole moiety, were evaluated for their cytotoxicity and their ability to bind to mammalian tubulin. Our results indicated that sertaconazole and econazole bound to goat brain tubulin with a dissociation constant of 9 and 19 µM respectively, while miconazole did not bind to goat brain tubulin. Econazole, miconazole, and sertaconazole inhibited the proliferation of HeLa cells with an IC50 of 28, 98, and 38 µM respectively with sertaconazole alone inducing a mitotic block in the treated cells. Since sertaconazole bound to goat brain tubulin with higher affinity and blocked the cells at mitosis, we hypothesized that its cytotoxic mechanism might involve inhibition of tubulin and econazole which did not block the cells at mitosis may have additional targets than tubulin. Sertaconazole inhibited the polymerization of tubulin in HeLa cells and the in vitro assembled goat brain tubulin. Competitive tubulin-binding assay using colchicine and computational simulation studies showed that sertaconazole bound closer to the colchicine site and induced the tubulin dimer to adopt a "bent" conformation which is incompetent for the polymerization. Results from RT-PCR analysis of the A549 cells treated with sertaconazole indicated activation of apoptosis. Sertaconazole significantly inhibited the migration of HeLa cells and showed synergistic antiproliferative potential with vinblastine. Collectively, the results suggest that sertaconazole which is already in clinical practice could be useful as a topical chemotherapy agent for the treatment of skin cancers in combination with other systemic anticancer agents.

Benserazide Perturbs Kif15-kinesin Binding Protein Interaction with Prolonged Metaphase and Defects in Chromosomal Congression: A Study Based on in silico Modeling and Cell Culture.

The interaction of Kif15 with kinesin binding protein (KBP) is critical for its microtubule localization, bundling of kinetochore microtubules and proper alignment of chromosomes at the metaphase plate. The Kif15-KBP structure was prepared from the crystal structure of Kif15 and nonhomologous model of KBP through docking. Benserazide was retrieved when we did a screening of the ZINC Drug Database using the pharmacophore model generated from the potential binding site on Kif15 in an effort to identify molecules for repurposing as Kif15 inhibitors. Live cell imaging of HeLa cells revealed that benserazide delayed metaphase to anaphase-onset by 47±10 min compared to control cells. Benserazide treatment perturbed the kinetochore and microtubule interaction and inhibited the proliferation of HeLa cells with an IC50 of 101 µM with a mitotic block of 12 %. It did not bind to tubulin in the in vitro assays suggesting that the observed effects could be due to its perturbation of Kif15-KBP interaction.

Zerumbone, a cyclic sesquiterpene, exerts antimitotic activity in HeLa cells through tubulin binding and exhibits synergistic activity with vinblastine and paclitaxel.

OBJECTIVES: The aim of this study was to elucidate the antimitotic mechanism of zerumbone and to investigate its effect on the HeLa cells in combination with other mitotic blockers. MATERIALS AND METHODS: HeLa cells and fluorescence microscopy were used to analyse the effect of zerumbone on cancer cell lines. Cellular internalization of zerumbone was investigated using FITC-labelled zerumbone. The interaction of zerumbone with tubulin was characterized using fluorescence spectroscopy. The Chou and Talalay equation was used to calculate the combination index. RESULTS: Zerumbone selectively inhibited the proliferation of HeLa cells with an IC50 of 14.2 ± 0.5 µmol/L through enhanced cellular uptake compared to the normal cell line L929. It induced a strong mitotic block with cells exhibiting bipolar spindles at the IC50 and monopolar spindles at 30 µmol/L. Docking analysis indicated that tubulin is the principal target of zerumbone. In vitro studies indicated that it bound to goat brain tubulin with a Kd of 4 µmol/L and disrupted the assembly of tubulin into microtubules. Zerumbone and colchicine had partially overlapping binding site on tubulin. Zerumbone synergistically enhanced the anti-proliferative activity of vinblastine and paclitaxel through augmented mitotic block. CONCLUSION: Our data suggest that disruption of microtubule assembly dynamics is one of the mechanisms of the anti-cancer activity of zerumbone and it can be used in combination therapy targeting cell division.

Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine.

Indirubin, a bis-indole alkaloid used in traditional Chinese medicine has shown remarkable anticancer activity against chronic myelocytic leukemia. The present work was aimed to decipher the underlying molecular mechanisms responsible for its anticancer attributes. Our findings suggest that indirubin inhibited the proliferation of HeLa cells with an IC50 of 40 µM and induced a mitotic block. At concentrations higher than its IC50, indirubin exerted a moderate depolymerizing effect on the interphase microtubular network and spindle microtubules in HeLa cells. Studies with goat brain tubulin indicated that indirubin bound to tubulin at a single site with a dissociation constant of 26 ±â€¯3 µM and inhibited the in vitro polymerization of tubulin into microtubules in the presence of glutamate as well as microtubule-associated proteins. Molecular docking analysis and molecular dynamics simulation studies indicate that indirubin stably binds to tubulin at the interface of the α-ß tubulin heterodimer. Further, indirubin stabilized the binding of colchicine on tubulin and promoted the cysteine residue modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating towards alteration of tubulin conformation upon binding. In addition, we found that indirubin synergistically enhanced the anti-mitotic and anti-proliferative activity of vinblastine, a known microtubule-targeted agent. Collectively our studies indicate that perturbation of microtubule polymerization dynamics could be one of the possible mechanisms behind the anti-cancer activities of indirubin.

Biochemical and Biophysical characterization of curcumin binding to human mitotic kinesin Eg5: Insights into the inhibitory mechanism of curcumin on Eg5.

In this study we have characterized the biochemical and biophysical interactions of curcumin with the mitotic kinesin Eg5 which plays a pivotal role in the separation of centrosomes during cell division. Curcumin bound to the purified Eg5 (Eg5-437H) with a Kd value of 7.8µM. The temperature dependent binding analysis and evaluation of thermodynamic parameters indicated the involvement of static quenching mechanism in the binding process. Evidences from competition experiment with monastrol indicated that curcumin bound to Eg5 at a novel druggable site. Using Förster resonance energy transfer the distance between curcumin and monastrol binding site from TRP127 on Eg5-437H was found to be 33Å and 17Å respectively. Curcumin inhibited the ATPase activity of Eg5 motor and perturbed the dynamic interactions between Eg5 and microtubules. Results from circular dichroism studies and molecular dynamics simulations suggest that curcumin binding might perturb the Eg5-437H secondary structure which could be the reason behind its inhibitory effects on Eg5. Cell culture studies performed in HeLa cells indicated that curcumin potentiated the mitotic arrest and monopolar spindle formation in synergism with monastrol, indicating that both ligands could bind simultaneously to the same target.

Dihydropyrazole and dihydropyrrole structures based design of Kif15 inhibitors as novel therapeutic agents for cancer.

Mitotic Kinesin motors, Eg5 and Kif15, have recently emerged as good targets for cancer as they play an inevitable role during mitosis. But, most of the Eg5 inhibitors were found ineffective when the cancer cells develop resistance to them by escalating the expression of Kif15 as alternative to Eg5. Therefore, the drugs that target Kif15 became necessary to be used either as a single or in combination with Eg5 inhibitors. The present study used 39 dihydropyrazole and 13 dihydropyrrole derivatives that were having in vitro inhibitory potential against kinesin motors to develop a common pharmacophore hypothesis AHRR and atom-based QSAR model. The model was used for virtual screening of ZINC database and the resultant hits were docked against Kif15. The four drug candidates with high docking score were examined for their activity and pharmacokinetic behaviour. Based on the results these drugs could be considered as lead candidates in further drug development for cancer.

Structure-Activity Relationship Study Reveals Benzazepine Derivatives of Luteolin as New Aldose Reductase Inhibitors for Diabetic Cataract.

Hyperglycaemia in diabetic patients causes diverse range of complications and the earliest among them is diabetic cataract. The role of aldose reductase, the key enzyme in polyol pathway, is well known in the genesis of cataract in chronic diabetic patients. Controlling of sorbitol flux into lens epithelial cells through aldose reductase inhibitors is an important treatment strategy. Due to the side effects of many drugs so far developed, the development of aldose reductase inhibitors from natural sources has gained considerable attention. This study was undertaken to identify suitable drugs for diabetic cataract using molecular modeling and simulation methods. A series of 18 luteolin derivatives having in vitro inhibitory potential against aldose reductase was used to develop a common pharmacophore hypothesis AHRRR and atom-based 3D-QSAR model. The model was used for virtual screening of ZINC database and the resultant hits were docked against aldose reductase. The two drug candidates which belonged to benzazepine class of drugs scored high in the molecular docking. They were further examined for their activity and pharmacokinetic behaviour. Their druglikeness behaviour was found suitable to be used as drugs as per Lipinski's rule of five criteria. Human intestinal absorption (HIA), skin permeability (SP), blood brain barrier (BBB) penetration and plasma protein binding (PPB) was found to be in the acceptable range. Based on the results, these drugs could be considered as potential candidates in further drug development against diabetic cataract.

Parmotrema tinctorum exhibits antioxidant, antiglycation and inhibitory activities against aldose reductase and carbohydrate digestive enzymes: an in vitro study.

This study evaluated the inhibitory potential of ethyl acetate extract of Parmotrema tinctorum (PTEE), an edible lichen, against aldose reductase (AR) and carbohydrate digestive enzymes such as α-glucosidase and α-amylase. It was also screened for antioxidant activities by using DPPH, ABTS, superoxide and hydroxyl radical-scavenging assays. PTEE exhibited α-glucosidase, α-amylase and AR inhibition along with significant antiglycation potential with an estimated IC50 value of 58.45 ± 1.24, 587.74 ± 3.27, 139.28 ± 2.6 and 285.78 ± 1.287 µg/mL, respectively. Antioxidant activity of PTEE against DPPH (IC50 396.83 ± 2.98 µg/mL), ABTS (151.34 ± 1.79 µg/mL), superoxide (30.29 ± 1.17 µg/mL) and hydroxyl (35.42 ± 1.22 µg/mL) radicals suggests the antioxidant potential of P. tinctorum. Significant antioxidant activity and inhibitory potential against carbohydrate digestive enzymes and AR suggest that P. tinctorum can be developed as functional food/nutraceuticals for diabetes after detailed study.