Wound Healing Activity of Topical Herbal Gels Containing Barringtonia acutangula Fruit Extract: In silico and In vivo Studies.

The current study describes the efficacy of B. acutangula fruit extract in wound healing via incorporation within topical gels. B. acutangula fruit extract was produced by solvent extraction method. The bioactive extract was incorporated within Carbopol 940-based topical gels, which were applied topically over the excision and incision wounds. The change in healing process was observed till 20 days. The percentages of closure of excision wound area were 92.89% and 93.43%, when treated with topical herbal gels containing B. acutangula fruit extract of 5% and 10%, respectively. The tensile strengths of incision area in rats treated with topical herbal gels containing 5% and 10% methanol extract of B. acutangula fruits were found to be 25 ± 5.12 g and 30 ± 4.10 g, respectively. The wound healing activity of topical herbal gels containing B. acutangula fruit extract in rats was found to be significant when compared with that of the reference standard and untreated groups. In addition, in silico studies suggested about good skin permeability and binding to the proteins responsible for delaying wound healing. It can be concluded that this topical herbal gels containing B. acutangula fruit extract could be used clinically for the treatment of wounds.

Exploring the antifilarial potential of an important medicinal plant Typhonium trilobatum (L. Schoot): Isolation, characterization, and structural elucidation of bioactive compounds against Brugia malayi.

ETHNOPHARMACOLOGY RELEVANCE: The plant Typhonium trilobatum has been utilized in traditional medicine for the treatment of many ailments, including parasitic infections. Recent examinations indicate that the bioactive substances from this plant may have antiparasitic activities against Brugia malayi, which have not been determined. PURPOSE: The parasitic nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti causing lymphatic filariasis, remain a significant challenge to global public health. Given the ongoing nature of this enduring menace, the current research endeavours to examine the efficacy of an important medicinal plant, Typhonium trilobatum. METHODS: Different extracts of the T. trilobatum tubers were evaluated for their antiparasitic activity. The most prominent extract was subjected to Gas Chromatography Mass Spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC) followed by Column Chromatography for isolating bioactive molecules. The major compounds were isolated and characterized based on different spectroscopic techniques (FTIR, NMR and HRMS). Further, the antiparasitic activity of the isolated compounds was evaluated against B. malayi and compared with clinically used antifilarial drugs like Diethylcarbamazine and Ivermectin. RESULTS: The methanolic extract of the tuber exhibited significant antiparasitic activity compared to the other extracts. The bioactive molecules isolated from the crude extract were identified as Linoleic acid and Palmitic acid. Antiparasitic activity of both the compounds has been performed against B. malayi and compared with clinically used antifilarial drugs, Ivermectin and DEC. The IC50 value of Linoleic acid was found to be 6.09 ± 0.78 µg/ml after 24 h and 4.27 ± 0.63 µg/ml after 48 h, whereas for Palmitic acid the value was 12.35 ± 1.09 µg/ml after 24 h and 8.79 ± 0.94 µg/ml after 48 h. The IC50 values of both the molecules were found to be similar to the standard drug Ivermectin (IC50 value of 11.88 ± 1.07 µg/ml in 24 h and 2.74 ± 0.43 µg/ml in 48 h), and much better compared to the DEC (IC50 values of 194.2 ± 2.28 µg/ml in 24 h and 101.8 ± 2.06 µg/ml in 48 h). Furthermore, it has been observed that both the crude extracts and the isolated compounds do not exhibit any detrimental effects on the J774.A.1 macrophage cell line. CONCLUSION: The isolation and characterization of bioactive compounds present in the methanolic tuber extract of Typhonium trilobatum were explored. Moreover, the antimicrofilarial activity of the crude extracts and its two major compounds were determined using Brugia malayi microfilarial parasites without any significant side effects.

Biochemical and in silico analysis of the binding mode of erastin with tubulin.

Erastin (ERN) is a small molecule that induces different forms of cell death. For example, it has been reported to induce ferroptosis by disrupting tubulin subunits that maintain the voltage-dependent anion channels (VDACs) of mitochondria. Although its possible binding to tubulin has been suggested, the fine details of the interaction between ERN and tubulin are poorly understood. Using a combination of biochemical, cell-model and in silico approaches, we elucidate the interactions of ERN with tubulin and their biological manifestations. After confirming ERN's antiproliferative efficacy (IC50, 20 ± 3.2 M) and induction of cell death in the breast cancer cell line MDA-MB-231, the binding interactions of ERN with tubulin were examined. ERN bound to tubulin in a concentration-dependent manner, disorganizing the structural integrity of the protein, as substantiated via the tryptophan-quenching assay and the aniline-naphthalene sulfonate binding assay, respectively. In silico studies based on molecular docking revealed a docking score of -5.863 kcal/mol, suggesting strong binding interactions of ERN with tubulin. Additionally, molecular dynamics simulation and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analyses evinced the binding free energy (ΔGbinding) of -31.235 kcal/mol, substantiating strong binding affinity of ERN with tubulin. Ligplot analysis showed hydrogen bonding with specific amino acids (Asn A226, Thr A223, Gln B247 and Val B355). QikProp-based ADME (absorption, distribution, metabolism and excretion) assessment showed considerable therapeutic potential for ERN.Communicated by Ramaswamy H. Sarma.

Variation in Yield, Chemical Composition and Biological Activities of Essential Oil of Three Curcuma Species: A Comparative Evaluation of Hydrodistillation and Solvent-Free Microwave Extraction Methods.

The essential oils of three medicinally important Curcuma species (Curcuma alismatifolia, Curcuma aromatica and Curcuma xanthorrhiza) were extracted using conventional hydro-distillation (HD) and solvent free microwave extraction (SFME) methods. The volatile compounds from the rhizome essential oils were subsequently analysed by GC-MS. The isolation of essential oils of each species was carried out following the six principles of green extraction and comparison was made between their chemical composition, antioxidant, anti-tyrosinase and anticancer activities. SFME was found to be more efficient than HD in terms of energy savings, extraction time, oil yield, water consumption and waste production. Though the major compounds of essential oils of both the species were qualitatively similar, there was a significant difference in terms of quantity. The essential oils extracted through HD and SFME methods were dominated by hydrocarbon and oxygenated compounds, respectively. The essential oils of all Curcuma species exhibited strong antioxidant activity, where SFME was significantly better than HD with lower IC50 values. The anti-tyrosinase and anticancer properties of SFME-extracted oils were relatively better than that of HD. Further, among the three Curcuma species, C. alismatifolia essential oil showed the highest rates of inhibition in DPPH and ABTS assay, significantly reduced the tyrosinase activity and exhibited significant selective cytotoxicity against MCF7 and PC3 cells. The current results suggested that the SFME method, being advanced, green and fast, could be a better alternative for production of essential oils with better antioxidant, anti-tyrosinase and anticancer activities for application in food, health and cosmetic industries.

Deciphering the antibiofilm potential of 2-Phenylethyl methyl ether (PEME), a bioactive compound of Kewda essential oil against Staphylococcus aureus.

Opportunistic pathogenic bacteria and their pathogenicity linked with biofilm infections become a severe issue as they resist the actions of multiple antimicrobial drugs. Naturally derived drugs having antibiofilm properties are more effective than chemically synthesized drugs. The plant derived essential oils are a rich source of phytoconstituents with widespread pharmacological values. In the present investigation, a major phytoconstituent, 2-Phenyl Ethyl Methyl Ether (PEME) of Kewda essential oil extracted from the flowers of Pandanus odorifer was explored for its prospective antimicrobial and anti-biofilm properties against ESKAPE pathogenic bacterial strains, Staphylococcus aureus and MTCC 740. The minimum inhibitory concentration (MIC) of PEME was found to be 50 mM against the tested bacterial strains. A gradual decrease in biofilm production was observed when PEME was treated with the sub-MIC concentration. The reduction in biofilm formation was noticeable from qualitative assay i.e., Congo Red Agar Assay (CRA) and further quantified by crystal violet staining assay. The decline in exopolysaccharides production was quantified, with the highest inhibition against MTCC 740 with a decrease of 71.76 ± 4.56% compared to untreated control. From the microscopic analysis (light and fluorescence microscopic method), PEME exhibited inhibitory effect on biofilm formation on the polystyrene surface. The in silico studies stated that PEME could invariably bind to biofilm associated target proteins. Further, transcriptomic data analysis suggested the role of PEME in the down-regulation of specific genes, agrA, sarA, norA and mepR, which are critically associated with bacterial virulence, biofilm dynamics and drug resistance patterns in S. aureus. Further, qRT-PCR analysis validated the role of PEME on biofilm inhibition by relative downregulation of agrA, sarA, norA and mepR genes. Further, advanced in silico methodologies could be employed in future investigations to validate its candidature as promising anti-biofilm agent.

Development of 9-(N-arylmethylamino) congeners of noscapine: the microtubule targeting drugs for the management of breast cancer.

Noscapine is a natural lead molecule with anticancer activity at a higher concentrations. So, there is an urge for the development of more potent derivatives of noscapine. In this study, we have approached for development of 9-N-arylmethylamino derivatives of noscapine that kills cancer cells without affecting the normal cells. They were designed by substituting N-aryl methyl pharmacophore at the C-9 position and screened out top-ranked three derivatives 13a-c using molecular docking. Further, their theoretical free energy of binding with tubulin was calculated followed by chemical synthesis and experimental validation. In vitro antiproliferative activity of noscapine and its 9-N-arylmethylamino derivatives (13a-c) was carried out using MCF-7 (a triple receptors positive) and MDA-MB-231 (a triple receptor negative) breast cancer cell lines. Further, cytotoxicity to normal cells was examined using human embryonic kidney cells (HEK cells). Inhibition to cell cycle progression and induction of apoptosis was monitored using FACS. The binding of noscapine and 13a-c with tubulin was examined using fluorescence quenching assay. The 9-N-arylmethylamino derivatives of noscapine (13a-c) were found to inhibit the proliferation of cancer cells at a much lower concentration (IC50 values range between 9.1 to 47.3 µM) compared to noscapine (IC50 value is 45.8-59.3 µM). Surprisingly, the proliferation of HEK cells was not inhibited even at a concentration of 100 µM (cytotoxicity is < 5%). These derivatives induced apoptosis by arresting cells at G2/M-phase and also bind to tubulin. The 9-(N-arylmethylamino) noscapinoids have the potential to be a novel therapeutic agent for the treatment of breast cancer. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03445-3.

Identification of potent EGFR-TKD inhibitors from NPACT database through combined computational approaches.

Cancer is the world's second leading cause of death, and there are no approved herbal therapies. The epidermal growth factor receptor tyrosine kinase (EGFR-TK) receptor is a transmembrane protein with eight domains that is found in almost every cancer type and plays an important role in abnormal cell cellular function and causes malignant outcomes. The current study aimed to virtually screen phytochemicals from the NPACT database against EGFR-TKD and also to identify potential inhibitors of this transmembrane protein among plant candidates for anticancer drug development. The docking scores of the chosen phytochemicals were compared with the control (erlotinib). Kurarinone, (2S)-2-methoxykurarnione, and Sophoraflavanone-G exhibited a stronger binding affinity of -18.102 kcal/mol, -14.243 kcal/mol, and -13.759 kcal/mol than erlotinib -12.783 kcal/mol. Moreover, several online search engines were used to predict ADME and toxicity. The drug-likeness of selected phytochemicals was higher than the reference (erlotinib). A 100 ns molecular dynamic (MD) simulation was also applied to the docked conformations to examine the stability and molecular mechanics of protein-ligand interactions. Furthermore, the calculated molecular mechanics Poisson Boltzmann surface area energy of (2S)-2-methoxykurarnione was found to be -129.555 ± 0.512 kJ/mol, which approximately corresponds to the free energy of the reference molecule -130.595 ± 0.908 kJ/mol. We identify phytoconstituents present in Sophora flavescens from the NPACT database, providing key insights into tyrosine kinase inhibition and may serve as better chemotherapeutic agents. Experimental validation is required to determine the anti-EGFR potency of the potent lead molecules discussed in this study.Communicated by Ramaswamy H. Sarma.

In vitro characterization and molecular dynamic simulation of shikonin as a tubulin-targeted anticancer agent.

Shikonin (SK), a naphthoquinone compound from the purple gromwell, Lithospermum erythrorhizon, possesses a considerable antiproliferative potential. By using a combination of biophysical techniques, cellular assays, immunofluorescence imaging, and molecular dynamic simulation, we identified a possible mechanism of action of SK. SK inhibited the viability of the triple negative breast cancer cells MDA-MB-231 (IC50 of 1 ± 0.1 µM), and its inhibitory effect was irreversible. It strongly suppressed the clonogenic and migratory potential of the cells. Although SK did not show any phase-specific inhibition of cell cycle progression, it induced apoptosis as confirmed by annexin-V-based flow cytometry and Western immunoblotting of PARP1. Probing further into its mechanism using a tryptophan-quenching assay, it was found that SK binds the microtubule-building protein tubulin with a dissociation constant (Kd) of 8 ± 2.7 µM, without grossly damaging the tertiary structure of the protein. The drug-bound tubulin could not assemble microtubules properly in vitro as confirmed by polymer mass analysis, turbidimetry analysis, and transmission electron microscopy, and in cells, as visualized by immunofluorescence imaging. In cells, SK also suppressed the dynamicity of microtubules as indicated by considerable acetylation of the cellular microtubules. The fine details of tubulin-SK interactions were then elucidated using molecular docking and molecular dynamic simulation. The free energy change of the interaction (ΔGbind,pred) was found to be -14.60 kcal/mol and the binding involved both the intermolecular van der Waals (ΔEvdw) and the electrostatic (ΔEele) interactions. Taken together, our data provide evidence for a possible mechanism of action of SK as a tubulin-targeted anticancer agent.

Biology and biotechnological aspect of sweet potato (Ipomoea batatas L.): a commercially important tuber crop.

MAIN CONCLUSION: This review highlights the economic importance of sweet potato and discusses new varieties, agronomic and cultivation practices, pest and disease control efforts, plant tissue culture protocols, and unexplored research areas involving this plant. Abstract Sweet potato is widely consumed in many countries around the world, including India, South Africa and China. Due to its valuable nutritional composition and highly beneficial bioactive compounds, sweet potato is considered a major tuber crop in India. Based on the volume of production, this plant ranks seventh in the world among all food crops. Sweet potato is considered a "Superfood" by the 'Centre for Science in the Public Interest' (CSPI), USA. This plant is mostly propagated through vegetative propagation using vine cuttings or tubers. However, this process is costly, labour-intensive, and comparatively slow. Conventional propagation methods are not able to supply sufficient disease-free planting materials to farmers to sustain steady tuber production. Therefore, there is an urgent need to use various biotechnological approaches, such as cell, tissue, and organ culture, for the large-scale production of healthy and disease-free planting material for commercial purposes throughout the year. In the last five decades, a number of tissue culture protocols have been developed for the production of in vitro plants through meristem culture, direct adventitious organogenesis, callus culture and somatic embryogenesis. Moreover, little research has been done on synthetic seed technology for the in vitro conservation and propagation of sweet potato. The current review comprehensively describes the biology, i.e., plant phenotypic description, vegetative growth, agronomy and cultivation, pests and diseases, varieties, and conventional methods of propagation, as well as biotechnological implementation, of this tuber crop. Furthermore, the explored and unexplored areas of research in sweet potato using biotechnological approaches have been reviewed.

Combination of docetaxel and newly synthesized 9-Br-trimethoxybenzyl-noscapine improve tubulin binding and enhances antitumor activity in breast cancer cells.

To strategically design and frame the novel 9-Br-Trimethoxybenzyl noscapine (BTN) with rigorous binding affinity with tubulin, the structure of noscapine (an antitussive plant alkaloid) was amended with a 3,4,5-trimethoxybenzyl group linked at the seventh position on the lower isobenzofuran unit. Molecular modelling and cellular studies were used to assess the single and combined effects of BTN and docetaxel (DOX). Based on MM-GBSA, the individual calculated free energies of binding (ΔGbind, pred) for BTN and DOX with tubulin was found to be -25.69 and -38.17 kcal/mol, respectively, and -29.11 and -36.60 kcal/mol based on MM-PBSA. Furthermore, the ΔGbind,pred of BTN was dramatically reduced (-30.02 and -33.54 kcal/mol using MM-GBSA and MM-PBSA) in presence of DOX on its binding pocket. Parenthetically, the ΔGbind,pred of DOX was substantially decreased (-39.17 and -35.80 kcal/mol using MM-GBSA and MM-PBSA) in the presence of BTN on its binding pocket. The synergistic activity of both compounds on tubulin dimmer was also analysed using purified tubulin, where a combined regimen of BTN and DOX attenuated tubulin intensity to a higher value (50%) particularly in comparison to the single regimen. In comparison to the single regimen, the combination of BTN and DOX effectively prevents cell cycle progression during the G2/M phase and induces breast cancer cell death. Female athymic nude mice were xenografted with MCF-7 cells and the efficacy of (150 mg/kg/day), DOX (1.5 mg/kg/week, i.v.), or in combination (BTN 300 mg/kg/day + DOX 1.0 mg/kg/week, i.v) were evaluated.

Synergistic interaction of N-3-Br-benzyl-noscapine and docetaxel abrogates oncogenic potential of breast cancer cells.

Noscapine, an opium alkaloid, was discovered to bind tubulin, arrest dividing cells at mitosis, and selectively induce apoptosis to cancer cells. N-3-Br-Benzyl-Noscapine (Br-Bn-Nos), one of the derivatives of noscapine, was demonstrated to have improved anticancer potential compared with noscapine. We approached to evaluate the single and combined effect of Br-Bn-Nos and docetaxel (DOX) based on molecular modeling and cellular study. The individual predicted free energy of binding (∆Gbind,pred ) for Br-Bn-Nos and DOX with tubulin was found to be -28.89 and -36.07 kcal/mol based on molecular mechanics generalized Born solvation area (MM-GBSA) as well as -26.21 and -34.65 kcal/mol based on molecular mechanics Poisson Boltzmann solvation area (MM-PBSA), respectively. However, the ∆Gbind,pred of Br-Bn-Nos was significantly reduced (-33.02 and -30.24 kcal/mol using MM-GBSA and MM-PBSA) in the presence of DOX on its binding pocket. Parenthetically, the ∆Gbind,pred of DOX was significantly reduced (-37.17 and -32.80 kcal/mol using MM-GBSA and MM-PBSA) in the presence of Br-Bn-Nos on its binding pocket. The reduced ∆Gbind,pred in the presence of Br-Bn-Nos and DOX together indicated a combination effect of both the ligands. The combined interaction of both the agents onto tubulin dimmer was also determined experimentally using purified tubulin, in which a combination regimen of Br-Bn-Nos and DOX reduced the fluorescence intensity of tubulin to a higher value (68%) compared with the single regimen. Further, isobologram analysis revealed the synergistic effect of Br-Bn-Nos and DOX in antiproliferative activity using MCF-7 cell line at 48 hr (sum FIC = 0.49) and at 72 hr (sum FIC = 0.62). The combination dose regimen of Br-Bn-Nos and DOX blocks the cell cycle progression at the G2/M phase and induces apoptosis to cancer cells more effectively compared with the single regimen. Taken together, our study provides compelling evidence that the anticancer potential of noscapine derivatives may be substantially improved when it is used in a combined application with DOX for breast cancer.

Rational design, chemical synthesis and cellular evaluation of novel 1,3-diynyl derivatives of noscapine as potent tubulin binding anticancer agents.

We present a new class of derivatives of noscapine, 1,3-diynyl-noscapinoids of an antitussive plant alkaloid, noscapine based on our in silico efforts that binds tubulin and displays anticancer activity against a panel of breast cancer cells. Structure-activity analyses pointed the C-9 position of the isoquinoline ring which was modified by coupling of 1,3-diynyl structural motifs to rationally design and screened a series of novel 1,3-diynyl-noscapinoids (20-22) with robust binding affinity with tubulin. The selected 1,3-diynyl-noscapinoids, 20-22 revealed improved predicted binding energy of -6.568 kcal/mol for 20, -7.367 kcal/mol for 21 and -7.922 kcal/mol for 22, respectively in comparison to the lead molecule (-5.246 kcal/mol). These novel derivatives were chemically synthesized and validated their anticancer activity based on cellular studies using two human breast adenocarcinoma, MCF-7 and MDAMB-231, as well as with a panel of primary breast cancer cells isolated from patients. Interestingly, all these derivatives inhibited cellular proliferation in all the cancer cells that ranged between 6.2 to 38.9 µM, which is 6.7 to 1.5 fold lower than that of noscapine. Unlike previously reported derivatives of noscapine that arrests cells in the S-phase, these novel derivatives effectively inhibit proliferation of cancer cells, arrests cell cycle in the G2/M-phase followed by apoptosis and appearance of apoptotic cells. Thus, we conclude that 1,3-diynyl-noscapinoids have great potential to be a novel therapeutic agent for breast cancers.

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.

Smart polymeric eye gear: A possible preventive measure against ocular transmission of COVID-19.

The angiotensin-converting enzyme 2(ACE-2) receptors with approx. 0.8% congestion in conjunctival surface, leads to increase susceptibility of Covid-19 transmission through ocular surface. It has been observed that prophylactic measures such as goggle or face shield are unable to offer complete protection against ocular transmission of SRS-CoV-2. Hence, it is hypothesized that topical ocular prophylaxis using biocompatible polymers with reported in-vitro and in-vivo evidence of ACE inhibition and antiviral activity appears to be a promising strategy for preventing ocular transmission of Covid-19 to healthcare workers. They are capable of binding to ACE-2 receptors which may provide highly potential trails to block virus entry to host cells. Further biopolymers imparting antiviral activities greatly improve their protective performance. They not only provide prolong protection but also are safe for long-term use. This article discusses the description of structural and functional attributes of ACE-2 to identify appropriate polymer with better binding affinity. Furthermore, potential polymers with appropriate concentration are suggested for evaluation through a hypothesis to consider them for Covid-19 implication.

Mucoadhesive formulations: innovations, merits, drawbacks, and future outlook.

Mucosa has now been recognized as a potential site for both local and systemic delivery of therapeutics. Mucoadhesive drug delivery systems with customizable release profiles have recently gained considerable interest among formulation scientists to improve clinical outcomes of drugs. This review summarizes the current development in the processing methods and polymers involved in mucoadhesive drug delivery systems. Mucoadhesive drug delivery systems are suitable for drugs that have a localized effect, undergo extensive pre-systemic metabolism, narrow absorption window, and narrow therapeutic index. Polymer characteristics like surface charge, hydrophilic surface groups, wettability, molecular weight, chain flexibility, molecular conformations, etc. are critical for assessing the extent of mucoadhesiveness and treatment response. The current review focuses on valuable principles, merits, drawbacks, and future outlooks of different mucoadhesive drug delivery systems.

Elucidation of the anticancer potential and tubulin isotype-specific interactions of ß-sitosterol.

Beta-sitosterol (ß-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the 'SITO-site') compared to the colchicine binding site at the interface of α- and ß-tubulin. In this study, we investigated the anticancer efficacy of ß-SITO against invasive breast carcinoma using MCF-7 cells. Since 'isotypes' of ß-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of ß-tubulin in αß-tubulin dimer. ß-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of ß-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to ß-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that ß-SITO preferentially binds with specific ß-tubulin isotypes (ßII and ßIII) in the αß-tubulin dimer. Both these ß-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of ß-SITO, and its potential clinical utility against ßII and ßIII isotype-overexpressing neoplasms.

Biochemical characterization and molecular dynamic simulation of ß-sitosterol as a tubulin-binding anticancer agent.

Βeta-sitosterol (ß-SITO), a phytosterol present in pomegranate, peanut, corn oil, almond, and avocado, has been recognized to offer health benefits and potential clinical uses. ß-SITO is orally bioavailable and, as a constituent of edible natural products, is considered to have no undesired side effects. It has also been considered as a potent anticancer agent. However, the molecular mechanism of action of ß-SITO as a tubulin-binding anticancer agent and its binding site on tubulin are poorly understood. Using a combination of biochemical analyses and molecular dynamic simulation, we investigated the molecular details of the binding interactions of ß-SITO with tubulin. A polymer mass assay comparing the effects of ß-SITO and of taxol and vinblastine on tubulin assembly showed that this phytosterol stabilized microtubule assembly in a manner similar to taxol. An 8-anilino-1-naphthalenesulfonic acid assay confirmed the direct interaction of ß-SITO with tubulin. Although ß-SITO did not show direct binding to the colchicine site on tubulin, it stabilized the colchicine binding. Interestingly, no sulfhydryl groups of tubulin were involved in the binding interaction of ß-SITO with tubulin. Based on the results from the biochemical assays, we computationally modeled the binding of ß-SITO with tubulin. Using molecular docking followed by molecular dynamic simulations, we found that ß-SITO binds tubulin at a novel site (which we call the 'SITO site') adjacent to the colchicine and noscapine sites. Our data suggest that ß-SITO is a potent anticancer compound that interferes with microtubule assembly dynamics by binding to a novel site on tubulin.

Molecular modeling reveals binding interface of γ-tubulin with GCP4 and interactions with noscapinoids.

The initiation of microtubule assembly within cells is guided by a cone shaped multi-protein complex, γ-tubulin ring complex (γTuRC) containing γ-tubulin and atleast five other γ-tubulin-complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ-tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/ß-tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ-tubulin and GCP4 through computational techniques. We simulated two complexes of γ-tubulin-GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of -158.82 and -170.19 kcal/mol for dimer1 and -79.53 and -101.50 kcal/mol for dimer2 using MM-PBSA and MM-GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ-tubulin. From the results of the free-energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ-tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ-tubulin-GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ-tubulin-GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo-noscapine followed by noscapine and amino-noscapine. This offers a novel chemical scaffold for γ-tubulin binding drugs near γ-tubulin-GCP4 interface.

Taking aim at a dynamic target: Noscapinoids as microtubule-targeted cancer therapeutics.

Noscapine and its synthetic derivatives called noscapinoids have been shown to possess potential anticancer properties. These alkaloids target microtubules and inhibit cell proliferation. Noscapinoids are microtubule poisons that induce minor alterations in the innate dynamic instability of microtubules leading to mitotic arrest and cell death. Over the past decade, a number of noscapine derivatives have been synthesized that, compared to the parent compound, show superior anticancer potential, enhanced tumor specificity and tumor regression, and little or no toxicity to normal tissues. Based on their successive synthetic modifications at different points in the scaffold structure of noscapine, aided by computational design and structure-activity relationship studies, the derivatives of noscapine have been classified into different "generations" based on modifications. Several studies have reported the potential to develop noscapinoids as anticancer drugs. Increasing their tumor specificity - either through antibody conjugation or nanoparticle-based carriers - may facilitate the progression of maytansinoid-based cancer drugs to the clinic.