Tubulin Polymerization Inhibitors Research Articles

P03.11.B MITOTIC ENRICHMENT AS AN EFFICIENT RADIOSENSITIZATION STRATEGY FOR INTRACRANIAL TUMORS

Abstract BACKGROUND Radiotherapy remains one of the most effective modalities for anticancer treatment and part of standard of care for many primary and metastatic brain tumors. Boosting the efficacy of radiotherapy is therefore a logical avenue to improve patient survival. We have developed a radiosensitization strategy called ‘induction of mitotic enrichment’. It has long been known that the radiosensitivity of a cell depends on the phase of the cell cycle and that mitotic cells are especially vulnerable. Enriching the tumor for mitotic cells by arresting them during division prior to each radiotherapy fraction should therefore render the tumor population more sensitive to irradiation. Ideally, induction of mitotic enrichment should be reversible and non-cytotoxic to prevent healthy tissue toxicity and be compatible with clinically applied fractionated radiotherapy regimens. MATERIAL AND METHODS Mitotic enrichment in vitro is established using flow cytometry and radiosensitization is assessed using colony formation assays. In vivo, several independent luciferase-tagged orthotopic brain tumor models are used to investigate mitotic enrichment (phospho-histone 3 immunohistochemistry), tumor growth, survival and healthy tissue toxicity (body weight, blood liver enzymes, γH2AX and cleaved-caspase 3 immunohistochemistry). Pharmacokinetic studies are quantified using LC-MS/MS. All experiments on primary brain tumors included serum-free neurosphere culture models. RESULTS We have now identified an orally available targeted tubulin polymerization inhibitor that can achieve repeated and reversible mitotic enrichment for up to 10 hours prior to radiotherapy, without causing cytotoxicity in vitro or healthy tissue toxicity in vivo. This tubulin inhibitor (ABT-751) efficiently radiosensitizes a range of preclinical tumor models representing adult and pediatric, primary and metastatic brain tumors. Importantly, ABT-751 also improves survival in mouse models of adult glioblastoma (GBM), but only in a mitotic enrichment setup when given several hours prior to radiotherapy to allow accumulation in mitosis. Of note, induction of mitotic enrichment does not exacerbate healthy tissue toxicity. CONCLUSION Induction of mitotic enrichment by ABT-751 radiosensitizes adult GBM to fractionated radiotherapy. We are currently expanding our preclinical development of mitotic enrichment as a radiosensitization strategy to other targets and cancers and have designed a phase 0/I trial for adult GBM to demonstrate induction of mitotic enrichment and tolerability when combined with radiotherapy. We expect to start enrollment in this trial soon.

Identification of 1,3,4-oxadiazoles as tubulin-targeted anticancer agents: a combined field-based 3D-QSAR, pharmacophore model-based virtual screening, molecular docking, molecular dynamics simulation, and density functional theory calculation approach

Cancer is one of the most prominent causes of death worldwide and tubulin is a crucial protein of cytoskeleton that maintains essential cellular functions including cell division as well as cell signalling, that makes an attractive drug target for cancer drug development. 1,3,4-oxadiazoles disrupt microtubule causing G2-M phase cell cycle arrest and provide anti-proliferative effect. In this study, field-based 3D-QSAR models were developed using 62 bioactive anti-tubulin 1,3,4-oxadiazoles. The best model characterized by PLS factor 7 was rigorously validated using various statistical parameters. Generated 3D-QSAR model having high degree of confidence showed favourable and unfavourable contours around 1,3,4-oxadiazole core that assisted in defining proper spatial positioning of desired functional groups for better bioactivity. A five featured pharmacophore model (AAHHR_1) was developed using same ligand library and validated through enrichment analysis (BEDROC160.9 value = 0.59, Average EF 1% = 27.05, and AUC = 0.74). Total 30,212 derivatives of 1,3,4-oxadiazole obtained from PubChem database was prefiltered through validated pharmacophore model and docked in XP mode on binding cavity of tubulin protein (PDB code: 1SA0) which led into the identification of 11 HITs having docking scores between −7.530 and −9.719 kcal/mol while the reference compound Colchicine exerted docking score of −7.046 kcal/mol. Following the analysis of MM-GBSA and ADME studies, HIT1 and HIT4 emerged as the two promising hits. To verify their thermodynamic stability at the target site, molecular dynamic simulations were carried out. Both HITs were further subjected to DFT analysis to determine their HOMO-LUMO energy gap for ensuring their biological feasibility. Finally, molecular docking based structural exploration for 1,3,4-oxadiazoles to set up a lead of Formula I for further advancements of tubulin polymerization inhibitors as anti-cancer agents. Communicated by Ramaswamy H. Sarma

Synthesis and biological evaluation of structurally diverse 6-aryl-3-aroyl-indole analogues as inhibitors of tubulin polymerization

The synthesis and evaluation of small-molecule inhibitors of tubulin polymerization remains a promising approach for the development of new therapeutic agents for cancer treatment. The natural products colchicine and combretastatin A-4 (CA4) inspired significant drug discovery campaigns targeting the colchicine site located on the beta-subunit of the tubulin heterodimer, but so far these efforts have not yielded an approved drug for cancer treatment in human patients. Interest in the colchicine site was enhanced by the discovery that a subset of colchicine site agents demonstrated dual functionality as both potent antiproliferative agents and effective vascular disrupting agents (VDAs). Our previous studies led to the discovery and development of a 2-aryl-3-aroyl-indole analogue (OXi8006) that inhibited tubulin polymerization and demonstrated low nM IC50 values against a variety of human cancer cell lines. A water-soluble phosphate prodrug salt (OXi8007), synthesized from OXi8006, displayed promising vascular disrupting activity in mouse models of cancer. To further extend structure-activity relationship correlations, a series of 6-aryl-3-aroyl-indole analogues was synthesized and evaluated for their inhibition of tubulin polymerization and cytotoxicity against human cancer cell lines. Several structurally diverse molecules in this small library were strong inhibitors of tubulin polymerization and of MCF-7 and MDA-MB-231 human breast cancer cells. One of the most promising analogues (KGP591) caused significant G2/M arrest of MDA-MB-231 cells, disrupted microtubule structure and cell morphology in MDA-MB-231 cells, and demonstrated significant inhibition of MDA-MB-231 cell migration in a wound healing (scratch) assay. A phosphate prodrug salt, KGP618, synthesized from its parent phenolic precursor, KGP591, demonstrated significant reduction in bioluminescence signal when evaluated in vivo against an orthotopic model of kidney cancer (RENCA-luc) in BALB/c mice, indicative of VDA efficacy. The most active compounds from this series offer promise as anticancer therapeutic agents.

Isothiocyanates as Tubulin Polymerization Inhibitors-Synthesis and Structure-Activity Relationship Studies.

Among the various substances that interfere with the microtubule formation process, isothiocyanates (ITCs) are the group of compounds for which the binding mode and mechanism of action have not yet been explained. To better understand the structure-activity relationship of tubulin-isothiocyanate interactions, we designed and synthesized a series of sixteen known and novel, structurally diverse ITCs, including amino acid ester-derived isothiocyanates, bis-isothiocyanates, analogs of benzyl isothiocyanate, and phosphorus analogs of sulforaphane. All synthesized compounds and selected natural isothiocyanates (BITC, PEITC, AITC, and SFN) were tested in vitro to evaluate their antiproliferative activity, tubulin polymerization inhibition potential, and influence on cell cycle progression. The antiproliferative activity of most of the newly tested compounds exceeded the action of natural isothiocyanates, with four structures being more potent as tubulin polymerization inhibitors than BITC. As a confirmation of anti-tubulin activity, the correlation between polymerization inhibition and cell cycle arrest in the G2/M phase was observed for the most active compounds. In light of the biological results indicating significant differences in the impact of structurally diverse isothiocyanate on tubulin polymerization, in silico analysis was conducted to analyze the possible mode of isothiocyanate-tubulin binding and to show how it can influence the polymerization reaction.

A Review on the Synthesis and Anticancer Potentials of Imidazothiazole Derivatives.

Cancer is one of the severe diseases in which abnormal cells divide and proliferate in an uncontrolled manner without any regulation. Globally cancer is among the leading causes of death; according to a recent report of by the WHO, around 10 million people died in 2018 due to cancer. It has also been reported that by 2040, approximately 30 million new cases will be reported every year. The increase in the incidences of cancer is taking a toll on the health care system worldwide. Considerable scientific literature is available on anticancer agents but newer therapeutic strategies are still required in this field to address novel approaches to drug design and discovery to counter this problem. Imidazothiazole represents a privileged scaffold in medicinal chemistry and provides the medicinal chemist the possibility to modulate the physiochemical properties of the lead compound. In recent times, imidazothiazole scaffold is broadly explored for its anticancer activity, which acts through various mechanisms such as EGFR, B-RAF, DHFR kinase inhibition and tubulin polymerization inhibition and other molecular mechanisms of action. Due to their feasible synthetic accessibility and promising pharmacological profile, it has attracted various medicinal chemists to explore and develop imidazothiazole derivatives as potent and safe anticancer agents. In the present article, we have reviewed various potent imidazothiazole scaffold-based derivatives reported as anticancer agents, their synthetic strategies, Structure Activity Relationship (SAR), mechanism of action, and molecular docking along with their future perspective. This review will be very useful for medicinal chemists for drug design and development of imidazothiazole-based potent antiproliferative agents.

Synthesis and antitumor activity of a 2-hydroxy substituted chalcone (C6)

Cancers account for approximately 13% of all deaths each year with breast cancer being one of the most common. Chemotherapeutic agents used in cancer usually have nonspecific toxicity which kills both tumor and normal cells and cause serious side effects that often limit their efficacy. This study aimed to synthesize chalcone and evaluate its antitumor activity. Chalcone was synthesized using Claisen-Schmidt condensation and characterized using spectroscopic techniques. The LD50 of the synthesized compound was estimated using OECD-425 guidelines in rats. A mammary tumor was induced with a single subcutaneous administration of 65 mg/kg of 1-methyl nitrosourea (MNU). The rats were palpated weekly to determine the size of the tumor. Eight weeks post MNU administration, the rats were divided into five groups of six rats each and treated with graded doses (12.5, 25, and 50 mg/kg) of the compound and paclitaxel (10 mg/kg) for six weeks. Before treatment, three rats were randomly selected and sacrificed, and mammary gland samples were subjected to histological assessment to confirm the induction of the tumor. At the end of the treatment, the rats were euthanized, and mammary glands were collected and subjected to histological evaluation. The possible mechanism of action of the synthesized compound was elucidated in silico using molecular docking. The compound was synthesized and named C6. C6 was found to be relatively safe with LD50 above 2000 mg/kg and exhibited remarkable antitumor activity. MNU-induced mammary tumor rats treated with C6 produced a significant decrease in tumor diameter when compared with the untreated group, histology slides displayed fewer signs of hyperplasia and small numbers of connective tissue with larger lobules when compared with the untreated group. In silico tubulin-binding interactions revealed that C6 binding to tubulin was like that of colchicine, and also has higher affinity to tubulin than colchicine. The synthesized chalcone demonstrated significant antitumor activities in MNU-induced mammary tumors in rats postulated via inhibition of tubulin polymerization.

Fused Imidazopyrazine-Based Tubulin Polymerization Inhibitors Inhibit Neuroblastoma Cell Function.

Targeting the colchicine binding site on tubulin is a promising approach for cancer treatment to overcome the limitations of current tubulin polymerization inhibitors. New classes of colchicine binding site inhibitors (CBSIs) are continually being uncovered; however, balancing metabolic stability and cellular potency remains an issue that needs to be resolved. Therefore, we designed and synthesized a series of novel fused imidazopyridine and -pyrazine CBSIs and evaluated their cellular activity, metabolic stability, and tubulin-binding properties. Evidence shows that the imidazo[1,2-a]pyrazine series are effective against neuroblastoma cell lines marked by MYCN amplification. Further assessment shows that a combination of an imidazo[1,2-a]pyrazine core with a trimethoxyphenyl ring D results in the highest cellular activity and binding characteristics compared with a dichloromethoxyphenyl or difluoromethoxyphenyl ring D. However, the metabolic stability of compounds with a dichloromethoxyphenyl or difluoromethoxyphenyl ring D is significantly higher than that of those containing a trimethoxyphenyl ring D, suggesting that improved metabolic stability is achieved with a moderate impact on potency.

Design, synthesis and evaluation of dihydro-1H-indene derivatives as novel tubulin polymerisation inhibitors with anti-angiogenic and antitumor potency

Angiogenesis plays an important role in tumour generation and progression, which is used to supply nutrients and metastasis. Herein, a series of novel dihydro-1H-indene derivatives were designed and evaluated as tubulin polymerisation inhibitors by binding to colchicine site, exhibiting anti-angiogenic activities against new vessel forming. Through structure-activity relationships study, compound 12d was found to be the most potent derivative possessing the antiproliferative activity against four cancer lines with IC50 values among 0.028−0.087 µM. Compound 12d bound to colchicine site on tubulin and inhibited tubulin polymerisation in vitro. In addition, compound 12d induced cell cycle arrest at G2/M phase, stimulated cell apoptosis, inhibited tumour metastasis and angiogenesis. Finally, the results of in vivo assay suggested that compound 12d could prevent tumour generation, inhibit tumour proliferation and angiogenesis without obvious toxicity. Collectively, all these findings suggested that compound 12d is a novel tubulin polymerisation inhibitor deserving further research.

Unleashing the potential of natural biological peptide Macropin: Hydrocarbon stapling for effective breast cancer treatment

The identification of novel candidate molecules with the potential to revolutionize the treatment of breast cancer holds profound clinical significance. Macropin (Mac)-1, derived from the venom of wild bees, emerges as an auspicious therapeutic agent for combating breast cancers. Nevertheless, linear peptides have long grappled with the challenges of traversing cell membranes and succumbing to protease hydrolysis. To address this challenge, the present study employed hydrocarbon stapling modification to synthesize a range of stapled Mac-1 peptides, which were comprehensively evaluated for their chemical and biological properties. Significantly, Mac-1-sp4 exhibited a remarkable set of improvements, including enhanced helicity, proteolytic stability, cell membrane permeability, induction of cell apoptosis, in vivo antitumor activity, and inhibition of tubulin polymerization. This study explores the significant impact of the hydrocarbon stapling technique on the secondary structure, hydrolase stability, and biological activity of Mac-1, shedding light on its potential as a revolutionary and potent anti-breast cancer therapy. The findings establish a strong basis for the development of innovative and highly effective anti-tumor treatments.

Discovery and optimization of 2,3-diaryl-1,3-thiazolidin-4-one-based derivatives as potent and selective cytotoxic agents with anti-inflammatory activity

Several studies have indicated the potential therapeutic outcomes of combining selective COX-2 inhibitors with tubulin-targeting anticancer agents. In the current study, a novel series of thiazolidin-4-one-based derivatives (7a–q) was designed by merging the pharmacophoric features of some COXs inhibitors and tubulin polymerization inhibitors. Compounds 7a–q were synthesized and evaluated for their cytotoxic activity against MCF7, HT29, and A2780 cancer cell lines (IC50 = 0.02–17.02 μM). The cytotoxicity of 7a–q was also assessed against normal MRC5 cells (IC50 = 0.47–13.46 μM). Compounds 7c, 7i, and 7j, the most active in the MTT assay, significantly reduced the number of HT29 colonies compared to the control. Compounds 7c, 7i, and 7j also induced significant decreases in the tumor volumes and masses in Ehrlich solid carcinoma-bearing mice compared to the control. The three compounds also exhibited significant anti-HT29 migration activity in the wound-healing assay. They have also induced cell cycle arrest in HT29 cells at the S and G2/M phases. In addition, they induced significant increases in both early and late apoptotic events in HT29 cells compared to the control, where 7j showed the highest effect. On the other hand, compound 7j (1 μM) displayed weak inhibitory activity against tubulin polymerization compared to colchicine (3 μM). On the other hand, compounds 7a–q inhibited the activity of COX-2 (IC50 = 0.42–29.11 μM) compared to celecoxib (IC50 = 0.86 μM). In addition, 7c, 7i, and 7j showed moderate inhibition of inflammation in rats compared to indomethacin, with better GIT safety profiles. Molecular docking analysis revealed that 7c, 7i, and 7j have higher binding free energies towards COX-2 than COX-1. These above results suggested that 7j could serve as a potential anticancer drug candidate.

Effect of Colchicine Binding Site Inhibitors on the Tubulin Intersubunit Interaction.

Microtubules are dynamic, non-covalent polymers consisting of α- and β-tubulin subunits that are involved in a wide range of intracellular processes. The polymerization and dynamics of microtubules are regulated by many factors, including small molecules that interact with different sites on the tubulin dimer. Colchicine binding site inhibitors (CBSIs) destabilize microtubules and inhibit tubulin polymerization, leading to cell cycle arrest. Because of their therapeutic potential, the molecular mechanism of CBSI function is an area of active research. Nevertheless, important details of this mechanism have yet to be resolved. In this study, we use atomistic molecular dynamics simulations to show that the binding of CBSIs to the tubulin heterodimer leads to the weakening of tubulin intersubunit interaction. Using atomistic molecular dynamics simulations and binding free energy calculations, we show that CBSIs act as protein-protein interaction inhibitors and destabilize interlinkage between α and β subunits, which is crucial for longitudinal contacts in the microtubule lattice. Our results offer new insight into the mechanisms of microtubule polymerization inhibition by colchicine and its analogs.

Discovery of chiral 1,4-diarylazetidin-2-one-based hydroxamic acid derivatives as novel tubulin polymerization inhibitors with histone deacetylase inhibitory activity

Tubulin and histone deacetylase have been clinically proven as promising targets for cancer therapy. Herein, we describe the design and synthesis of chiral 1,4-diarylazetidin-2-one-based hydroxamic acids as novel tubulin/HDAC dual inhibitors. Among them, compound 12a was validated to effectively disrupt tubulin polymerization, and exhibited potent HDAC1/8 inhibitory activities. Meanwhile, 12a showed good antiproliferative activities against four tumor cell lines. Further studies showed 12a works through blocking cellular cycle, inducing apoptosis and inhibiting colony formation. In addition, 12a has suitable physicochemical properties and high liver microsomal metabolic stability. Importantly, compound 12a was found to exhibit significant antitumor efficacy in vivo, thus warranting it as a promising tubulin/HDAC dual inhibitor for further development.

Semicarbazides carrying indole core: Synthesis, cytotoxicity evaluation against human breast cancer cell lines, and molecular modeling studies.

In this paper, we report the synthesis and cytotoxicity evaluation of novel indole-carrying semicarbazide derivatives (IS1-IS15). The target molecules were obtained by the reaction of aryl/alkyl isocyanates with 1H-indole-2-carbohydrazide that was in-house synthesized from 1H-indole-2-carboxylic acid. Following structural characterization by 1H NMR, 13C NMR, and HRMS, IS1-IS15 were investigated for their cytotoxic activity against human breast cancer cell lines, MCF-7 and MDA-MB-231. According to the data obtained from the MTT assay, phenyl ring with a lipophilic group at its para-position and alkyl moiety were preferential substituents on the indole-semicarbazide scaffold for antiproliferative activity. The effect of IS12 (N-(4-chloro-3-(trifluoromethyl)phenyl)-2-(1H-indole-2-carbonyl)hydrazine-1-carboxamide), the compound that demonstrated remarkable antiproliferative activity on both cell lines, was also evaluated on the apoptotic pathway. Moreover, the calculation of critical descriptors constituting drug-likeness confirmed the position of the selected compounds in the anticancer drug development process. Finally, molecular docking studies suggested the inhibition of tubulin polymerization as the potential activity mechanism of this class of molecules.

Synthesis and anti-tumor activity of piperonal substituted chalcone

Objectives: Chalcones have been identified as potential antitumor agents with a novel target, the tubulin. The aim of the study was to synthesize a piperonal substituted chalcone and evaluate its in vivo antitumor activity. Materials and Methods: Piperonal substituted chalcone was synthesized using Claisen-Schmidt condensation and characterized using various spectroscopic techniques. The lethal dose (LD50) of the synthesized compound was estimated using OECD-425 guidelines in rats. Antitumor activity of the synthesized compound was evaluated on 1-methyl nitrosourea (MNU)-induced mammary tumor in female Wistar rats. Histological evaluation was used to confirm tumor induction and assess treatment with the synthesized compound. The possible mechanism of action of the synthesized compound was elucidated in silico using molecular docking. Results: The compound was synthesized and named C2. C2 was found to be relatively safe with LD50 >2000 mg/kg orally. Moreover, C2 exhibited remarkable antitumor activity, at all the tested doses in a dose dependent manner. Histological evaluation of the MNU-induced mammary tumor rats treated with C2 displayed fewer signs of hyperplasia and small numbers of connective tissue with larger lobules when compared with the untreated group. In silico tubulin-binding interactions revealed that the kinetics of C2 binding to tubulin was like that of colchicine. Comparison of crystal structures of tubulin-C2 and tubulin-colchicine complexes showed that the binding mode of C2 to tubulin was like that of colchicine to tubulin and produced the same conformational changes on the tubulin structure as colchicine. Conclusion: The synthesized chalcone demonstrated remarkable antitumor activities in MNU-induced mammary tumors in rats possibly through inhibition of tubulin polymerization.

Discovery of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerisation with potent liver cancer inhibitory activity

In this work, a series of novel arylamide derivatives containing piperazine moiety were designed and synthesised as tubulin polymerisation inhibitors. Among 25 target compounds, compound 16f (MY-1121) exhibited low nanomolar IC50 values ranging from 0.089 to 0.238 μM against nine human cancer cells. Its inhibitory effects on liver cancer cells were particularly evident with IC50 values of 89.42 and 91.62 nM for SMMC-7721 and HuH-7 cells, respectively. Further mechanism studies demonstrated that compound 16f (MY-1121) could bind to the colchicine binding site of β-tubulin and directly act on β-tubulin, thus inhibiting tubulin polymerisation. Additionally, compound 16f (MY-1121) could inhibit colony forming ability, cause morphological changes, block cell cycle arrest at the G2 phase, induce cell apoptosis, and regulate the expression of cell cycle and cell apoptosis related proteins in liver cancer cells. Overall, the promising bioactivities of compound 16f (MY-1121) make the novel arylamide derivatives have the value for further development as tubulin polymerisation inhibitors with potent anticancer activities.

Synthesis and anthelmintic activity of novel thiosemicarbazide and 1,2,4-triazole derivatives: In vitro, in vivo, and in silico study

IntroductionIntestinal parasitic infections are neglected diseases and, due to the increasing resistance of parasites to available drugs, they pose an increasing therapeutic challenge. Therefore, there is a great need for finding new compounds with antiparasitic activity. Objectives: In this work, new thiosemicarbazide and 1,2,4-triazole derivatives were synthesized and tested for their anthelmintic activity. MethodsThe synthesis was carried out by classical methods of organic chemistry. Anthelmintic activity tests were carried out in vitro (Rhabditis sp., Haemonchus contortus, Strongylidae sp.) in vivo (Heligmosomoides polygyrus/bakeri), and in silico analysis was performed. ResultsQuinoline-6-carboxylic acid derivative compounds were designed and synthesized. The highest activity in the screening tests in the Rhabditis model was demonstrated by compound II-1 with a methoxyphenyl substituent LC50 = 0.3 mg/mL. In the next stage of the research, compound II-1 was analyzed in the H. contortus model. The results showed that compound II-1 was active and had ovicidal (percentage of dead eggs > 45 %) and larvicidal (percentage of dead larvae > 75 %) properties. Studies in the Strongylidae sp. model confirmed the ovicidal activity of compound II-1 (percentage of dead eggs ≥ 55 %). In vivo studies conducted in the H. polygyrus/bakeri nematode model showed that the number of nematodes decreased by an average of 30 % under the influence of compound II-1. In silico studies have shown two possible modes of action of compound II-1, i.e. inhibition of tubulin polymerization and SDH. The test compound did not show any systemic toxic effects. Its influence on drug metabolism related to the activity of cytochrome CYP450 enzymes was also investigated. ConclusionThe results obtained in the in vitro, in vivo, and in silico studies indicate that the test compound can be described as a HIT, which in the future may be used in the treatment of parasitic diseases in humans and animals.

Design, synthesis, and biological evaluation of 4-aryl-9H-carbazoles as tubulin polymerization inhibitors with potent anticancer activities

Some novel 4-aryl-9H-carbazoles were designed as potential tubulin polymerization inhibitors through structural optimization and bioisosteric strategy. These compounds showed effective antiproliferative activities against a diverse set of cancer cell lines, including HeLa, HepG2, and SGC-7901 cells. Among them, compound 13k exerted the strongest inhibitory effect on HeLa cells with an IC50 of 1.2 ± 0.09 μM. Mechanism studies indicated that 13k significantly inhibited tubulin polymerization in vitro and disrupted the dynamic homeostasis of microtubules in HeLa cells. Furthermore, 13k concentration-dependently caused G2/M phase cell cycle arrest and apoptosis in HeLa cells. Molecular docking analysis demonstrated the interaction of 13k at the colchicine binding site of tubulin. In addition, the prediction of physicochemical properties studies shows that 13k has good pharmacokinetic properties. These preliminary results suggested that 13k is a new tubulin polymerization inhibitor and worthy of further investigation.

Design, synthesis and tubulin polymerization inhibition activity of newly synthesized hydrazone-linked to combretastatin analogues as potential anticancer agents

A new series of hydrazone derivatives were synthesized by varying the active part (hydrazide group) of cis-vinyl amide derivatives, characterized by 1H NMR, 13C NMR spectral data and screened for their cytotoxic activity against MCF-7 breast cancer cell line. Results indicated that compounds 3 g, 3k and 4 were potent cytotoxic agents (IC50 = 4.02–5.36 μM) relative to CA-4 as a reference compound. The most active hydrazinyl compounds were further evaluated for the β-tubulin inhibition activity. Cellular cycle analysis of the inhibitory effect of N-(furan-2-ylmethylene)hydrazinyl 4 on each phase of the cell cycle of breast adenocarcinoma MCF-7 cells was investigated using flow cytometric analysis. Test compound elicited cellular cycle arrest at G2/M phase. Annexin V dual staining analysis results revealed that treatment of MCF-7 cells with N-(furan-2-ylmethylene)hydrazinyl 4 increase the percentage of apoptosis compared to negative untreated control. Besides, N-(furan-2-ylmethylene)hydrazinyl 4 induced apoptosis via decreased the level of mitochondrial membrane potential relative to untreated control cells as revealed by flow cytometry analysis. Moreover, molecular modeling investigations were conducted to clarify the manner in which the most potent compound binds within the tubulin's colchicine-binding pocket, thereby elucidating the reasons behind its exceptional cytotoxicity and its superior ability to inhibit tubulin when compared to CA-4.

Exploration of quinoxaline-benzimidazole hybrids as apoptosis-inducing agents and tubulin polymerisation inhibitors

We herein report the design and synthesis of a new class of quinoxaline-benzimidazole hybrids based on the molecular hybridization concept and evaluation of their in vitro cytotoxicity profile against human cancer cell lines, i.e., colorectal, lung, skin, and mouse melanoma cell line. Among all the screened compounds, compound 5l (2-(1-(3, 4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)quinoxaline) exhibited potent cytotoxicity against lung cancer cell line (A549) with an IC50 of 4.37 ± 0.09 µM with a cytospecificity towards cancer cells. Further, AO/EB, DAPI, DCFDA, and JC-1 staining techniques confirmed that 5l induced apoptosis in the A549 cell line. In addition, 5l in annexin V-FITC/PI assay induced early apoptosis. The clonogenic assay revealed that 5l inhibited colony formation in a dose-dependent manner. Cell cycle distribution analysis unveiled that 5l caused the arrest of G2/M phase of cell cycle. Moreover, 5l inhibited tubulin polymerization with an IC50 value of < 2.19 µM. Docking studies revealed that 5l has a prominent binding affinity towards colchicine binding site of α/β- tubulin with good protein-ligand interactions with a binding energy of -45.139 kcal/mol. In silico ADME/T prediction studies disclosed the favourable drug-like properties of 5l.

A mouse model of sensory neuropathy induced by a long course of monomethyl-auristatin E treatment

Antibody-drug conjugates (ADCs) are anticancer drugs consisting of a monoclonal antibody, targeting selective tumor antigens, to which has been frequently associated a highly potent cytotoxic agent, the monomethyl auristatin E (MMAE) using a chemical linker. MMAE is a tubulin polymerization inhibitor derived from dolastin-10. These MMAE-ADCs are responsible for peripheral nerve toxicities. Our objective was to develop and characterize a mouse model of MMAE-induced peripheral neuropathy induced by free MMAE injections.MMAE was injected in Swiss mice at 50 μg/kg i.p. every other day for 7 weeks. Assessments of motor and sensory nerve functions were performed once a week on MMAE and Vehicle-treated mice. Sciatic nerve and paw skin were removed at the end of experiment for subsequent immunofluorescence and morphological analysis.MMAE did not affect motor coordination, muscular strength and heat nociception, but significantly induced tactile allodynia in MMAE-treated mice compared with Vehicle-treated mice from day 35 to day 49. MMAE significantly reduced myelinated and unmyelinated axon densities in sciatic nerves and led to a loss of intraepidermal nerve fiber in paw skin.In summary, long course of low dose of MMAE induced a peripheral sensory neuropathy associated with nerve degeneration, without general state alteration. This model may represent a ready accessible tool to screen neuroprotective strategies in the context of peripheral neuropathies induced by MMAE-ADCs.