A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent.

Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical-grade rigosertib, which is free of this impurity, does not exhibit tubulin-binding activity. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.

Bioprocessing of Epothilone B from Aspergillus fumigatus under solid state fermentation: Antiproliferative activity, tubulin polymerization and cell cycle analysis.

Epothilone derivatives have been recognized as one of the most powerful anticancer drugs towards solid tumors, for their unique affinity to bind with ß-tubulin microtubule arrays, stabilizing their disassembly, causing cell death. Sornagium cellulosum is the main source for Epothilone, however, the fermentation bioprocessing of this myxobacteria is the main challenge for commercial production of Epothilone. The metabolic biosynthetic potency of epothilone by Aspergillus fumigatus, an endophyte of Catharanthus roseus, raises the hope for commercial epothilone production, for their fast growth rate and feasibility of manipulating their secondary metabolites. Thus, nutritional optimization of A. fumigatus for maximizing their epothilone productivity under solid state fermentation process is the objective. The highest yield of epothilone was obtained by growing A. fumigatus on orange peels under solid state fermentation (2.2 µg/g), bioprocessed by the Plackett-Burman design. The chemical structure of the extracted epothilone was resolved from the HPLC and LC-MS/MS analysis, with molecular mass 507.2 m/z and identical molecular fragmentation pattern of epothilone B of S. cellulosum. The purified A. fumigatus epothilone had a significant activity towards HepG2 (IC50 0.98 µg/ml), Pancl (IC50 1.5 µg/ml), MCF7 (IC50 3.7 µg/ml) and WI38 (IC50 4.6 µg/ml), as well as a strong anti-tubulin polymerization activity (IC50 0.52 µg/ml) compared to Paclitaxel (2.0 µg/ml). The effect of A. fumigatus epothilone on the immigration ability of HepG2 cells was assessed, as revealed from the wound closure of the monolayer cells that was estimated by ~ 63.7 and 72.5%, in response to the sample and doxorubicin, respectively, compared to negative control. From the Annexin V-PI flow cytometry results, a significant shift of the normal cells to the apoptosis was observed in response to A. fumigatus epothilone by ~ 20 folds compared to control cells, with the highest growth arrest of the HepG2 cells at the G0-G1 stage.

Collective Synthesis of Sarpagine and Macroline Alkaloid-Inspired Compounds.

Design strategies that can access natural-product-like chemical space in an efficient manner may facilitate the discovery of biologically relevant compounds. We have employed a divergent intermediate strategy to construct an indole alkaloid-inspired compound collection derived from two different molecular design principles, i.e. biology-oriented synthesis and pseudo-natural products. The divergent intermediate was subjected to acid-catalyzed or newly discovered Sn-mediated conditions to selectively promote intramolecular C- or N-acylation, respectively. After further derivatization, a collection totalling 84 compounds representing four classes was obtained. Morphological profiling via the cell painting assay coupled with a subprofile analysis showed that compounds derived from different design principles have different bioactivity profiles. The subprofile analysis suggested that a pseudo-natural product class is enriched in modulators of tubulin, and subsequent assays led to the identification of compounds that suppress in vitro tubulin polymerization and mitotic progression.

Synthesis and anticancer properties of novel dolastatin 10 analogs featuring five-membered heterocyclic rings with a linkable group at the C-terminus.

Dolastatin 10 (Dol-10), a natural marine-source pentapeptide, is a powerful antimitotic agent regarded as one of the most potent anticancer compounds found to date. Dol-10 however, lacks chemical conjugation capabilities, which restricts the feasibility of its application in targeted drug therapy. This limitation has spurred the prospect that chemical structure of the parent molecule might allow conjugation of the derivatives to drug carriers such as antibodies. By first employing docking studies, we designed and prepared a series of novel Dol-10 analogs with a modified C-terminus, preserving high potency of the parent compound while enhancing conjugation capability. The modifications involved the introduction of a methyleneamine functionality at position 4 of the 1,3-thiazole ring, along with the substitution of the thiazole ring with a 1,2,3-triazole moiety, furnished with methylenehydroxy, carboxy, methyleneamine, and N(Me)-methyleneamine tethering functionalities at position 4. Among the synthesized pentapeptides, DA-1 exhibited the highest potency in prostate cancer (PC-3) cells, eliciting apoptosis (IC50 0.2 ± 0.1 nm) and cell cycle arrest at the mitotic stage after at least 6 days of culture. This delayed response suggests the accumulation of cellular stress or significant physiological alterations that profoundly impact the cell cycle. We believe that these novel Dol-10 derivates represent a new and straightforward route for the development of C-terminus modified Dol-10-based microtubule inhibitors, thereby advancing targeted anticancer therapy.

Thiostrepton induces spindle abnormalities and enhances Taxol cytotoxicity in MDA-MB-231 cells.

BACKGROUND: Thiostrepton (TST) is a known inhibitor of the transcription factor Forkhead box M1 (FoxM1) and inducer of heat shock response (HSR) and autophagy. TST thus may be one potential candidate of anticancer drugs for combination chemotherapy. METHODS AND RESULTS: Immunofluorescence staining of mitotic spindles and flow cytometry analysis revealed that TST induces mitotic spindle abnormalities, mitotic arrest, and apoptotic cell death in the MDA-MB-231 triple-negative breast cancer cell line. Interestingly, overexpression or depletion of FoxM1 in MDA-MB-231 cells did not affect TST induction of spindle abnormalities; however, TST-induced spindle defects were enhanced by inhibition of HSP70 or autophagy. Moreover, TST exhibited low affinity for tubulin and only slightly inhibited in vitro tubulin polymerization, but it severely impeded tubulin polymerization and destabilized microtubules in arrested mitotic MDA-MB-231 cells. Additionally, TST significantly enhanced Taxol cytotoxicity. TST also caused cytotoxicity and spindle abnormalities in a Taxol-resistant cell line, MDA-MB-231-T4R. CONCLUSIONS: These results suggest that, in addition to inhibiting FoxM1, TST may induce proteotoxicity and autophagy to disrupt cellular tubulin polymerization, and this mechanism might account for its antimitotic effects, enhancement of Taxol anticancer effects, and ability to overcome Taxol resistance in MDA-MB-231 cells. These data further imply that TST may be useful to improve the therapeutic efficacy of Taxol.

Design, synthesis, and biological evaluation of novel 2,3-Di-O-Aryl/Alkyl sulfonate derivatives of l-ascorbic acid: Efficient access to novel anticancer agents via in vitro screening, tubulin polymerization inhibition, molecular docking study and ADME predictions.

A series of novel l-ascorbic acid derivatives bearing aryl and alkyl sulfonate substituents were synthesized and characterized. In vitro anticancer evaluation against MCF-7 (breast) and A-549 (lung) cancer cell lines revealed potent activity for most of the compounds, with 2b being equipotent to the standard drug colchicine against MCF-7 (IC50 = 0.04 µM). Notably, compound 2b displayed 89-fold selectivity for MCF-7 breast cancer over MCF-10A normal breast cells. Derivatives with two sulfonate groups (2a-g, 3a-g) exhibited superior potency over those with one sulfonate (4a-c,5g, 6b). Compounds 2b and 2c potently inhibited tubulin polymerization in A-549 cancer cells (73.12 % and 62.09 % inhibition, respectively), substantiating their anticancer potential through microtubule disruption. Molecular docking studies showed higher binding scores and affinities for these compounds at the colchicine-binding site of α, ß-tubulin compared to colchicine itself. In-silico ADMET predictions indicated favourable drug-like properties, with 2b exhibiting the highest binding affinity. These sulfonate derivatives of l-ascorbic acid represents promising lead scaffolds for anticancer drug development.

Synthesis and characterization of bis-amide SSE1917 as a microtubule-stabilizing anticancer agent.

Microtubule dynamics are critical for spindle assembly and chromosome segregation during cell division. Pharmacological inhibition of microtubule dynamics in cells causes prolonged mitotic arrest, resulting in apoptosis, an approach extensively employed in treating different types of cancers. The present study reports the synthesis of thirty-two novel bis-amides (SSE1901-SSE1932) and the evaluation of their antiproliferative activities. N-(1-oxo-3-phenyl-1-(phenylamino)propan-2-yl)benzamide (SSE1917) exhibited the most potent activity with GI50 values of 0.331 ± 0.01 µM in HCT116 colorectal and 0.48 ± 0.27 µM in BT-549 breast cancer cells. SSE1917 stabilized microtubules in biochemical and cellular assays, bound to taxol site in docking studies, and caused aberrant mitosis and G2/M arrest in cells. Prolonged treatment of cells with the compound increased p53 expression and triggered apoptotic cell death. Furthermore, SSE1917 suppressed the growth of both mouse and patient-derived human colon cancer organoids, highlighting its potential therapeutic value as an anticancer agent.

Design, synthesis, and biological evaluation of new biaryl derivatives of cycloalkyl diacetamide bearing chalcone moiety as type II c-MET kinase inhibitors.

Many human cancers have been associated with the deregulation of the mesenchymal-epithelial transition factor tyrosine kinase (MET) receptor, a promising drug target for anticancer drug discovery. Herein, we report the discovery of a novel structure of potent chalcone-based derivatives type II c-Met inhibitors which are comparable to Foretinib (IC50 = 14 nM) as a potent reference drug. Based on our design strategy, we also expected an anti-tubulin activity for the compounds. However, the weak inhibitory effects on microtubules were confirmed by cell cycle analyses implicated that the observed cytotoxicity against HeLa cells probably was not derived from tubulin inhibition. Compounds 14q and 14k with IC50 values of 24 nM and 45 nM, respectively, demonstrated favorable inhibition of MET kinase activity, and desirable bonding interactions in the ligand-MET enzyme complex stability in molecular docking studies.

From Sea to Science: Coral Aquaculture for Sustainable Anticancer Drug Development.

Marine natural products offer immense potential for drug development, but the limited supply of marine organisms poses a significant challenge. Establishing aquaculture presents a sustainable solution for this challenge by facilitating the mass production of active ingredients while reducing our reliance on wild populations and harm to local environments. To fully utilize aquaculture as a source of biologically active products, a cell-free system was established to target molecular components with protein-modulating activity, including topoisomerase II, HDAC, and tubulin polymerization, using extracts from aquaculture corals. Subsequent in vitro studies were performed, including MTT assays, flow cytometry, confocal microscopy, and Western blotting, along with in vivo xenograft models, to verify the efficacy of the active extracts and further elucidate their cytotoxic mechanisms. Regulatory proteins were clarified using NGS and gene modification techniques. Molecular docking and SwissADME assays were performed to evaluate the drug-likeness and pharmacokinetic and medicinal chemistry-related properties of the small molecules. The extract from Lobophytum crassum (LCE) demonstrated potent broad-spectrum activity, exhibiting significant inhibition of tubulin polymerization, and showed low IC50 values against prostate cancer cells. Flow cytometry and Western blotting assays revealed that LCE induced apoptosis, as evidenced by the increased expression of apoptotic protein-cleaved caspase-3 and the populations of early and late apoptotic cells. In the xenograft tumor experiments, LCE significantly suppressed tumor growth and reduced the tumor volume (PC3: 43.9%; Du145: 49.2%) and weight (PC3: 48.8%; Du145: 7.8%). Additionally, LCE inhibited prostate cancer cell migration, and invasion upregulated the epithelial marker E-cadherin and suppressed EMT-related proteins. Furthermore, LCE effectively attenuated TGF-ß-induced EMT in PC3 and Du145 cells. Bioactivity-guided fractionation and SwissADME validation confirmed that LCE's main component, 13-acetoxysarcocrassolide (13-AC), holds greater potential for the development of anticancer drugs.

Synthesis of Estrone Heterodimers and Evaluation of Their In Vitro Antiproliferative Activity.

Directed structural modifications of natural products offer excellent opportunities to develop selectively acting drug candidates. Natural product hybrids represent a particular compound group. The components of hybrids constructed from different molecular entities may result in synergic action with diminished side effects. Steroidal homo- or heterodimers deserve special attention owing to their potentially high anticancer effect. Inspired by our recently described antiproliferative core-modified estrone derivatives, here, we combined them into heterodimers via Cu(I)-catalyzed azide-alkyne cycloaddition reactions. The two trans-16-azido-3-(O-benzyl)-17-hydroxy-13α-estrone derivatives were reacted with 3-O-propargyl-D-secoestrone alcohol or oxime. The antiproliferative activities of the four newly synthesized dimers were evaluated against a panel of human adherent gynecological cancer cell lines (cervical: Hela, SiHa, C33A; breast: MCF-7, T47D, MDA-MB-231, MDA-MB-361; ovarian: A2780). One heterodimer (12) exerted substantial antiproliferative activity against all investigated cell lines in the submicromolar or low micromolar range. A pronounced proapoptotic effect was observed by fluorescent double staining and flow cytometry on three cervical cell lines. Additionally, cell cycle blockade in the G2/M phase was detected, which might be a consequence of the effect of the dimer on tubulin polymerization. Computational calculations on the taxoid binding site of tubulin revealed potential binding of both steroidal building blocks, mainly with hydrophobic interactions and water bridges.

Antiproliferative and Pro-Apoptotic Activity and Tubulin Dynamics Modulation of 1H-Benzimidazol-2-yl Hydrazones in Human Breast Cancer Cell Line MDA-MB-231.

(1) Background: The aim of the work is the evaluation of in vitro antiproliferative and pro-apoptotic activity of four benzimidazole derivatives containing colchicine-like and catechol-like moieties with methyl group substitution in the benzimidazole ring against highly invasive breast cancer cell line MDA-MB-231 and their related impairment of tubulin dynamics. (2) Methods: The antiproliferative activity was assessed with the MTT assay. Alterations in tubulin polymerization were evaluated with an in vitro tubulin polymerization assay and a docking analysis. (3) Results: All derivatives showed time-dependent cytotoxicity with IC50 varying from 40 to 60 µM after 48 h and between 13 and 20 µM after 72 h. Immunofluorescent and DAPI staining revealed the pro-apoptotic potential of benzimidazole derivatives and their effect on tubulin dynamics in living cells. Compound 5d prevented tubulin aggregation and blocked mitosis, highlighting the importance of the methyl group and the colchicine-like fragment. (4) Conclusions: The benzimidazole derivatives demonstrated moderate cytotoxicity towards MDA-MB-231 by retarding the initial phase of tubulin polymerization. The derivative 5d containing a colchicine-like moiety and methyl group substitution in the benzimidazole ring showed potential as an antiproliferative agent and microtubule destabilizer by facilitating faster microtubule aggregation and disrupting cellular and nuclear integrity.

Avermectin B1a Shows Potential Anti-Proliferative and Anticancer Effects in HCT-116 Cells via Enhancing the Stability of Microtubules.

Avermectins are a group of macrocyclic lactones that are commonly used as pesticides to treat pests and parasitic worms. Some members of the avermectin family, such as ivermectin, have been found to exhibit anti-proliferative activity toward cancer cells. This study aimed to investigate the potential anti-cancer activities of avermectin B1a using the HCT-116 colon cancer cell line. The MTT assay was used to calculate the IC50 by incubating cells with increasing doses of avermectin B1a for 24, 48, and 72 h. Flow cytometry was used to evaluate apoptosis following the 24 h incubation of cells. The migration capacity of the HCT-116 cells in the absence or presence of avermectin B1a was also investigated. Finally, tubulin polymerization in the presence of avermectin B1a was evaluated. Avermectin B1a presented anti-proliferative activity with an IC50 value of 30 µM. Avermectin B1a was found to promote tubulin polymerization at 30 µM. In addition, avermectin B1a induced apoptosis in HCT-116 cells and substantially diminished their ability to migrate. Avermectin B1a exhibits significant anti-cancer activity and enhances tubulin polymerization, suggesting that it can be used as a promising microtubule-targeting agent for the development of future anticancer drugs.

A review on Millepachine and its derivatives as potential multitarget anticancer agents.

Chalcones have a long history of being used for many medical purposes. These are the most prestigious scaffolds in medicine. The potential of Millepachine and its derivatives to treat various malignancies has been demonstrated in this review. The anticancer effects of Millepachine and its derivatives on ovarian cancer, hepatocellular carcinoma, breast, liver, colon, cervical, prostate, stomach, and gliomas are highlighted in the current review. Several genes that are crucial in reducing the severity of the disease have been altered by these substances. They mainly work by preventing tubulin polymerizing. They also exhibit apoptosis and cell cycle arrest at the G2/M phase. Additionally, these compounds inhibit invasion and migration and have antiproliferative effects. Preclinical studies have shown that Millepachine and its derivatives offer exceptional potential for treating a number of cancers. These results need to be confirmed in clinical research in order to develop viable cancer therapies.

Structure Guided Design, Synthesis, and Biological Evaluation of Oxetane-Containing Indole Analogues.

The oxetane functional group offers a variety of potential advantages when incorporated within appropriate therapeutic agents as a ketone surrogate. OXi8006, a 2-aryl-3-aroyl-indole analogue, functions as a small-molecule inhibitor of tubulin polymerization that has a dual mechanism of action as both an antiproliferative agent and a tumor-selective vascular disrupting agent. Replacement of the bridging ketone moiety in OXi8006 with an oxetane functional group has expanded structure activity relationship (SAR) knowledge and provided insights regarding oxetane incorporation within this class of molecules. A new synthetic method using an oxetane-containing tertiary alcohol subjected to Lewis acid catalyzed conditions led to successful Friedel-Crafts alkylation and yielded fourteen new oxetane-containing indole-based molecules. This synthetic approach represents the first method to successfully install an oxetane ring at the 3-position of a 2-aryl-indole system. Several analogues showed potent cytotoxicity (micromolar GI50 values) against human breast cancer cell lines (MCF-7 and MDA-MB-231) and a pancreatic cancer cell line (PANC-1), although they proved to be ineffective as inhibitors of tubulin polymerization. Molecular docking studies comparing colchicine with the OXi8006-oxetane analogue 5m provided a rationale for the differential interaction of these molecules with the colchicine site on the tubulin heterodimer.

Probing a distinct druggable tubulin binding site with gatorbulins 1-7, their metabolic and physicochemical properties, and pharmacological consequences.

Microtubules, consisting of α/ß-tubulin heterodimers, are prime targets for anticancer drug discovery. Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2-7 (1b-1g) of this highly modified cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and ß-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDR1/P-gp, showing similar permeability with and against transporter gradient, indicating that GB1 (1a) is a poor P-gp substrate.

Development of QTMP: A promising anticancer agent through NP-Privileged Motif-Driven structural modulation.

In this study of creating new molecules from clinical trial agents, an approach of Combretastatin structural modulation with the installation of NP-privileged motifs was considered, and a series of trimethoxyphenyl-2-aminoimidazole with functionalized quinolines and isoquinolines was investigated. An exciting method of quinoline C3-H iodination coupled with imidazopyridine-C3-H arylation and hydrazine-mediated fused-ring cleavage enabled synthesizing a class of compounds with two specific unsymmetric aryl substitutions. Interestingly, three compounds (6, 11, and 13) strongly inhibited HeLa cell proliferation with a half-maximal inhibitory concentration (10-46 nM). Among the compounds, compound 6 (QTMP) showed stronger antiproliferative ability than CA-4 (a clinical trial agent) in various cancer cell lines, including cervical, lung, breast, highly metastatic breast, and melanoma cells. QTMP inhibited the assembly of purified tubulin, depolymerized microtubules of A549 lung carcinoma cells, produced defective spindles, and arrested the cells in the G2/M phase. Further, QTMP binds to the colchicine site in tubulin with a dissociation constant of 5.0 ± 0.6 µM. QTMP displayed higher aqueous stability than CA-4 at 37 °C. Further, in silico analysis of QTMP indicated excellent drug-like properties, including good aqueous solubility, balanced hydrophilicity-lipophilicity, and high GI-absorption ability. The results together suggest that QTMP has anticancer potential.

Design, synthesis, cytotoxic activities, and molecular docking of chalcone hybrids bearing 8-hydroxyquinoline moiety with dual tubulin/EGFR kinase inhibition.

The present study established thirteen novel 8-hydroxyquinoline/chalcone hybrids3a-mof hopeful anticancer activity. According to NCI screening and MTT assay results, compounds3d-3f, 3i,3k,and3ldisplayed potent growth inhibition on HCT116 and MCF7 cells compared to Staurosporine. Among these compounds,3eand3fshowed outstanding superior activity against HCT116 and MCF7 cells and better safety toward normal WI-38 cells than Staurosporine. The enzymatic assay revealed that3e,3d, and3ihad goodtubulin polymerization inhibition (IC50 = 5.3, 8.6, and 8.05 µM, respectively) compared to the reference Combretastatin A4 (IC50 = 2.15 µM). Moreover,3e,3l, and3fexhibited EGFR inhibition (IC50 = 0.097, 0.154, and 0.334 µM, respectively) compared to Erlotinib (IC50 = 0.056 µM). Compounds3eand3fwere investigated for their effects on the cell cycle, apoptosis induction, andwnt1/ß-cateningene suppression. The apoptosis markers Bax, Bcl2, Casp3, Casp9, PARP1, and ß-actin were detected by Western blot. In-silico molecular docking, physicochemical, and pharmacokinetic studies were implemented for the validation of dual mechanisms and other bioavailability standards. Hence, Compounds3eand3fare promising antiproliferative leads with tubulin polymerization and EGFR kinase inhibition.

New antiproliferative 3-substituted oxindoles inhibiting EGFR/VEGFR-2 and tubulin polymerization.

New 3-substituted oxindole derivatives were designed and synthesized as antiproliferative agents. The antiproliferative activity of compounds 6a-j was evaluated against 60 NCI cell lines. Among these tested compounds, compounds 6f and 6g showed remarkable antiproliferative activity, specifically against leukemia and breast cancer cell lines. Compound 6f was the most promising antiproliferative agent against MCF-7 (human breast cancer) with an IC50 value of 14.77 µM compared to 5-fluorouracil (5FU) (IC50 = 2.02 µM). Notably, compound 6f hampered receptor tyrosine EGFR fundamentally with an IC50 value of 1.38 µM, compared to the reference sunitinib with an IC50 value of 0.08 µM. Moreover, compound 6f afforded anti-tubulin polymerization activity with an IC50 value of 7.99 µM as an outstanding observable activity compared with the reference combretastatin A4 with an IC50 value of 2.64 µM. In silico molecular-docking results of compound 6f in the ATP-binding site of EGFR agreed with the in vitro results. Besides, the investigation of the physicochemical properties of compound 6f via the egg-boiled method clarified good lipophilicity, GIT absorption, and blood-brain barrier penetration properties.

New Combretastatin Analogs as Anticancer Agents: Design, Synthesis, Microtubules Polymerization Inhibition, and Molecular Docking Studies.

A new series of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol derivatives were synthesized as analogs for the anticancer drug combretastatin A-4 (CA-4) and characterized using FT-IR, 1 H-NMR, 13 CNMR, and HR-MS techniques. The new CA-4 analogs were designed to meet the structural requirements of the highest expected anticancer activity of CA-4 analogs by maintaining ring A 3,4,5-trimethoxyphenyl moiety, and at the same time varying the substituents effect of the triazole moiety (ring B). In silico analysis indicated that compound 3 has higher total energy and dipole moment than colchicine and the other analogs, and it has excellent distribution of electron density and is more stable, resulting in an increased binding affinity during tubulin inhibition. Additionally, compound 3 was found to interact with three apoptotic markers, namely p53, Bcl-2, and caspase 3. Compound 3 showed strong similarity to colchicine, and it has excellent pharmacokinetics properties and a good dynamic profile. The in vitro anti-proliferation studies showed that compound 3 is the most cytotoxic CA-4 analog against cancer cells (IC50 of 6.35 µM against Hep G2 hepatocarcinoma cells), and based on its selectivity index (4.7), compound 3 is a cancer cytotoxic-selective agent. As expected and similar to colchicine, compound 3-treated Hep G2 hepatocarcinoma cells were arrested at the G2/M phase resulting in induction of apoptosis. Compound 3 tubulin polymerization IC50 (9.50 µM) and effect on Vmax of tubulin polymerization was comparable to that of colchicine (5.49 µM). Taken together, the findings of the current study suggest that compound 3, through its binding to the colchicine-binding site at ß-tubulin, is a promising microtubule-disrupting agent with excellent potential to be used as cancer therapeutic agent.

Bex1 is essential for ciliogenesis and harbours biomolecular condensate-forming capacity.

BACKGROUND: Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia. RESULTS: Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system. CONCLUSIONS: Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization.