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IMB5046 binds to tubulin at the colchicine pocket.: (a) Limited proteolysis assay. Tubulin treated with IMB5046 showed similar patterns of proteolysis with that of colchicine. Representative result of three independent experiments is shown. The histogram shows the relative density of each fragment. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01 versus Ctrl. (b) Colchicine competition assay. IMB5046 and nocodazole decreased the intrinsic colchicine fluorescence, while vincristine did not. Data are expressed as mean ± SD (n = 3). (c) The superimposition of the predicted binding mode of colchicine (in stick, carbon in orange) with the crystal structure (in stick, carbon in grey), as well as the predicted binding mode of IMB5046 (in stick, carbon in purple). (d) The detailed interactions between IMB5046 and tubulin.
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Multidrug resistance is a major limitation for microtubule-binding agents in cancer treatment. Here we report a novel microtubule inhibitor (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester, IMB5046), its cytotoxicity against multidrug-resistant cell lines and its antitumor efficacy in animal models. IMB5046 disrupted microtubule...
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... there are two established binding sites on tubulin, the vinca site and the colchicine site. A limited proteolysis assay is widely used to investigate the binding site of drugs on tubulin 10,11 . Binding of drugs to tubulin dimer will cause protein confor- mational changes, resulting in different patterns of proteolysis by trypsin. As shown in Fig. 3a, pre-incubation of tubulin with 100 μM IMB5046 followed by limited trypsin digestion produced an enhanced βcol fragment which was similar to that caused by colchicine. The vinca site agent vincristine substantially decreased the intensity of the βcol, αN and αC fragments. These results indicate that IMB5046 binds to tubulin at the ...
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... further confirm the result, a colchicine competition assay was performed. As reported, competi- tion between the test compound and colchicine for the binding site will decrease the intrinsic fluorescence of colchicine-tubulin complex by reducing the amount of colchicine bound 12 . As shown in Fig. 3b, IMB5046 decreased the intrinsic colchicine fluorescence in a concentration-dependent manner. As a positive control, noc- odazole which is known to bind at the colchicine site decreased the fluorescence, while vincristine had no effect on the fluorescence. These results further support that IMB5046 binds to tubulin at the colchicine ...
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... study was conducted to explore the interaction between tubulin and IMB5046. In a docking study, colchicine, the original ligand in the crystal structure, was selected as the reference compound, and the predicted binding mode generated through molecular docking was quite closed to that in the crystal structure, with an RMSD value of 0.50 Å (Fig. 3c), which indicates the docking results are reasonable. Therefore, IMB5046 was docked into the binding site of the catalytic domain in the same protocol, and the docking score of IMB5046 (−21.7 kcal/mol) was comparable to that of colchicine (−21.9 kcal/mol). In particular, IMB5046 forms a hydrogen bond interaction with Lys254, as well as ...
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... IMB5046 was docked into the binding site of the catalytic domain in the same protocol, and the docking score of IMB5046 (−21.7 kcal/mol) was comparable to that of colchicine (−21.9 kcal/mol). In particular, IMB5046 forms a hydrogen bond interaction with Lys254, as well as hydrophobic interactions with Leu248, Ala317 of the β subunit at the site (Fig. 3d), exhibiting a different binding mode from colchicine, which mainly forms hydrophobic interactions with α and β subunits. Thus, IMB5046, with a novel scaffold, targets the colchicine-binding site of tubulin in a divergent mode, and may provide a type of promising lead compound. (Table 1a). As shown, A431, HT-1080 and HT29 cells were ...
Citations
... We wished to investigate the preclinical potential of a panel of related (E)-9-(2-nitrovinyl)anthracenes as antiproliferative compounds in CLL, which is a more common but related B-cell malignancy. Nitro-group-containing compounds may induce selective cancer cell toxicity by diverse mechanisms [46] such as topoisomerase inhibition [47], histone deacetylase inhibition [48], DNA alkylation [49] or tubulin polymerisation inhibition [50][51][52], while anthracene-containing compounds are reported to interact selectively with G-quadruplex structures and inhibit telomerase [53]. The objective of this research was the investigation of a series of (E)-9-(2-nitrovinyl)anthracenes and related nitrostyrene compounds for antiprolifertive evaluation in BL and CLL cell lines and is arranged as follows: ...
... The reduction of (E)-9-(2-nitrovinyl)anthracene was also investi- The nitrostyrene-containing compounds such as 10a-c reduced cell viability effectively in both BL and CLL cell lines and were superior to the clinical drugs fludarabine phosphate and taxol [39,45]. The IC 50 values in the CLL cell lines were in the low-micromolar range (2-5 µM) irrespective of IGVH mutational status (I83 and PGA-1: mutated-IGVH; HG-3 and CII: unmutated-IGVH). This result compared favourably with IC 50 values of 20-50 µM obtained for fludarabine phosphate (a current CLL frontline treatment) in these cell lines. ...
... We wished to investigate the preclinical potential of a panel of related (E)-9-(2-nitrovinyl)anthracenes as antiproliferative compounds in CLL, which is a more common but related B-cell malignancy. Nitro-group-containing compounds may induce selective cancer cell toxicity by diverse mechanisms [46] such as topoisomerase inhibition [47], histone deacetylase inhibition [48], DNA alkylation [49] or tubulin polymerisation inhibition [50][51][52], while anthracene-containing compounds are reported to interact selectively with G-quadruplex structures and inhibit telomerase [53]. ...
Chronic lymphocytic leukaemia (CLL) is a malignancy of the immune B lymphocyte cells and is the most common leukaemia diagnosed in developed countries. In this paper, we report the synthesis and antiproliferative effects of a series of (E)-9-(2-nitrovinyl)anthracenes and related nitrostyrene compounds in CLL cell lines and also in Burkitt’s lymphoma (BL) cell lines, a rare form of non-Hodgkin’s immune B-cell lymphoma. The nitrostyrene scaffold was identified as a lead structure for the development of effective compounds targeting BL and CLL. The series of structurally diverse nitrostyrenes was synthesised via Henry–Knoevenagel condensation reactions. Single-crystal X-ray analysis confirmed the structure of (E)-9-chloro-10-(2-nitrobut-1-en-1-yl)anthracene (19f) and the related 4-(anthracen-9-yl)-1H-1,2,3-triazole (30a). The (E)-9-(2-nitrovinyl)anthracenes 19a, 19g and 19i–19m were found to elicit potent antiproliferative effects in both BL cell lines EBV−MUTU-1 (chemosensitive) and EBV+ DG-75 (chemoresistant) with >90% inhibition at 10 μM. Selected (E)-9-(2-nitrovinyl)anthracenes demonstrated potent antiproliferative activity in CLL cell lines, with IC50 values of 0.17 μM (HG-3) and 1.3 μM (PGA-1) for compound 19g. The pro-apoptotic effects of the most potent compounds 19a, 19g, 19i, 19l and 19m were demonstrated in both CLL cell lines HG-3 and PGA-1. The (E)-nitrostyrene and (E)-9-(2-nitrovinyl)anthracene series of compounds offer potential for further development as novel chemotherapeutics for CLL.
... In our previous work, a QSAR predictive model was developed using 213 CBS inhibitors against the A549 cancer cell line. The inhibitor molecules were taken from PubMed literature data from the last 10 years of literature [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. The evaluation of the applicability domain (AD) defines that the QSAR model is reliable for making predictions within the defined chemical space. ...
... 18,50 The cytotoxicities of compounds containing this functional group have been described through various mechanisms, such as inhibition of topoisomerase, 51 alkylation of DNA, 52 or inhibition of tubulin polymerization. 53,54 In line with previous studies in the development of curcumin analogue-fused heterocycle, herein, we reported MAC-fused 1-Aryl-1H-pyrazole derivatives as desired anticancer agents. ...
Addressing the growing burden of cancer and the shortcomings of chemotherapy in cancer treatment are the current research goals. Research to overcome the limitations of curcumin and to improve its anticancer activity via its heterocycle-fused monocarbonyl analogues (MACs) has immense potential. In this study, 32 asymmetric MACs fused with 1-aryl-1H-pyrazole (7a-10h) were synthesized and characterized to develop new curcumin analogues. Subsequently, via initial screening for cytotoxic activity, nine compounds exhibited potential growth inhibition against MDA-MB-231 (IC50 2.43-7.84 μM) and HepG2 (IC50 4.98-14.65 μM), in which seven compounds showing higher selectivities on two cancer cell lines than the noncancerous LLC-PK1 were selected for cell-free in vitro screening for effects on microtubule assembly activity. Among those, compounds 7d, 7h, and 10c showed effective inhibitions of microtubule assembly at 20.0 μM (40.76-52.03%), indicating that they could act as microtubule-destabilizing agents. From the screening results, three most potential compounds, 7d, 7h, and 10c, were selected for further evaluation of cellular effects on breast cancer MDA-MB-231 cells. The apoptosis-inducing study indicated that these three compounds could cause morphological changes at 1.0 μM and could enhance caspase-3 activity (1.33-1.57 times) at 10.0 μM in MDA-MB-231 cells, confirming their apoptosis-inducing activities. Additionally, in cell cycle analysis, compounds 7d and 7h at 2.5 μM and 10c at 5.0 μM also arrested MDA-MB-231 cells in the G2/M phase. Finally, the results from in silico studies revealed that the predicted absorption, distribution, metabolism, excretion, and the toxicity (ADMET) profile of the most potent MACs might have several advantages in addition to potential disadvantages, and compound 7h could bind into (ΔG -10.08 kcal·mol-1) and access wider space at the colchicine-binding site (CBS) than that of colchicine or nocodazole via molecular docking studies. In conclusion, our study serves as a basis for the design of promising synthetic compounds as anticancer agents in the future.
... Nitrobenzoate-based compounds have been reported to exert various biological activities, including antimalarial [19], antibiotic [20], and anticancer [21,22] activities. Nitrobenzoate-based compounds have been reported to suppress metastatic activity by inhibiting the mucin-type, transmembrane glycoprotein, podoplanin-stimulated tumorcell-induced platelet aggregation [22] and inhibit multidrug-resistant cancer by inhibiting tubulin polymerization [23]. Although some biological activities of nitrobenzoate-derived compounds have been studied, the angiogenesis-related biological activity of nitrobenzoatederived compounds is much less studied. ...
Proper growth and patterning of blood vessels are critical for embryogenesis. Chemicals or environmental hormones may interfere with vascular growth and cause developmental defects. Nitrobenzoate-based compounds have been demonstrated to have a wide range of biological and pharmacological functions, leading to the development of numerous 4-nitrobenzoate derivatives for clinical application. In this study, we tested a novel nitrobenzoate-derived compound, X8, and investigated its effects on vascular development using zebrafish as a model organism. We first determined the survival rate of embryos after the addition of exogenous X8 (0.5, 1, 3, 5, and 10 μM) to the fish medium and determined a sublethal dose of 3 μM for use in further assays. We used transgenic fish to examine the effects of X8 treatment on vascular development. At 25-32 h postfertilization (hpf), X8 treatment impaired the growth of intersegmental vessels (ISVs) and caudal vein plexuses (CVPs). Moreover, X8-treated embryos exhibited pericardial edema and circulatory defects at 60-72 hpf, suggesting the effects of X8 in vasculature. Apoptosis tests showed that the vascular defects were likely caused by the inhibition of proliferation and migration. To investigate the molecular impacts underlying the defects in the vasculature of X8-treated fish, the expression levels of vascular markers, including ephrinb2, mrc1, and stabilin, were assessed, and the decreased expression of those genes was detected, indicating that X8 inhibited the expression of vascular genes. Finally, we showed that X8 treatment disrupted exogenous GS4012-induced angiogenesis in Tg(flk:egfp) zebrafish embryos. In addition, vascular defects were enhanced during cotreatment with X8 and the VEGFR2 inhibitor SU5416, suggesting that X8 treatment causes vascular defects mediated by disruption of VEGF/VEGFR2 signaling. Collectively, our findings indicate that X8 could be developed as a novel antiangiogenic agent.
... Pf-tubulin polymerization was carried out, as described previously, in 200 μL of ice-cold re-assembly buffer (100 mM PIPES, pH 6.9, 10 % glycerol, 2 mM MgCl 2 ⋅H 2 O and 0.5 mM EGTA), containing 1 mM GTP and different equimolar concentrations (0.5, 1.0, 2.5, 5, 10, 20 and 40 μM) of PfαI-and Pfβ-tubulins [53][54][55][56][57]. The reaction mixture was prepared in clear, flat bottom 96-well plates. ...
Development of resistance to current antimalarial therapies remains a significant source of concern. To address this risk, new drugs with novel targets in distinct developmental stages of Plasmodium parasites are required. In the current study, we have targeted P. falciparum Tubulin (PfTubulin) proteins which represent some of the potential drug targets for malaria chemotherapy. Plasmodial microtubules (MTs) play crucial role during parasite proliferation, growth, and transmission, which render them highly desirable targets for the development of next-generation chemotherapeutics. Towards this, we have evaluated the antimalarial activity of Tubulin targeting compounds received from the Medicines for Malaria Venture (MMV) “Pathogen Box” against the human malaria parasite, P. falciparum (including 3D7 (chloroquine and artemisinin sensitive strain), RKL-9 (chloroquine resistant strain) and R539T (artemisinin resistant strains). At nanomolar concentrations, the filtered out compounds exhibited pronounced multistage antimalarial effects across the parasite life cycle, including intra-erythrocytic blood stages, liver stage parasites, gametocytes and ookinetes. Concomitantly, these compounds were found to impede male gamete ex-flagellation, thus showing their transmission-blocking potential . Target mining of these potent compounds, by combining in silico, biochemical and biophysical assays, implicated PfTubulin as their molecular target, which may possibly act by disrupting MT assembly dynamics by binding at the interface of α-βTubulin-dimer. Further, promising ADME profile of the parent scaffold supported its consideration as a lead compound for further development. Thus, our work highlights the potential of targeting PfTubulin proteins in discovering and developing next-generation, multistage antimalarial agents for treating Multi-Drug Resistant (MDR) malaria parasites.
... A total of 213 colchicine binding inhibitors against the A549 cancer cell line were collected from the PubMed literature of the last 10 years (Fig. 2). All of these compounds were divided into three groups, Sublib-I, Sublib-II, and Sublib-III, [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] based on the types of molecules. All of the molecules with their IC 50 values are presented in Table S1-S17, which are available in supplementary material. ...
Microtubule targeting agents often have limitations to the development of resistance. Colchicine binding site agents have several advantages as compared to other tubulin inhibitors. Numerous medications in this class are less susceptible to multidrug resistance that restricts the viability of different inhibitors. In the present study, molecules that bind to the colchicine binding site of tubulin are collected from PubMed literature against the A549 cancer cell line. Regression models were established between the descriptor and IC50 value of all the compounds present in the training set based on significant molecular fingerprints using Multiple Linear Regression (MLR). Fifteen most significant descriptors selected include – Burden Modified Eigenvalues Descriptors, PaDEL Weighted Path Descriptor, Auto-correlation Descriptor, Topological Distance Matrix Descriptor, MLFER Descriptor, Barysz Matrix Descriptor, Chi-Path Cluster Descriptor, and validated using internal and external validation parameters. The selected MLR-GA model has R2adjusted = 0.7895, Q2CV= 0.76577, R2pred= 0.7419, R2tes=0.77373). An applicability domain is also defined so that it defines the chemical space that the model can predict. The above details suggest a good predictive model for colchicine binding site inhibitors that can predict the IC50 value of the unknown chemical compound. Keywords: IC50, Microtubule, Colchicine, Descriptor, Regression, QSAR
... Mice were treated with PST-3 in 0.1% Captex200/Tween80 (1/4) at 20, 40 or 60 mg/kg by intraperitoneal injection. Under the same conditions, Colchicine at 0.05 mg/ kg/day (maximum tolerated dose) was a positive control [37]. Carry the rotating experience two times a week. ...
The composition of microtubules involving several steps, including the polymerization and depolymerization of α-tubulin and β-tubulin heterodimers. Microtubule-targeting agents can increase or inhibit microtubule polymerization, thereby disrupting the dynamic process and stalling cells in G2/M phase. Microtubule-targeting agents are generally cytotoxic, which neurological toxicity being one of the significant adverse events associated. We recently reported a novel 5-arylalkynyl-2-benzoyl thiophene (PST-3) that exhibited broad-spectrum cellular cytotoxicity and in vivo potency with high safety. PST-3 was a substrate of p-gp, which could not cross the blood-brain barrier and lead to less neurotoxicity. The antitumor activities in vitro demonstrated that PST-3 combined with the colchicine-binding site on microtubule, induces morphological changes, disrupts microtubule networks, inhibits polymerization of tubulin, arrests breast cancer cells in the G2/M phase of the cell cycle and induces apoptosis. Evaluation of the antitumor effect in vivo demonstrated that PST-3 elicited MDA-MB-468 tumor %T/C of 11.75%, whereas elicited MCF7 tumor %T/C of 44.38% in breast cancer xenograft models. Besides, in vivo experiments of a higher dose (60 mg/kg) of PST-3 treatment for 21 days did not produce any significant neurotoxicity. These results provide evidence that PST-3 might possess the potential to be developed into a new microtubule inhibitor without neurological toxicity.
... So far, there are varying reports of resistance to spindle poisons because of the evasive properties conferred on tumor cells via the actions of CAPs. This has led to discoveries of new drugs, such as IMB5046 and epothilones, which confer significant antitumor effects on cells resistant to colchicine, vincristine, and paclitaxel [26,27]. IMB5046 is a newly developed MT-depolymerizing agent that exhibits potent cytotoxicity against multi-drug-resistant cell lines and in mice at well-tolerated doses. ...
... IMB5046 possesses a unique chemical structure and does not bind to P-glycoprotein (P-gp) unlike most MT-binding agents; hence, IMB5046 can circumvent P-gp-mediated multidrug resistance in cancers. Moreover, this feature distinguishes it in its mechanism of action as it functions by binding to the colchicine pockets on tubulins to inhibit MT polymerization in cancer cells [26]. ...
The traditional functions of cytoskeletal-associated proteins (CAPs) in line with polymerization and stabilization of the cytoskeleton have evolved and are currently underrated in oncology. Although therapeutic drugs have been developed to target the cytoskeletal components directly in cancer treatment, several recently established therapeutic agents designed for new targets block the proliferation of cancer cells and suppress resistance to existing target agents. It would seem like these targets only work toward inhibiting the polymerization of cytoskeletal components or hindering mitotic spindle formation in cancer cells, but a large body of literature points to CAPs and their culpability in cell signaling, molecular conformation, organelle trafficking, cellular metabolism, and genomic modifications. Here, we review those underappreciated functions of CAPs, and we delineate the implications of cellular signaling instigated by evasive properties induced by aberrant expression of CAPs in response to stress or failure to exert normal functions. We present an analogy establishing CAPs as vulnerable targets for cancer systems and credible oncotargets. This review establishes a paradigm in which the cancer machinery may commandeer the conventional functions of CAPs for survival, drug resistance, and energy generation; an interesting feature overdue for attention.
... First, since the expression level of STIM1 and EB1 in different types of cells could be variable (Roos et al., 2005;DeHaven et al., 2007), the different cell lines applied in those studies might lead to distinct results. Moreover, the treatment duration of taxol in Grigoriev's study last for only 2 h, and which is much shorter than the 24 h in other study (Dowdy et al., 2006;Zheng et al., 2016). Insufficient treatment might influence the effect of microtubule stabilization. ...
Store-operated Ca²⁺ entry (SOCE) is an essential pathway for Ca²⁺ signaling, and regulates various vital cellular functions. It is triggered by the endoplasmic reticulum Ca²⁺ sensor stromal interaction molecule 1 (STIM1). Illustration of STIM1 spatiotemporal structure at the nanometer scale during SOCE activation provides structural and functional insights into the fundamental Ca²⁺ homeostasis. In this study, we used direct stochastic optical reconstruction microscopy (dSTORM) to revisit the dynamic process of the interaction between STIM1, end-binding protein (EB), and microtubules to the ER-plasma membrane. Using dSTORM, we found that“powder-like”STIM1 aggregates into “trabecular-like” architectures toward the cell periphery during SOCE, and that an intact microtubule network and EB1 are essential for STIM1 trafficking. After thapsigargin treatment, STIM1 can interact with EB1 regardless of undergoing aggregation. We generated STIM1 variants adapted from a real-world database and introduced them into SiHa cells to clarify the impact of STIM1 mutations on cancer cell behavior. The p.D76G and p.D84Y variants locating on the Ca²⁺ binding domain of STIM1 result in inhibition of focal adhesion turnover, Ca²⁺ influx during SOCE and subsequent cell migration. Inversely, the p.R643C variant on the microtubule interacting domain of STIM1 leads to dissimilar consequence and aggravates cell migration. These findings imply that STIM1 mutational patterns have an impact on cancer metastasis, and therefore could be either a prognostic marker or a novel therapeutic target to inhibit the malignant behavior of STIM1-mediated cancer cells. Altogether, we generated novel insight into the role of STIM1 during SOCE activation, and uncovered the impact of real-world STIM1 variants on cancer cells.
... 37 As a cytoskeleton, the microtubule has a unique morphological structure, after fixation and staining, the morphological characteristics of a microtubule can be observed under a high-resolution microscope. 38 By using a transmission electron microscope, Zheng Y B et al. 39 and S. Ez-Calvo G et al. 40 have successfully observed the collapse of the microtubule network caused by tubulin inhibitors, which provided an effective method for the screening of tubulin inhibitors. However, this method is usually not used for early screening of inhibitors, only for verifying their microtubule inhibiting activity. ...
... On the contrary, the molecules acting on the vinblastine site make the region fold more tightly, 105 thus eliminating the accessibility of the site to be hydrolyzed by trypsin and significantly reducing the intensity of βcol, αN and αC fragments. 39 For the molecules acting on the tubulin paclitaxel site, a limited number of trypsin can induce another new cleavage mode of tubulin, which not only produces αN and αC fragments, but also produces another cleavage band different from βcol. 106 This principle provides a new method for screening new candidates for tubulin inhibitor. ...
Tubulin, an important target in tumor therapy, is one of the hotspots in the field of antineoplastic drugs in recent years, and it is of great significance to design and screen new inhibitors for this target. Natural products and chemical synthetic drugs are the main sources of tubulin inhibitors. However, due to the variety of compound structure types, it has always been difficult for researchers to screen out polymerization inhibitors with simple operation, high efficiency and low cost. A large number of articles have reported the screening methods of tubulin inhibitors and their biological activity. In this article, the biological activity detection methods of tubulin polymerization inhibitors are reviewed. Thus, it provides a theoretical basis for the further study of tubulin polymerization inhibitors and the selection of methods for tubulin inhibitors.
