The compound millepachine and its derivatives inhibit tubulin polymerization by irreversibly binding to the colchicine-binding site in β-tubulin.
ABSTRACT: Inhibitors that bind to the paclitaxel- or vinblastine-binding sites of tubulin have been part of the pharmacopoeia of anticancer therapy for decades. However, tubulin inhibitors that bind to the colchicine-binding site are not used in clinical cancer therapy, because of their low therapeutic index. To address multidrug resistance to many conventional tubulin-binding agents, numerous efforts have attempted to clinically develop inhibitors that bind the colchicine-binding site. Previously, we have found that millepachine (MIL), a natural chalcone-type small molecule extracted from the plant Millettia pachycarpa, and its two derivatives (MDs) SKLB028 and SKLB050 have potential antitumor activities both in vitro and in vivo However, their cellular targets and mechanisms are unclear. Here, biochemical and cellular experiments revealed that the MDs directly and irreversibly bind β-tubulin. X-ray crystallography of the tubulin-MD structures disclosed that the MDs bind at the tubulin intradimer interface and to the same site as colchicine and that their binding mode is similar to that of colchicine. Of note, MDs inhibited tubulin polymerization and caused G2/M cell-cycle arrest. Comprehensive analysis further revealed that free MIL exhibits an s-cis conformation, whereas MIL in the colchicine-binding site in tubulin adopts an s-trans conformation. Moreover, introducing an α-methyl to MDs to increase the proportion of s-trans conformations augmented MDs' tubulin inhibition activity. Our study uncovers a new class of chalcone-type tubulin inhibitors that bind the colchicine-binding site in β-tubulin and suggests that the s-trans conformation of these compounds may make them more active anticancer agents.
Project description:Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays.
Project description:We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: (1) in silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; (2) the molecular mechanics-generalized Born/surface area (MM-GB/SA) scoring results ??G(bind-cald) for both noscapine and Br-noscapine (3.915 and 3.025 kcal/mol) are in reasonably good agreement with our experimentally determined binding affinity (??G(bind-Expt) of 3.570 and 2.988 kcal/mol, derived from K(d) values); and (3) Br-noscapine competes with colchicine binding to tubulin. The simplest interpretation of these collective data is that Br-noscapine binds tubulin at a site overlapping with, or very close to colchicine-binding site of tubulin. Although we cannot rule out a formal possibility that Br-noscapine might bind to a site distinct and distant from the colchicine-binding site that might negatively influence the colchicine binding to tubulin.
Project description:AIM:Discovery of novel series of colchicine binding site inhibitors (CBSIs). MATERIALS & METHODS:Isoxazoline 3a-d, pyrazoline 4a-b, 7a-f and 8a-f, cyclohexenone 9a-b and 10a-b or pyridine derivatives 11a-b were synthesized and evaluated for their inhibition of tubulin polymerization and cytotoxicity. Most of the compounds displayed potent to moderate antitumor activity against leukemia SR cell line.7c, 7e and 11a were more potent than colchicine with IC50 of 0.09, 0.05 and 0.06 ?M, and percentage inhibition in tubulin polymerization of 95.2, 96.0 and 96.3%, respectively. Compounds 7c and 11a showed cell-cycle arrest at G2/M phase and induced apoptosis and were able to bind the colchicine binding site of tubulin with comparable affinity to colchicine. Docking study showed that these compounds may interact with tubulin exploiting a binding cavity not commonly reported in the binding of CBSI. CONCLUSION:Compounds 7c and 11a may be considered as promising CBSI based on their excellent activity and favorable drug likeness profile.
Project description:Agents targeting colchicine-binding pocket usually show a minimal drug-resistance issue, albeit often associated with high toxicity. Chalcone-based compounds, which may bind to colchicine-binding site, are found in many edible fruits, suggesting that they can be effective drugs with less toxicity. Therefore, we synthesized and examined 24 quinolone chalcone compounds, from which we identified ((E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-17) and ((E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-20) as promising leads. In particular, CTR-20 was effective against 65 different cancer cell lines originated from 12 different tissues, largely in a cancer cell-specific manner. We found that both CTR-17 and CTR-20 reversibly bind to the colchicine-binding pocket on ?-tubulin. Interestingly however, both the CTRs were highly effective against multidrug-resistant cancer cells while colchicine, paclitaxel and vinblastine were not. Our study with CTR-20 showed that it overcomes multidrug-resistance through its ability to impede MRP1 function while maintaining strong inhibition against microtubule activity. Data from mice engrafted with the MDA-MB-231 triple-negative breast cancer cells showed that both CTR-17 and CTR-20 possess strong anticancer activity, alone or in combination with paclitaxel, without causing any notable side effects. Together, our data demonstrates that both the CTRs can be effective and safe drugs against many different cancers, especially against multidrug-resistant tumors.
Project description:A series of naphthalene-chalcone derivatives (<b>3a-3t</b>) were prepared and evaluated as tubulin polymerisation inhibitor for the treatment of breast cancer. All compounds were evaluated for their antiproliferative activity against MCF-7 cell line. The most of compounds displayed potent antiproliferative activity. Among them, compound <b>3a</b> displayed the most potent antiproliferative activity with an IC<sub>50</sub> value of 1.42?±?0.15?µM, as compared to cisplatin (IC<sub>50</sub>?=?15.24?±?1.27?µM). Additionally, the promising compound <b>3a</b> demonstrated relatively lower cytotoxicity on normal cell line (HEK293) compared to tumour cell line. Furthermore, compound <b>3a</b> was found to induce significant cell cycle arrest at the G<sub>2</sub>/M phase and cell apoptosis. Compound <b>3a</b> displayed potent tubulin polymerisation inhibitory activity with an IC<sub>50</sub> value of 8.4?µM, which was slightly more active than the reference compound colchicine (IC<sub>50</sub>?=?10.6?µM). Molecular docking analysis suggested that <b>3a</b> interact and bind at the colchicine binding site of the tubulin.
Project description:Compounds that bind at the colchicine site of tubulin have drawn considerable attention with studies indicating that these agents suppress microtubule dynamics and inhibit tubulin polymerization. Data for 18 polysubstituted pyrrole compounds are reported, including antiproliferative activity against human MDA-MB-435 cells and calculated free energies of binding following docking the compounds into models of alphabeta-tubulin. These docking calculations coupled with HINT interaction analyses are able to represent the complex structures and the binding modes of inhibitors such that calculated and measured free energies of binding correlate with an r(2) of 0.76. Structural analysis of the binding pocket identifies important intermolecular contacts that mediate binding. As seen experimentally, the complex with JG-03-14 (3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester) is the most stable. These results illuminate the binding process and should be valuable in the design of new pyrrole-based colchicine site inhibitors as these compounds have very accessible syntheses.
Project description:The crucial role of the microtubule in cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular, tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the alphabeta-tubulin colchicine site. Computational docking along with HINT (Hydropathic INTeractions) score analysis fitted these inhibitors into the colchicine site and revealed detailed structure-activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3 nM to 100 muM) such that calculated and measured free energies of binding correlate with an r(2) of 0.89 (standard error +/- 0.85 kcal mol(-1)). This correlation suggests that the activity of unknown compounds may be predicted.
Project description:Tubulin dynamics is a promising target for new chemotherapeutic agents. The colchicine binding site is one of the most important pockets for potential tubulin polymerization destabilizers. Colchicine binding site inhibitors (CBSI) exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation. A large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. CBSIs have been modified as to chemical structure as well as pharmacokinetic properties, and tested in order to find a highly potent, low toxicity agent for treatment of cancers. CBSIs are believed to act by a common mechanism via binding to the colchicine site on tubulin. The present review is a synopsis of compounds that have been reported in the past decade that have provided an increase in our understanding of the actions of CBSIs.
Project description:Displaying a strong antiproliferative activity on a wide variety of cancer cells, EAPB0203 and EAPB0503 belong to the imidazo[1,2-a]quinoxalines family of imiquimod structural analogues. EAPB0503 has been shown to inhibit tubulin polymerization. The aim of the present study is to characterize the interaction of EAPB0203 and EAPB0503 with tubulin. We combine experimental approaches at the cellular and the molecular level both in vitro and in silico in order to evaluate the interaction of EAPB0203 and EAPB0503 with tubulin. We examine the influence of EAPB0203 and EAPB0503 on the cell cycle and fate, explore the binding interaction with purified tubulin, and use a computational molecular docking model to determine the binding modes to the microtubule. We then use a drug combination study with other anti-microtubule agents to compare the binding site of EAPB0203 and EAPB0503 to known potent tubulin inhibitors. We demonstrate that EAPB0203 and EAPB0503 are capable of blocking human melanoma cells in G2 and M phases and inducing cell death and apoptosis. Second, we show that EAPB0203 and EAPB0503, but also unexpectedly imiquimod, bind directly to purified tubulin and inhibit tubulin polymerization. As suggested by molecular docking and binding competition studies, we identify the colchicine binding site on ?-tubulin as the interaction pocket. Furthermore, we find that EAPB0203, EAPB0503 and imiquimod display antagonistic cytotoxic effect when combined with colchicine, and disrupt tubulin network in human melanoma cells. We conclude that EAPB0203, EAPB0503, as well as imiquimod, interact with tubulin through the colchicine binding site, and that the cytotoxic activity of EAPB0203, EAPB0503 and imiquimod is correlated to their tubulin inhibiting effect. These compounds appear as interesting anticancer drug candidates as suggested by their activity and mechanism of action, and deserve further investigation for their use in the clinic.
Project description:Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. Tubulin isotype betaVI, however, is significantly divergent from the other isotypes in sequence, assembly properties, and function. It is the major beta-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes betaI, betaII, betaIII, and betaIotaV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype betaVI. In this investigation, we characterize the activity of various colchicine site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is approximately 95% betaVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (K(a) = 0.18 x 10(5) M(-1) vs 5.0 x 10(5) M(-1) for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 A of the colchicine binding site compared to betaI, betaII, and betaIV; three of these amino acids are also altered in betaIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb isotype betaVI is almost identical to that of its human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells.