Colchicine Site Of Tubulin Research Articles

Binding of Indanocine to the Colchicine Site on Tubulin Promotes Fluorescence, and Its Binding Parameters Resemble Those of the Colchicine Analogue AC

Indanocine, a synthetic indanone, has shown potential antiproliferative activity against several tumor types. It is different from many other microtubule-disrupting drugs, because it displays toxicity toward multidrug resistance cells. We have examined the interaction of indanocine with tubulin and determined their binding and thermodynamic parameters using isothermal titration calorimetry (ITC). Indanocine is weakly fluorescent in aqueous solution, and the binding to tubulin enhances fluorescence with a large blue shift in the emission maxima. Indanocine binds to the colchicine site of tubulin, although it bears no structural similarity with colchicine. Nevertheless, like colchicine analogue AC, indanocine is a flexible molecule in which two halves of the molecule are connected through a single bond. Also, like AC, indanocine binds to the colchicine binding site of tubulin in a reversible manner and the association reaction occurs at a faster rate compared to that of colchicine-tubulin binding. The binding kinetics was studied using stopped-flow fluorescence. The association process follows biphasic kinetics similar to that of the colchicine-tubulin interaction. The activation energy of the reaction was 10.5 +/- 0.81 kcal/mol. Further investigation using ITC revealed that the enthalpy of association of indanocine with tubulin is negative and occurs with a negative heat capacity change (DeltaC(p) = -175.1 cal mol(-1) K(-1)). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen bonding forces. Finally, we conclude that even though indanocine possesses no structural similarity with colchicine, it recognizes the colchicine binding site of tubulin and its binding properties resemble those of the colchicine analogue AC.

Carbendazim Inhibits Cancer Cell Proliferation by Suppressing Microtubule Dynamics

Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G(2)/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC(50), 10 microM) and half-maximally arrests mitosis at a similar concentration (8 microM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 microM, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (K(d), 42.8 +/- 4.0 microM). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest.

Inhibition of the Ras-Net (Elk-3) Pathway by a Novel Pyrazole that Affects Microtubules

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

Docking and hydropathic scoring of polysubstituted pyrrole compounds with antitubulin activity

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 αβ-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.

A novel class of tubulin inhibitors that exhibit potent antiproliferation and in vitro vessel-disrupting activity

Since anticancer agents that interfere with microtubule function are in widespread use and have a broad spectrum of activity against both hematological malignancies and solid tumors, there is an urgent need to develop novel tubulin inhibitors with broader activities and avoiding drug resistance. In this study, we describe the characterization of select lead compounds from a novel class of indazole-based tubulin inhibitors. Three lead compounds, TH-337, TH-482 and TH-494, exhibit potent antiproliferative activity against cell lines derived from human pancreatic carcinoma, human breast adenocarcinoma and human colorectal adenocarcinoma cells. The three compounds were also tested for cytotoxicity against a panel of clinically relevant drug resistant cancer cell lines that either overexpress the drug resistance pumps MDR-1, MRP-1 and BCRP-1 or have altered Topoisomerase II activity. TH-482 and -494 retained cytotoxic activities against all of the resistant cell lines tested; however, TH-337 exhibited decreased cytotoxicity in the cell line overexpressing BCRP-1, indicating that TH-337 is a substrate of that pump. We show that TH-482's antiproliferative activity is due to cell cycle arrest at the G(2)/M phase. We demonstrate that TH-482 binds specifically to the colchicine site of tubulin and that it inhibits tubulin polymerization in vitro in a concentration-dependent manner. The in vitro anti-vascular activities of TH-482 were assessed using the HUVEC-C cell line. TH-482 inhibits in vitro neovessel formation and disrupts pre-established vessels using HUVEC-C cells. TH-482 also increases permeability of vascular endothelial cells in a concentration- and time-dependent manner. TH-482 demonstrates potent in vitro efficacy as a novel tubulin-targeted anti-proliferative and anti-vascular agent and notably is more potent in antiproliferative assays than the benchmark compound combretastatin A-4. These results identify TH-482 as a potent tubulin inhibitor, and support the investigation of its in vivo efficacy and pharmacokinetic properties as the prototype of a new class of anti-tubulin agents.

Ligands of the colchicine site of tubulin: A common pharmacophore and new structural classes

Structure-activity relationships for ligands of the colchicine site of tubulin were analyzed based on their common pharmacophore. The role of the elucidation of the three-dimensional structure of the colchicine site of tubulin on the development of studies aimed at the search for the ligands of this site is analyzed.

Novel imidazole-based combretastatin A-4 analogues: Evaluation of their in vitro antitumor activity and molecular modeling study of their binding to the colchicine site of tubulin

The in vitro antitumor activity of novel combretastatin-like 1,5- and 1,2-diaryl-1 H-imidazoles was evaluated against the NCI 60 human tumor cell lines panel. Compounds 2d and 2g proved to be more cytotoxic than CA-4 in tests involving their evaluation over a 10 −4–10 −8 range. Docking experiments showed a good correlation between the MG_MID Log GI 50 values of all these compounds and their calculated interaction energies with the colchicine binding site of αβ-tubulin.

Novel Cytotoxic Agents for Non-small Cell Lung Cancer

Cytotoxic agents have unequivocal activity in non-small cell lung cancer. Currently available agents have demonstrated the ability to prolong life and improve quality of life in advanced disease, and to increase the rate of cure when used in stage I and II disease in the adjuvant setting and as part of chemoradiotherapy in stage III disease. These agents have served as the core regimen to which agents effecting newly discovered molecular targets are added. However, there is little question that there is much room for improvement. A number of new agents have been identified that either derive from older drugs or affect their targets in novel ways. In particular, tubulin has been an attractive target since the dawn of medical oncology. A number of new antitubulin agents are currently in development. Epothilones are novel agents that bind to the same site as taxanes, yet are structurally distinct. Xyotax and abraxane are agents in which paclitaxel is delivered in a novel fashion that may both reduce toxicity and enhance activity. ABT 751 is an agent that targets the colchicine site of tubulin. Another approach is to enhance the activity of currently available agents by targeting detoxification and resistance pathways. TLK-286 is a novel agent that may enhance the activity of previously available agents by inhibiting GST-pi, a detoxifying mechanism that may be of particular relevance to platinum agents. It has also demonstrated some single-agent cytotoxic activity.

In vivo synergy combining oxaliplatin with AVE8062, a vascular-disrupting agent

13074 Background: AVE8062 is a synthetic, water-soluble compound, analog of Combretastatin A4, currently in Phase I clinical trials. It inhibits tubulin polymerization through binding to the colchicine site of tubulin. In vivo, it induces rapid tumor blood flow shutdown and necrosis, leading to tumor regressions (Proc AACR, 2002). Previously (Proc AACR, 2003), we reported a high synergy resulting from the combination of AVE8062 with either cisplatin or carboplatin in a preclinical in vivo model. We report here the combination data of AVE8062 with oxaliplatin. Methods: A 3-arm dose-response study was performed comparing the single agents and their combination. Synergy was defined as a log cell kill (lck) of at least 1 log superior to the lck of the best single agent. The AVE8062 / oxaliplatin simultaneous combination was evaluated in female BALB/c mice bearing advanced stage (250mg) SC murine colon C51 adenocarcinoma. Drugs were administered IV with 2 intermittent injections, 4 days apart. The combination arm was evaluated over 7 dose levels. Results: At the Highest nontoxic dose (HNTD - 58 mg/kg/inj), AVE8062 was found active: 0.9 lck, 1/5 partial regression (PR) and no complete regression (CR). At the HNTD (7 mg/kg/inj), oxaliplatin was found highly active [5.8 lck, 5/5 CR, no tumor free survivors (TFS)]. The HNTD for the combination (58 mg/kg/inj of AVE8062 with 7 mg/kg/inj of oxaliplatin) was well tolerated with 11.7% body weight loss at nadir. The combination was highly active over the 2 highest dose levels with TFS on day 148 post tumor implantation (4/6 TFS at combination HNTD and 6/6 TFS at 80% HNTD). A high antitumor activity was maintained at 60% HNTD (5.9 lck, 6/6 CR and no TFS). The lowest dose tested, corresponding to 1/10 of the HNTD fractions, was found active with a lck of 1.0 and no regression. Conclusions: The combination of AVE8062 with oxaliplatin was found highly synergistic (superior activity than either of the single agents alone). The combination was well tolerated with a combination toxicity index of 2, indicating that there was no overlap in host toxicity. These data provide a good rationale to perform Phase I combination trials combining AVE8062 with oxaliplatin. [Table: see text]

Synthesis and Biological Evaluation of 2-Amino-3-(3‘,4‘,5‘-trimethoxybenzoyl)-5-aryl Thiophenes as a New Class of Potent Antitubulin Agents

A new series of compounds in which the 2-amino-5-chlorophenyl ring of phenstatin analogue 7 was replaced with a 2-amino-5-aryl thiophene was synthesized and evaluated for antiproliferative activity and for inhibition of tubulin polymerization and colchicine binding to tubulin. 2-Amino-3-(3',4',5'-trimethoxybenzoyl)-5-phenyl thiophene (9f) as well as the p-fluoro-, p-methyl-, and p-methoxyphenyl substituted analogues (9i, j, and l, respectively) displayed high antiproliferative activities with IC50 values from 2.5 to 6.5 nM against the L1210 and K562 cell lines. Compounds 9i and j were more active than combretastatin A-4 as inhibitors of tubulin polymerization. Molecular docking simulations to the colchicine site of tubulin were performed to determine the possible binding mode of 9i. The results obtained demonstrated that antiproliferative activity correlated well with the inhibition of tubulin polymerization and the lengthening of the G2/M phase of the cell cycle. Moreover, a good correlation was found between these inhibitory effects and the induction of apoptosis in cells treated with the compounds.

Sulfonamide Drugs Binding to the Colchicine Site of Tubulin: Thermodynamic Analysis of the Drug−Tubulin Interactions by Isothermal Titration Calorimetry

The discovery of several sulfonamide drugs paved the way toward the synthesis of 6 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide, E7010) and 7 (N-(3-fluoro-4-methoxyphenyl)pentafluorobenzenesulfonamide, T138067), both of which inhibit tubulin polymerization and are under clinical development. A series of diarylsulfonamides containing an indole scaffold was also found to have antimitotic properties, but their mode of interactions with tubulin has remained unidentified so far. In this study, we demonstrate that these sulfonamide drugs bind to the colchicine site of tubulin in a reversible manner. They quenched intrinsic tryptophan fluorescence of tubulin presumably due to drug-induced conformational changes in the protein, but were unable to modulate GTPase activity of tubulin in contrast to colchicine that enhances the same enzymatic activity. Further investigation using isothermal titration calorimetry (ITC) revealed that 5 (N-(5-chloro-7-indolyl)-4-methoxybenzenesulfonamide) afforded a large positive value of heat capacity change (DeltaC(p)() = +264 cal mol(-1) K(-1)) on binding to tubulin, suggesting a substantial conformational transition in the protein along with partial enthalpy-entropy compensation. On the other hand, the 2-chloro regioisomer 2 gave a large negative value of DeltaC(p)() (-589 cal mol(-1) K(-1)) along with complete enthalpy-entropy compensation. This thermodynamic profile was thought to be attributable to a prominent contribution of van der Waals interaction and hydrogen bonding between specific groups in the drug-tubulin complex. These results indicate that a mere alteration in the position of a single substituent chlorine on the indole scaffold has a great influence on the drug-tubulin binding thermodynamics.

Synthesis of B-ring homologated estradiol analogues that modulate tubulin polymerization and microtubule stability.

2-Methoxyestradiol is a cytotoxic human metabolite of estradiol with the ability to bind to the colchicine site of tubulin and inhibit its polymerization, and its 2-ethoxy analogue is even more potent. On the basis of a hypothetical relationship between the structures of colchicine and 2-methoxyestradiol, a B-ring-expanded 2-ethoxyestradiol analogue was synthesized in which the B-ring of the steroid is replaced by the B-ring of colchicine. The synthesis relied on the B-ring expansion of available 6-keto estradiol derivatives as opposed to a total synthesis of the homologated steroid framework. The relative configurations of the acetamido substituents in both epimers of the final product were determined by NOESY NMR and confirmed by X-ray crystallography. The epimer having the 6alpha-acetamido substituent was more active as an inhibitor of tubulin polymerization, and it was also more cytotoxic than the 6beta-epimer. These results are consistent with the proposed structural resemblance of 2-methoxyestradiol and colchicine. Several of the synthetic intermediates proved to be potent inhibitors of tubulin polymerization. On the other hand, a 3,17beta-diacetylated, B-ring-expanded analogue of 2-ethoxyestradiol having a ketone at C-6 resembled paclitaxel (Taxol) in its ability to enhance tubulin polymerization and stabilize microtubules. The corresponding 3-acetate and the 17beta-acetate were both synthesized, and it was determined that the 17beta-acetate, but not the 3-acetate, conferred on the steroid derivative its paclitaxel-like activity.

Multiple flexible alignment with SEAL: a study of molecules acting on the colchicine binding site.

An extension of the steric and electrostatic alignment alignment (SEAL) method (MultiSEAL) is described that allows the overlay of multiple molecules and conformations. The method is well-suited for the systematic study of possible alignments, also revealing information about the conformational energies associated with a given overlay. It has been tested on three examples: angiotensin II antagonists, 5-HT3 antagonists, and dopaminergic compounds. The utility of the method is further demonstrated in an analysis of molecules that putatively bind to the colchicine site of tubulin. On the basis of its overlay with colchicine, allocolchicine, 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone, and combretastatin A-4, it appears that 2-methoxyestradiol (2-ME) is unlikely to fit the colchine site properly. The weak antimitotic activity of 2-ME may be explained by its partial fit in the site.

Antitumor agents. 199. Three-dimensional quantitative structure-activity relationship study of the colchicine binding site ligands using comparative molecular field analysis.

Inhibitors of tubulin polymerization interacting at the colchicine binding site are potential anticancer agents. We have been involved in the synthesis of a number of colchicine site agents, such as thiocolchicinoids and allocolchicinoids, which are colchicine analogues, and 2-phenyl-quinolones and 2-aryl-naphthyridinones, which are the amino analogues of cytotoxic antimitotic flavonoids. The most cytotoxic of the latter compounds strongly inhibit binding of radiolabeled colchicine to tubulin, and these agents therefore probably bind in the colchicine site of tubulin. We have applied conventional CoMFA and q(2)-GRS CoMFA to identify the essential structural requirements for increasing the ability of these compounds to form tubulin complexes. The CoMFA model for the training set of 51 compounds yielded cross-validated R(2) (q(2)) values of 0.637 for conventional CoMFA and 0.692 for q(2)-GRS CoMFA. The predictive power of this model was confirmed by successful activity prediction for a test set of 53 compounds with known potencies as inhibitors of tubulin polymerization. The activities of 88% of the compounds were predicted with absolute value of residuals of less than 0.5. The predictive q(2) values were 0.546 for conventional CoMFA and 0.426 for q(2)-GRS CoMFA. The conventional CoMFA model with the highest predictive q(2) (0.546) was analyzed in detail in terms of underlying structure-activity relationships.

Biosynthesis of Radiolabeled Curacin A and Its Rapid and Apparently Irreversible Binding to the Colchicine Site of Tubulin

Curacin A is a potent competitive inhibitor of colchicine binding to tubulin, and it inhibits the growth of tumor cells. We prepared [14C]curacin A biosynthetically to investigate its interaction with tubulin. Binding was rapid, even at 0°C, with a minimum kf of 4.4 × 103 M−1 s−1. We were unable to demonstrate any dissociation of the [14C]curacin A from tubulin. Consistent with these observations, the Ka value was so high that an accurate determination by Scatchard analysis was not possible. The [14C]curacin A was released from tubulin following urea treatment, indicating that covalent bond formation does not occur. We concluded that curacin A binds more tightly to tubulin than does colchicine. Besides high-affinity binding to the colchicine site, we observed significant superstoichiometric amounts of the [14C]curacin A bound to tubulin, and Scatchard analysis confirmed the presence of two binding sites of relatively low affinity with a Ka of 3.2 × 10−5 M−1.

Oncocidin A1: a novel tubulin-binding drug with antitumor activity against human breast and ovarian carcinoma xenografts in nude mice

We identified a structural analog of thyroid hormone, methyl-3,5-diiodo-4-(4′-methoxyphenoxy)benzoate (Oncocidin A1™), that inhibits human carcinoma cell proliferation and the growth of human breast (MDA MB-231) and ovarian (OVCAR-3) carcinoma xenografts in nude mice. This novel antitumor agent is orally bioavailable and well tolerated by animals. Exposure of MCF-7 and MDA MB-231 breast carcinoma cells to Oncocidin A1 in vitro caused a cell-cycle arrest in prometaphase (a G 2/M arrest) and apoptosis, suggesting a cytotoxic mechanism involving mitotic spindle function. The interaction of Oncocidin A1 with microtubules was demonstrated by: 1) immunofluorescence studies of microtubule assembly in the presence of the drug in cell-free and in cellular assays; and 2) in vitro binding inhibition studies involving radiolabeled Oncocidin A1 or colchicine and tubulin monomers. Taken together, these experiments indicate that Oncocidin A1 perturbs cellular microtubule assembly, possibly by binding to the colchicine site on tubulin. Three-dimensional structural modelling of Oncocidin A1 revealed that it can adopt a twisted conformation similar to that of combretastatin A-4, which binds to the colchicine site of tubulin. The novel structural features of Oncocidin A1 could guide the design of a new class of microtubule-binding antitumor agents having substantially reduced normal tissue toxicity upon oral administration.

Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells.

Originally purified as a major lipid component of a strain of the cyanobacterium Lyngbya majuscula isolated in Curaçao, curacin A is a potent inhibitor of cell growth and mitosis, binding rapidly and tightly at the colchicine site of tubulin. Because its molecular structure differs so greatly from that of colchicine and other colchicine site inhibitors, we prepared a series of curacin A analogs to determine the important structural features of the molecule. These modifications include reduction and E-to-Z transitions of the olefinic bonds in the 14-carbon side chain of the molecule; disruption of and configurational changes in the cyclopropyl moiety; disruption, oxidation, and configurational reversal in the thiazoline moiety; configurational reversal and substituent modifications at C13; and demethylation at C10. Inhibitory effects on tubulin assembly, the binding of colchicine to tubulin, and the growth of MCF-7 human breast carcinoma cells were examined. The most important portions of curacin A required for its interaction with tubulin seem to be the thiazoline ring and the side chain at least through C4, the portion of the side chain including the C9-C10 olefinic bond, and the C10 methyl group. Only two modifications totally eliminated the tubulin-drug interaction. The inactive compounds were a segment containing most of the side chain, including its two substituents, and analogs in which the methyl group at the C13 oxygen atom was replaced by a benzoate residue. Antiproliferative activity comparable with that observed with curacin A was only reproduced in compounds that were potent inhibitors of the binding of colchicine to tubulin. Molecular modeling and quantitative structure-activity relationship studies demonstrated that most active analogs overlapped extensively with curacin A but failed to provide an explanation for the apparent structural analogy between curacin A and colchicine.

Limitations in the use of tubulin polymerization assays as a screen for the identification of new antimitotic agents: The potent marine natural product curacin A as an example

AbstractCuracin A is a newly isolated lipid natural product that binds in the colchicine site of tubulin and inhibits mitosis. We have examined its effects on tubulin polymerization, studied by turbidimetry, under three reaction conditions. In 1.0 M glutamate + 1.0 mM MgCl2, with a 37°C reaction temperature, we could find no concentration of curacin A that completely inhibited polymerization. A maximal inhibitory effect on turbidity development (about 50%) was observed with 5 m̈M drug. Higher drug concentrations induced an abnormal polymerization reaction, which at 40 m̈M differed little from the control reaction except for slightly delayed depolymerization in response to reducing the temperature to 0°C. In 0.8 M glutamate (no MgCl2), with a 30°C reaction temperature, complete inhibition did occur at 3–5 m̈M drug, but higher drug concentrations again induced an abnormal polymerization reaction. With 40 m̈M curacin A this reaction was also similar to the control reaction, except that cold‐induced depolymerization was slightly enhanced relative to the control. When polymerization was induced by microtubule‐associated proteins, maximal inhibition of turbidity development (about 70%) occurred with 2 m̈M drug, with higher curacin A concentrations inducing abnormal polymerization reactions that reached about 60% of the control turbidity readings. Under all three reaction conditions the polymer induced by high concentrations of curacin A consisted of large aggregates of tightly coiled spiral fibers that had a 2–3 filament substructure. The implications of these findings with curacin A are discussed in terms of the use of tubulin polymerization assays as a screen for identifying new antimitotic drugs. © 1995 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.

A metabolite of estradiol, 2-methoxyestradiol (2ME), inhibits angiogenesis in the chicken embryo chorioallantoic membrane assay. Since 2ME causes mitotic perturbations, we examined its interactions with tubulin. In our standard 1.0 M glutamate system (plus 1.0 mM MgCl2 at 37 degrees C), superstoichiometric concentrations (relative to tubulin) of 2ME inhibited the nucleation and propagation phases of tubulin assembly but did not affect the reaction extent. Although polymer formed in the presence of 2ME was more cold-stable than control polymer, morphology was little changed. Under suboptimal reaction conditions (0.8 M glutamate/no MgCl2 at 26 degrees C), substoichiometric 2ME totally inhibited polymerization. No other estrogenic compound was as effective as 2ME as an inhibitor of polymerization or of the binding of colchicine to tubulin. Inhibition of colchicine binding was competitive (Ki, 22 microM). Thus, a mammalian metabolite of estradiol binds to the colchicine site of tubulin and, depending on reaction conditions, either inhibits assembly or seems to be incorporated into a polymer with altered stability properties.

Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin: equilibrium studies.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)