Taxol Binding Site Research Articles

Heterogeneous and homogeneous nucleation of Taxol™ crystals in aqueous solutions and gels: Effect of tubulin proteins

In this study we report crystallization of Taxol in pure water, aqueous solutions containing tubulin proteins and tubulin-containing agarose gels. We show that crystallization of Taxol in tubulin-free aqueous solutions occurs by the formation of sheaf-like crystals, while in the presence of tubulin Taxol crystallizes in the form of spherulites. Whereas sheaves are characteristic for crystals formed by homogeneous nucleation, the spherical symmetry of the Taxol crystal formed in the presence of tubulin suggests they result from heterogeneous nucleation. To explain the formation of tubulin–Taxol nuclei we suggest a new, secondary Taxol-binding site within the tubulin heterodimer. Contrary to the known binding site, where the Taxol molecule is almost completely buried in the protein, the Taxol molecule in the secondary binding site is partially exposed to the solution and may serve as a bridge, connecting other Taxol molecules. Results presented in this work are important for in vivo and in vitro microtubule studies due to the possibility of mistaking these Taxol spherulites for microtubule asters, moreover a novel variable is proposed in the study of cells treated with Taxol for cancer treatment via sequestration of tubulin.

Structure-Activity Relationship of Taxol Inferring from Docking Taxol Analogues to Microtubule Binding Site

In order to find the minimal structural requirements to maintain microtubule binding, 12 taxol analogues have been docked to the taxol binding site of tubulin. By comparing the interactions of each analogue with beta-tubulin, the structure-activity relationships are summarized as follow: C-2 benzoyl and taxane ring systems are the essential groups for microtubule binding, the improvements of bioactivity and bioavailability are dependent on the substituents at positions C-1, C-4, C-7, C-9, C-10, and C-14, whereas the C-13 side chain mainly provides a specific binding.

Identification and Characterization of an Intermediate Taxol Binding Site Within Microtubule Nanopores and a Mechanism for Tubulin Isotype Binding Selectivity

Tubulin, the primary subunit of microtubules, is remarkable for the variety of small molecules to which it binds. Many of these are very useful or promising agents in cancer chemotherapy. One of the most useful of these is paclitaxel. The tubulin molecule is itself an alpha/beta heterodimer, both alpha- and beta-tubulin monomers existing as multiple isotypes. Despite the success of paclitaxel as an anticancer drug, resistance often occurs in cancer cells and has been associated with variations in tubulin isotype expression, most notably with the increased expression of betaIII-tubulin. Paclitaxel is thought to reach its binding site on beta-tubulin by diffusion through nanopores in the microtubule wall. It has been suggested that a transitional step in this process may be the binding of paclitaxel to an intermediate site within a nanopore, from which it moves directly to its binding site in the microtubule interior facing the lumen. To test this hypothesis, we have computationally docked paclitaxel within a microtubule nanopore and simulated its passage to the intermediate binding site. Targeted molecular dynamics was then used to test the hypothesis that paclitaxel utilizes the H6/H7 loop as a hinge to move directly from this intermediate binding site to its final position in the luminal binding site. We observed that this motion appears to be stabilized by the formation of a hydrogen bond involving serine 275 in beta-tubulin isotypes I, IIa, IIb, IVa, IVb, V, VII, and VIII. Interestingly, this residue is replaced by alanine in the betaIII and VI isotypes. This observation raises the possibility that the observed isotype difference in paclitaxel binding may be a kinetic effect arising from the isotype difference at this residue. We are now able to suggest derivatives of paclitaxel that may reverse the isotype-specificity or lead to an alternate stabilizing hydrogen-bond interaction with tubulin, thus increasing the rate of passage to the luminal binding site and hopefully offering a therapeutic advantage in paclitaxel resistant cases.

Binding affinity of fluorochromes and fluorescent proteins to Taxol™ crystals

The ability of Taxol (paclitaxel) to bind and stabilize microtubules is the basis for its use as an anti-mitotic drug as well as an additive for in vivo and in vitro studies of microtubules. The low solubility of Taxol in aqueous solutions, however, facilitates the formation of Taxol crystals that can be decorated with fluorescent tubulin. In cells treated with Taxol, these decorated Taxol crystals may be mistaken for self-assembled tubulin asters when observed with a fluorescent microscope. We confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated not only with fluorescent tubulin but also with other fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Using computational docking studies we identified a new, potential Taxol binding site within the tubulin dimmer, allowing the interaction between crystalline Taxol and tubulin. Our calculations, however, show that fluorescent tubulin binding to Taxol crystals is more favorable via the fluorochromes covalently linked to the tubulin dimmers, rather than via the new Taxol-binding site, what is in accordance with our experimental data.

An Inhibitor of FtsZ with Potent and Selective Anti-Staphylococcal Activity

FtsZ is an essential bacterial guanosine triphosphatase and homolog of mammalian beta-tubulin that polymerizes and assembles into a ring to initiate cell division. We have created a class of small synthetic antibacterials, exemplified by PC190723, which inhibits FtsZ and prevents cell division. PC190723 has potent and selective in vitro bactericidal activity against staphylococci, including methicillin- and multi-drug-resistant Staphylococcus aureus. The putative inhibitor-binding site of PC190723 was mapped to a region of FtsZ that is analogous to the Taxol-binding site of tubulin. PC190723 was efficacious in an in vivo model of infection, curing mice infected with a lethal dose of S. aureus. The data validate FtsZ as a target for antibacterial intervention and identify PC190723 as suitable for optimization into a new anti-staphylococcal therapy.

Tau-isoform dependent enhancement of taxol mobility through microtubules

Tau, a family of microtubule-associated proteins (MAPs), stabilizes microtubules (MTs) and regulates their dynamics. Tau isoforms regulate MT dynamic instability differently: 3-repeat tau is less effective than 4-repeat tau at suppressing the disassembly of MTs. Here, we report another tau-isoform-dependent phenomenon, revealed by fluorescence recovery after photobleaching measurements on a BODIPY-conjugated taxol bound to MTs. Saturating levels of recombinant full-length 3-repeat and 4-repeat tau both cause taxol mobility to be remarkably sensitive to taxol concentration. However, 3-repeat tau induces 2.5-fold faster recovery (∼450 s) at low taxol concentrations (∼100 nM) than 4-repeat tau (∼1000 s), indicating that 3-repeat tau decreases the probability of taxol rebinding to its site in the MT lumen. Finding no tau-induced change in the MT-binding affinity of taxol, we conclude that 3-repeat tau either competes for the taxol binding site with an affinity of ∼1 μM or alters the MT structure so as to facilitate the passage of taxol through pores in the MT wall.

A Photoaffinity Analogue of Discodermolide Specifically Labels a Peptide in β-Tubulin

Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.

Multi‐sited mutations of beta‐tubulin are involved in benzimidazole resistance and thermotolerance of fungal biocontrol agent Beauveria bassiana

Fungicide resistance and thermotolerance of biocontrol agents in mitosporic fungi are of merits for enhancing fungal formulations against insect pests in the field. Among 20 wild strains of Beauveria bassiana (a well-known fungal biocontrol agent) tested in this study, 19 were sensitive or highly sensitive to carbendazim (methyl 2-benzimidazole carbamate), a typical benzimidazole fungicide, despite low resistance found in one strain. Sequential mutagenesis of a carbendazim-sensitive wild strain [minimal inhibitory concentration (MIC) = 1.32 microg ml(-1)] under artificial selection pressure generated 11 mutants sharing a common MIC of > 1000 microg ml(-1) without visible variation in colony growth and conidiation capacity. This represents at least 758-fold enhancement of the resistance among the mutants. However, accompanied with the enhanced resistance, all the mutants became less thermotolerable. Stressed at 48 degrees C, conidial LT(50)s of the mutants varied from 1.8 to 9.6 min and were lower than the parental LT(50) (36 min). Moreover, the contents of hydrophobin-like proteins in conidial walls declined significantly among the mutants compared with that of the wild parent. Mutations commonly relating to benzimidazole resistance in fungi were located at Q134, F167 and/or E198 around the taxol-binding site of beta-tubulin by sequencing the beta-tubulin of the mutants. Also, mutations of other 37 amino acid residues in the sequences (each having one to five residues mutated) were found for the first time and they were diverse in spatial structure. All mutations restricted to the half of beta-tubulin close to alpha-tubulin were likely involved in variation in each of the traits concerned but their interactions were complicated.

Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.

Taxol Anticancer Activity and DNA Binding

The interaction of taxol with DNA has major biological importance since it is shown the presence of higher concentration of taxol in the nucleus, than in the human lung tumor cell. Therefore, in this report we examine the interaction of taxol with calf-thymus DNA in aqueous solution at physiological pH, using constant DNA concentration (25 or 1.25 mM phosphate) and various taxol/DNA (phosphate) ratios 1/200 to 1/2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods are used to characterize the nature of drug-DNA interaction and to determine the taxol binding site, the binding constant, sequence selectivity, helix stability and biopolymer secondary structure in the taxol-DNA complexes in vitro. Structural analysis showed that taxol is an external DNA binder with no affinity towards DNA intercalation. The major target of taxol is A-T, G-C bases and the backbone PO(2) groups. Two bindings were observed for taxol-DNA complexes with K(1)= 1.4 x 10(4) M(-1) and K(2)=3.5 X 10(3) M(-1). The taxol-DNA interaction is associated with a partial helix stabilization and no major alterations of B-DNA structures.

Taxol interaction with DNA and RNA — Stability and structural features

Taxol (paclitaxel) is an anticancer drug that interacts with microtubule proteins in a manner that catalyzes their formation from tubulin and stabilizes the resulting structures. However, in the human lung tumor cell, the concentration of paclitaxel is highest in the nucleus. Therefore, it was of interest to examine the interaction of taxol with DNA and RNA in aqueous solution at physiological pH. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to characterize the nature of drug–DNA and drug–RNA interactions and to determine the taxol binding site, the binding constant, the sequence selectivity, the helix stability, and the biopolymer secondary structure in the taxol–polynucleotide complexes in vitro. The FTIR spectroscopic studies were conducted with taxol/polynucleotide (phosphate) ratios of 1/80, 1/40, 1/20, 1/10, 1/4, and 1/2 with a final DNA(P) or RNA(P) concentration of 12.5 mmol/L, and capillary electrophoresis was performed after incubation of taxol with polynucleotides at ratios of 1/200 to 1/12 with a final polynucleotide concentration of 1.25 mmol/L. Taxol was shown to bind to DNA and RNA at G–C, A–T, or A–U bases and the backbone PO2group. Two types of binding were observed for taxol–DNA with K1 = 1.3 × 104L mol–1and K2 = 3.5 × 103L mol–1, whereas taxol–RNA complexes showed one type of binding with K = 1.3 × 104L mol–1. The taxol–polynucleotide complexation is associated with a partial helix stabilization and no major alterations of B-DNA or A-RNA structure. Key words: DNA, RNA, taxol, binding site, binding constant, conformation, helix stability, electrophoresis, FTIR spectroscopy.

Fast Kinetics of Taxol Binding to Microtubules: EFFECTS OF SOLUTION VARIABLES AND MICROTUBULE-ASSOCIATED PROTEINS

The kinetics of Taxol association to and dissociation from stabilized microtubules has been measured by competition with the reference fluorescent derivative Flutax-1 (Diaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., and Andreu, J. M. (2000) J. Biol. Chem. 275, 26265-26276). The association rate constant at 37 degrees C is k(+) = (3.6 +/- 0.1) x 10(6) m(-1) s(-1). The reaction profile is similar to that of the first step of Flutax-1 binding, which probably corresponds to the binding of the Taxol moiety. The rate constant of the initial binding of Flutax-1 is inversely proportional to the viscosity of the solution, which is compatible with a diffusion-controlled reaction. Microtubule-associated proteins bound to the microtubule outer surface slow down the binding of Flutax-1 and Flutax-2 10-fold. The binding site is fully accessible to Flutax-2 in native cytoskeletons of PtK2 cells; the observed kinetic rates of Flutax-2 microtubule staining and de-staining are similar to the reaction rates with microtubule associated proteins-containing microtubules. The kinetic data prove that taxoids bind directly from the bulk solution to an exposed microtubule site. Several hypotheses have been analyzed to potentially reconcile these data with the location of a Taxol-binding site at the model microtubule lumen, including dynamic opening of the microtubule wall and transport from an initial Taxol-binding site at the microtubule pores.

Repeat motifs of tau bind to the insides of microtubules in the absence of taxol.

The tau family of microtubule-associated proteins has a microtubule-binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co- assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three-dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between alpha- and beta-tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on beta-tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in alpha-tubulin equivalent to the taxol-binding pocket in beta-tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.

Location and properties of the taxol binding center in microtubules: a picosecond laser study with fluorescent taxoids.

The interaction of two bioactive, fluorescent analogues of the anticancer drug Taxol, Flutax1 [7-O-[N-(fluorescein-4'-carbonyl)-L-alanyl]taxol] and Flutax2 [7-O-[N-(2,7-difluorofluorescein-4'-carbonyl)-L-alanyl]taxol], with microtubules in solution has been studied with picosecond laser methods. As shown here, although a mixture of the fluorescein mono- and dianion species of Flutax1 is present in solution, the bound taxoid contains only the dianion form of the dye. This indicates strong electrostatic interactions at the microtubule lattice with the appending dye, most likely with charged residues of the M-loop of the beta-tubulin subunit. Moreover, analysis of the dynamic depolarization of microtubule-bound Flutax at low binding site occupancy was consistent with a protein active center with significant conformational flexibility. On the other hand, for microtubules fully saturated with the taxoid, a new, additional depolarizing process was observed, with relaxation times of 14 ns (Flutax1) and 8 ns (Flutax2), which is due to Förster resonance energy homotransfer (FREHT) between neighboring dye molecules. Application of a detailed analysis of FREHT-induced depolarization in a circular array of dye molecules presented here yielded a separation between nearest-neighbor Flutax moieties of 40 +/- 5 A, for microtubules made up of between 12 and 14 protofilaments, a value that is only compatible with the Taxol binding site being located at the inner wall of the microtubule. The internal position of the drug molecular target as measured here is also consistent with other spectroscopic observations and confirms existing predictions based on microtubule structures modeled from high-resolution, electron density maps of alphabeta-tubulin.

The interaction of baccatin III with the taxol binding site of microtubules determined by a homogeneous assay with fluorescent taxoid.

The ubiquitous Taxol binding site of microtubules also binds newly discovered ligands. We have designed a homogeneous assay for the high throughput detection of Taxol biomimetics, based on the displacement of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]Taxol from its binding site in diluted solutions of preserved microtubules. The state of this reference ligand is measured by fluorescence anisotropy in a microplate reader, with varying concentrations of nonfluorescent competitors. The binding equilibrium constant of Taxol has a value K(b) = 3.7 x 10(7) M(-1). We have found that baccatin III, an analogue of Taxol without the C-13 side chain, binds with K(b) = 1.5 x 10(5) M(-1), whereas the side chain methyl ester is inactive. This was unexpected from the structure-activity relationship of taxoids but compatible with models of Taxol docked at the microtubule site. Baccatin III binding has been confirmed by displacement of [(3)H]Taxol and by direct HPLC measurements of its cosedimentation with microtubules, among other methods. Consequently, baccatin III induces microtubule bundles and multipolar spindles in PtK2 and U937 cells, and mitotic arrest and apoptotic death of the U937 cells, at concentrations 200-500-fold larger than Taxol. The simplest analysis of these results strongly suggests that the interaction of the C-2 C-4 substituted taxane ring system with the microtubule binding site provides most (ca. 75%) of the free energy change of Taxol binding and is sufficient to activate microtubule stabilization and transmit the antitumor effects of Taxol, whereas the C-13 side chain provides a weak specific anchor.

The relationship between Taxol and (+)-discodermolide: synthetic analogs and modeling studies

Background: During the past decade, Taxol has assumed an important role in cancer chemotherapy. The search for novel compounds with a mechanism of action similar to that of Taxol, but with greater efficacy particularly in Taxol-resistant cells, has led to the isolation of new natural products. One such compound, (+)-discodermolide, although structurally distinct from Taxol, has a similar ability to stabilize microtubules. In addition, (+)-discodermolide is active in Taxol-resistant cell lines that overexpress P-glycoprotein, the multidrug-resistant transporter. Interestingly, (+)-discodermolide demonstrates a profound enhancement of the initiation process of microtubule polymerization compared to Taxol. Results: The synthesis of (+)-discodermolide analogs exploiting our highly efficient, triply convergent approach has permitted structure–activity relationship (SAR) studies. Small changes to the (+)-discodermolide structure resulted in a dramatic decrease in the ability of all four discodermolide analogs to initiate tubulin polymerization. Two of the analogs also demonstrated a decrease in total tubulin polymerization, while a change in the olefin geometry at the C8 position produced a significant decrease in cytotoxic activity. Conclusions: The availability of (+)-discodermolide and the analogs, and the resultant SAR analysis, have permitted an exploration of the similarities and differences between (+)-discodermolide and Taxol. Docking of the X-ray/solution structure of (+)-discodermolide into the Taxol binding site of β-tubulin revealed two possible binding modes (models I and II). The preferred pharmacophore model (I), in which the C19 side chain of (+)-discodermolide matches with the C2 benzoyl group of Taxol and the δ-lactone ring of (+)-discodermolide overlays with the C13 side chain of Taxol, concurred with the results of the SAR analysis.

Tubulins in the primate retina: evidence that xanthophylls may be endogenous ligands for the paclitaxel-binding site

The xanthophylls—lutein, zeaxanthin, and meso-zeaxanthin (L&Z)—are found in the central region of the primate retina, which is called the macula lutea (yellow spot). How they are anchored there and what their function is has been debated for over 50 years. Here, we present evidence that they may be bound to the paclitaxel (Taxol) binding site of the β-tubulin subunit of microtubules and that a major function may be to modulate the dynamic instability of microtubules in the macula. Also, we compare nucleic acid and amino acid sequences of tubulins that are in human brain with those we have isolated from human-retina and monkey-macula cDNA libraries. In so doing, we suggest that in primates, class I β-tubulin consists of at least two subtypes (β Ia and β Ib). Alignment analysis of the sequences of the genes for β Ia and β Ib indicates that the corresponding mRNAs may have other functions in addition to that of coding for proteins. Furthermore, we show that there are at least five different types of β-tubulin in the macula lutea of rhesus monkey.

A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells.

To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers. A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of [3H]-taxol, a known substrate of P-gp. The apparent affinities (K(I) values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [3H]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting. [3H]-Taxol (0.04 microM) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [3H]-taxol flux could be totally abolished by inclusion of (+/-)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium. However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of [3H]-taxol under the same conditions. These results suggest that P-gp, not MRP, was involved in taxol transport. Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of [3H]-taxol with resulting K(I) values of 1.5+/-0.8, 2.5+/-1.0, 3.0+/-0.3, 7.3+/-0.7, 8.5+/-2.8, 36.5+/-1.5, 276+/-69, and 313+/-112 microM, respectively. With the exception of that of quinidine, these K(I) values were comparable with literature values. This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp, however, this assay does not permit the differentiation of substrates and inhibitors. The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.

Additional transporter characterization may lead to new pharmaceutical opportunities.

Additional transporter characterization may lead to new pharmaceutical opportunities.

Molecular Recognition of Taxol by Microtubules: KINETICS AND THERMODYNAMICS OF BINDING OF FLUORESCENT TAXOL DERIVATIVES TO AN EXPOSED SITE

We have determined the kinetic scheme and the reaction rates of binding to microtubules of two fluorescent taxoids, 7-O-[N-(4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-1) and 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (K(a) approximately 10(7) m(-1), 37 degrees C). The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques. The kinetic constants of the bimolecular reaction were 6.10 +/- 0.22 x 10(5) m(-1) s(-1) and 13.8 +/- 1.8 x 10(5) m(-1) s(-1) at 37 degrees C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The reversal of this reaction is the rate-limiting step of dissociation. A third step has been detected using small angle x-ray scattering and involves a 2-nm increase in the diameter of microtubules. It is suggested that the first step entails the binding of the Taxol moiety and the second a relative immobilization of the fluorescent probe. The equilibrium and some kinetic measurements required the use of stabilized cross-linked microtubules, which preserved taxoid binding. The results indicate that the Taxol binding site is directly accessible, in contrast with its location at lumen in the current model of microtubules. An alternative structural model is considered in which the binding site is located between protofilaments, accessible from the microtubule surface.