Syntheses and biological activity of C-3'-difluoromethyl-taxoids

A series of new taxoids bearing difluoromethyl group at the C-3' position and modifications at the C-10 and C-14 positions has been synthesized and their biological activities studied. The in vitro cytotoxicity assay results indicate that these newly developed taxoids exhibit comparable to several times better activity against drug-sensitive cell line LCC6-WT, and 40-70 times better activity against the corresponding drug-resistant cancer cell line LCC6-MDR as compared to that of paclitaxel. Apoptosis analysis has revealed the exceptional activity of SB-T-12843 (1e) in inducing apoptosis in both MDR-bearing and MDR-negative cancer cells.     

Structure-activity relationship study of taxoids for their ability to activate murine macrophages as well as inhibit the growth of macrophage-like cells

A series of new taxoids modified at the C-3', C-3'N, C-10, C-2 and C-7 positions has been designed, synthesized and evaluated for their potency to induce NO and TNF production by peritoneal murine macrophages (Mphi) from LPS-responsive C3H/HeN and LPS-hyporesponsive C3H/HeJ strains and human blood cells, and for their ability to inhibit the growth of Mphi-like cell lines J774.1 and J7.DEF3. The SAR-study has shown that the nature of the substituents at these positions have critical effect on the induction of TNF and NO production by Mphi. Positions C-3' and C-10 are the most flexible and an intriguing effect of the length of the substituents at the C-10 position is observed for taxoids bearing a straight chain alkanoyl moiety. An aromatic group at the C-3'N and C-2 positions is required for the activity, while only hydroxyl or acetyl substituents seem to be tolerated at the C-7 position. The natural stereochemistry in the C-13 isoserine side chain of the taxoids is an absolute requirement for macrophage activation. It has also been clearly shown that there is no correlation between the ability of the taxoids to induce TNF/NO production in C3H/HeN Mphi and the cytotoxicity against Mphi-like cells.     

Synthesis of potent taxoids for tumor-specific delivery using monoclonal antibodies

The targeted delivery of taxoids, in the form of taxane-antibody immunoconjugates, requires the preparation of taxoids containing moieties suitable for their conjugation to monoclonal antibodies. A series of taxoids incorporating a disulfide-containing linker at various positions of the taxoid framework have been prepared to investigate the most suitable position for conjugation. A second series of taxoids modified at the C-2 position aimed at increasing the potency of these taxanes has also been prepared.     

Synthesis and structure-activity relationships of new second-generation taxoids

A series of second-generation taxoids bearing a substitutent on the C-2-benzoyl group and modifications at C-3′/C-10 positions was synthesized. These taxoids exhibited 2–3 orders of magnitude higher potency than that of paclitaxel against drug-resistant human breast cancer cell lines. It is also noteworthy that three taxoids showed almost no difference in activity against drug-resistant and drug-sensitive cell lines, which are categorized as “advanced second generation taxoids“.     

Design, synthesis and biological activity of novel C2-C3' N-Linked macrocyclic taxoids

A series of novel macrocyclic taxoids was designed and synthesized by connecting the C-2 and C-3' N positions of the taxoid framework with various tethers. Cytotoxicity of these macrocyclic taxoids was evaluated against a human breast cancer cell line LCC6-WT, and a couple of the taxoids exhibited 0.09-0.3 microM IC(50) values.     

Influence of microtubule-associated proteins on the differential effects of paclitaxel and docetaxel

Microtubules are complex structures arising in part from the polymerization of tubulin dimers. Tubulin binds to a wide range of drugs which have been used as probes for tubulin conformation and assembly properties. There is some evidence that taxol and taxotere have differing effects on tubulin conformation. Previous work has shown that MAP2 and Tau, although they both induce microtubule assembly, have qualitatively different effects on tubulin's behavior. Since most microtubulesin vivo are likely to be associated with MAPs, we decided to characterize the differential effects of MAP2, Tau, taxol, and taxotere on tubulin polymerization with the aim of understanding the mechanisms through which these agents stimulate microtubule assembly. Furthermore, the inhibitive effect of calcium has been used to elucidate the ability of the two drugs to force tubulin assembly. These observations suggest that docetaxel, in addition to its greater efficiency in tubulin assembly, may have the capacity to differently alter certain classes of microtubules. Tau and MAP2 accessory proteins may represent important cofactors modulating the effects of taxoids.     

Analysis of biosynthetic fluctuations of cultured Taxus seedlings using a metabolomic approach

Fluctuations in the biosynthesis of taxoids in 1–5 year old cultured seedlings of Taxus chinensis var. mairei were investigated using LC–IT-TOF-MS and a metabolomics approach. In the total ion chromatogram (TIC) of the extracts, 16 prominent peaks were observed. Ten compounds were identified by comparison of retention times and MS/MS spectra with those of reference compounds. An additional 6 taxoids were isolated by preparative HPLC and identified by comparison of their spectroscopic data with those reported in the literature. It was clarified that the relative concentrations of taxoids with 4(20) double bonds are high at early stages of cultivation. On the other hand, relatively higher amounts of 5-acetoxy taxoids oxidized at the 4- and 10- positions and taxoids having 5(20)-oxetane rings were found at later stages of cultivation. This approach provides practical information on the biosynthetic flow of taxoids in cultured yew seedlings.     

Effect of guanine nucleotides on the hydrophobic interaction of tubulin

The influence of guanine nucleotides on the binding of tubulin to hydrophobic components is investigated. Tubulin binds to a hydrophobic phenyl-Sepharose gel in a reversible, nucleotide-dependent way. Assembly-competent tubulin is released with ion-free water as eluent. It contains one guanosine triphosphate per dimer. More denatured tubulin needs a mixture of ethanol-water to elute. Consequently, hydrophobic interaction chromatography over phenyl-Sepharose represents an easy method for preparing polymerizable tubulin free of nucleotides at the exchangeable sites. While, in the absence of guanine nucleotide, the binding of tubulin to phenyl-Sepharose is rapid and immediately reversible on nucleotide addition, the binding of the nucleotide-dependent hydrophobic sites of tubulin to 1,8-ANS is slow, and its dissociation on nucleotide addition is poor. No differences are observed between the shielding of hydrophobic sites in the presence of GTP or GDP. Neither inorganic phosphate nor A1F4- is found to directly influence guanine nucleotides in their ability to shield hydrophobic sites.     

Regioselectivity of taxoid-O-acetyltransferases: heterologous expression and characterization of a new taxadien-5alpha-ol-O-acetyltransferase

In addition to the anticancer drug Taxol, yew (Taxus) species produce a large variety of other taxane diterpenoids which differ mainly in the type of acyl and aroyl groups appended to the many hydroxyl functions on the taxane core; acetate esters are particularly common. Taxol bears an acetate at C10 and another at C4 thought to originate by intramolecular migration of a C5 acetate function in the process of oxetane ring formation, but many other naturally occurring taxoids bear acetate groups at C1, C2, C7, C9, and C13, in addition to C5 and C10. cDNAs encoding a taxoid 5alpha-O-acetyltransferase (taxadien-5alpha-ol as substrate) and a taxoid 10beta-O-acetyltransferase (10-deacetylbaccatin III as substrate) have been acquired from a recently isolated family of Taxus acyl/aroyltransferase clones. To explore the origins of other acetylated taxoids, the group of recombinant Taxus acyltransferases was investigated with a range of polyhydroxylated taxoids as substrates. From this survey, a new acetyltransferase clone (denoted TAX19) was identified that was capable of acetylating taxadien-5alpha-ol with activity comparable to that of the previously identified 5alpha-O-acetyltransferase (clone TAX1). However, when these two recombinant enzymes were presented with taxadien-triol and tetraol substrates, they exhibited different regiospecificities. The TAX1 enzyme preferentially acetylates the "northern" hemisphere hydroxyls at C9 and C10, whereas the TAX19 enzyme preferentially acetylates the "east-west" pole positions at C5 and C13. The TAX1 enzyme possesses the lowest KM value with taxadien-5alpha-ol (an early pathway metabolite) as substrate, with much higher KM values for the polyhydroxylated taxoid substrates, whereas the TAX19 enzyme possesses lower KM values (than the TAX1 transferase) for all taxoid substrates tested. These results suggest that both TAX1 and TAX19 acyltransferases may function at the early C5 acetylation step of taxoid metabolism, and that the TAX19 acyltransferase, because of its broader specificity for polyhydroxylated taxoids, may also function later in metabolism and be responsible for the production of many other acetylated taxoids.     

Production of taxoids with biological activity by plants and callus culture from selected Taxus genotypes

Twenty seven different yew trees belonging to various genotypes and hybrids have been screened for their capacity to produce significant amounts of taxoids provided with biological activity in the tubulin test. From the three best genotypes selected, Taxus x media "Sargentii" proved to be able to produce viable calluses from excised roots placed in vitro. Taxoid composition at various times of the in vitro culture was determined and the carcinostatic efficiency of the extracts was established using the KB cell cytotoxicity test. In leaves and calluses, respectively, 0.069 and 0.032% paclitaxel (taxol) contents were found. These contents were significantly higher than those previously reported for other genotypes.     

Regio- and stereoselective hydroxylation of taxoids by filamentous fungi

Paclitaxel (Taxol), is one of the most promising chemotherapeutic agents developed for cancer treatment in past two decades. Microorganisms such as filamentous fungi are known to perform regio- and stereoselective hydroxylation of taxoids. Herein, we describe highly regio- and stereoselective hydroxylation at the 1beta and 9alpha positions of the taxane skeleton. Such hydroxylation reactions proceed readily for the taxadienes as substrates rather than taxoids having an oxetane ring. The presence of different oxygen substituents on the taxane nucleus, such as 5-acetoxy, has a significant effect on the selectivity and yield of the hydroxylation catalyzed by the microbial oxidases.     

The relationship of hydrophobic tubulin with membranes in neural tissue

Brain membrane preparations contain tubulin that can be extracted with Triton X-114. After the extract is allowed to partition, 8% of the total brain tubulin is isolated as a hydrophobic compound in the detergent-rich phase. Cytosolic tubulin does not show this hydrophobic behaviour since it is recovered in the aqueous phase. Membrane tubulin can be released by 0.1 M Na₂CO₃ treatment at pH11.5 in such a way that the hydrophobic tubulin is converted into the hydrophilic form. These results suggest that tubulin exists associated with some membrane component that confers the hydrophobic behaviour to tubulin. If the tissue is homogenized in microtubule-stabilizing buffer containing Triton X-100, the hydrophobic tubulin is isolated from the microtubule fraction. This result indicates that the hydrophobic tubulin isolated from membrane preparations belongs to microtubules thatin vivo are associated to membranes. Therefore, hydrophobic tubulin (tubulin-membrane component complex) can be obtained from membranes or from microtubules depending on the conditions of brain homogenization.Abbreviations TBS Tris-buffered saline - Mes 2-(N-morpholine) ethane sulfonic acid     

Molecular dynamics simulation study of tubulin dimer interaction with cytostatics

Colchicine, podophylotoxin, and indibulin are natural cytostatics that are used in the treatment of neoplasms. However, application of the compounds is restricted due to their high toxicity and low specificity. Computational experiments modeling tubulin interactions with the cytostatics seem a promising approach to design new analogues of the above-mentioned drugs with higher cytostatic activity and lower toxicity. Therefore, the CHARMM software was used to examine the macromolecules using molecular dynamics and mechanics methods. Particularly, a procedure was applied according to which molecules of each studied cytostatics were placed at several various random positions around the predicted binding site on tubulin. As a result, cytostatic binding regions were identified on the tubulin molecule. It was shown that, during the interaction, structural alterations occurred in these regions that may be responsible for tubulin polymerization. Thus, alterations have been revealed for the first time in the structure of tubulin in the regions of cytostatic binding that can substantially affect its function.     

Brain plasmamembrane NA+:,K+-ATPase is inhibited by acetylated tubulin

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the -subunit of Na⁺,K⁺-ATPase. In the present work we show that the interaction of tubulin with Na⁺K⁺-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na⁺K⁺-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.     

‘Rings’ of F-actin form around the nucleus in cultured human MCF7 adenocarcinoma cells upon exposure to both taxol and taxotere

The anti-cancer taxoids, Taxol® (paclitaxel) and Taxotere® (docetaxel), are the most promising anti-mitotic agents developed for cancer treatment in the past decade. The effectiveness of this new class of compounds lies in their unique mechanism of action on the cytoskeleton. Both taxol and taxotere bind to microtubules and shift the normal equilibrium between monomeric and polymerized tubulin to favor the polymerized form by strongly promoting tubulin assembly and inhibiting microtubule depolymerization. Although very similar in structure, these two compounds have recently demonstrated different in vitro, in vivo, and clinical activities; however, no study to date has effectively compared specific cytoskeletal alterations induced by taxol and taxotere in cultured cells. Using specific staining techniques for both F-actin and α-tubulin, this study provides the first detailed immunohistochemical comparison of the effects of equimolar concentrations of taxol and taxotere on both the microfilament and microtubule networks in a cultured cell line. Using human MCF7 breast adenocarcinoma cells, new observations of taxotere/taxol alterations of the cytoskeleton include: an increased abundance of parallel microtubule ‘bundles’ in taxotere treated cells and a definitive reorganization of the microfilament network which results in novel ring-like formations of F-actin condensed exclusively in the perinuclear zone. Reorganization of the actin cytoskeleton induced by a taxoid disruption of the microtubule equilibrium is indicative of the interdependence between microtubules and microfilaments in this transformed cell line and suggests that the indirect role of the taxoids on the microfilament network may have been overlooked in their mechanism of action as chemotherapeutic agents.     

Stabilization of tubulin by deuterium oxide

Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve the disruption of hydrophobic forces. The conformational change is followed by an aggregation process that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also observed that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbon structures and incompletely folded microtubules. When these polymers were disassembled and reassembled in H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of ribbon structures rather than microtubules.     

Molecular dynamics simulation of the tubulin dimer with cytostatics

Colchicin, podophylotoxin and indibulin are natural cytostatics that are used in the treatment of neoplasms. But applications of those compounds are rather restricted due to the high toxicity and low specificity. It seems very promising to investigate possibility to design new analogs of the above mentioned drugs that will possess higher cytostatic activity and less toxicity. For this purpose we see computer modeling experiments of tubulin and above mentioned compounds interaction as a powerful approach to design new artificial cytostatics with desired properties. In the current study the CHARMM software of protein-ligand interaction molecular dynamics method has been used. Particularly the following strategy has been applied. Molecules of the cytostatits have been positioned at several random positions around binding sites of tubulin and after energy minimization several binding sites have been identified on the tubulin macromolecule. In these binding sites structural changes that may be responsible for tubulin polymerization have been detected.     

Interaction of tubulin with the macromolecular apolar probe, octyl sepharose

Binding of the microtubule protein, tubulin, to hydrophobic groups immobilized on octyl sepharose has been investigated. The results indicate that tubulin binds to octyl sepharose in a time-, temperature-, and concentration-dependent manner. Binding is multiphasic, with one fast phase and at least two slow phases, and is influenced by the presence of antimitotic drugs. Colchicine, vinblastine and podophyllotoxin enhance the fast binding of tubulin with very little effect on the slow binding. Pre-incubation of tubulin with the apolar probe, bis(1,8-anilinonaphthalenesulfonate) (BisANS) enhances both the rapid and slow phases of binding of tubulin to octyl sepharose. 1,8-Anilinonaphthalenesulfonate, the monomer of BisANS, has no effect. These results are consistent with a model for tubulin decay which involves the appearance of hydrophobic sites with time.     

NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.     

Nucleotide-dependent bisANS binding to tubulin.

Non-covalent hydrophobic probes such as 5, 5'-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5'-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5'-diphosphate, guanosine 5'-monophosphate and adenosine 5'-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes.