Taxol binds to polymerized tubulin in vitro.

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5'-guanylyl-alpha,beta-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.

Taxol binds to cellular microtubules.

Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in cells. Tritium-labeled taxol binds directly to microtubules in vitro with a stoichiometry approaching one (Parness, J., and S. B. Horwitz, 1981, J. Cell Biol. 91:479-487). We now report studies in cells on the binding of [3H]taxol and the formation of microtubule bundles. [3H]Taxol binds to the macrophagelike cell line, J774.2, in a specific and saturable manner. Scatchard analysis of the specific binding data demonstrates a single set of high affinity binding sites. Maximal binding occurs at drug concentrations which produce maximal growth inhibition. Conditions which depolymerize microtubules in intact and extracted cells as determined by tubulin immunofluorescence inhibit the binding of [3H]taxol. This strongly suggests that taxol binds specifically to cellular microtubules. Extraction with 0.1% Nonidet P-40 or depletion of cellular ATP by treatment with 10 mM NaN3 prevents the characteristic taxol-induced bundle formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there must be specific cellular mechanisms which are required for bundle formation, in addition to the direct binding of taxol to cytoplasmic microtubules.

Trifluoperazine inhibits phagocytosis in a macrophagelike cultured cell line.

Trifluoperazine, a drug that binds to Ca2+-calmodulin and inhibits its interaction with other proteins, was found to inhibit growth and phagocytosis in a macrophagelike cell line, J774.16. Both effects were reversible and occurred at the same concentrations of drug (25--50 microM) that inhibited the activation of cyclic nucleotide phosphodiesterase by calmodulin in vitro. Fc-mediated phagocytosis was also depressed by W-7, a sulfonamide derivative that inhibits the activity of Ca2+-calmodulin. In contrast, taxol, a drug that stabilizes cellular microtubules, had no effect on Fc-mediated phagocytosis although it inhibited cell growth at nanomolar concentrations. The inhibitory effects of trifluoperazine and W-7 on phagocytosis suggest that calmodulin may be involved in this complex cellular function.

Chemosterilant action of anthramycin: a proposed mechanism.

The activity of anthramycin and structurally related analogs as chemosterilants of the housefly, Musca domestica L., correlates closely with the action of these compounds as inhibitors of Escherichia coli RNA polymerase. Since inhibition of RNA polymerase by anthramycin reflects binding of this antibiotic to the DNA primer required for enzyme activity, we propose that the interaction of anthramycin with DNA may also account for its action as a chemosterilant.

Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes.

The treatment of advanced ovarian cancer with taxol is hindered by the development of drug resistance. The cellular target for taxol is the microtubule that is stabilized by the drug. Taxol preferentially binds to the beta subunit of tubulin of which there are six distinct isotypes in mammalian cells. We have used highly specific oligonucleotides and polymerase chain reaction to analyze expression of all six beta-tubulin genes. Human lung cancer cells (A549) were selected in 12 and 24 nM taxol resulting in cell lines that were 9- and 17-fold resistant, respectively. These cells displayed an altered ratio of classes I, II, III, and IVa beta-tubulin isotypes. Ovarian tumors, seven untreated primary and four taxol- resistant tumor-bearing ascites, displayed significant increases (P < 0.005) in classes I (3.6-fold), III (4.4-fold), and IVa (7.6-fold) isotypes in the taxol-resistant samples as compared with untreated primary ovarian tumors. The increased expression appears to be related to the resistance phenotype, as the basal levels of the class III and IVa isotypes in the untreated tumors were extremely low. This is the first report of altered expression of specific beta-tubulin genes in taxol-resistant ovarian tumors and we propose that the latter may play a role in clinical resistance to taxol.

Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells.

In multidrug-resistant mouse J774.2 cells, the differential overproduction of functionally distinct phosphoglycoprotein isoforms reflects the amplification or transcriptional activation or both of two mdr gene family members, mdr1a and mdr1b. The mdr1a gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes. Independently selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6- and 5.0-kilobase (kb) or 4.7- and 5.1-kb mdr1a transcripts. However, abundant overproduction of the mdr1a gene product was observed only in cell lines which overexpressed the 4.6- and 5.0-kb mRNAs. In order to determine the basis for mdr1a transcript heterogeneity and the relationship between transcript size and steady-state mdr1a protein levels, genomic and cDNA sequence analyses of the 5' and 3' ends of the mdr1a gene were carried out. Promoter sequence analysis and primer extension mapping indicated that mdr1a transcripts were differentially initiated from two putative promoters to generate either 5.1- and 4.7-kb or 5.0- and 4.6-kb transcripts in four multidrug-resistant J774.2 cell lines. Sequence analysis of 3' cDNA variants and a 3' genomic fragment revealed that the 5.1- and 5.0-kb mRNAs had identical 3'-untranslated regions which differed from those of the 4.7- and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal. Transcript initiation from the putative upstream promoter correlated with a 70 to 85% decrease in steady-state mdr1a protein levels relative to transcript levels. In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdr1a. The implications of these findings for mdr gene expression and regulation are discussed.

Studies with RP 56976 (taxotere): a semisynthetic analogue of taxol.

RP 56976 (taxotere), a new semisynthetic analogue of taxol, is a potentially important chemotherapeutic agent for the treatment of cancer. We report here that this drug is a potent inhibitor of cell replication and, like taxol, promotes the in vitro assembly of stable microtubules in the absence of guanosine triphosphate and induces microtubule-bundle formation in cells. Compared with taxol, RP 56976 is slightly more active as a promoter of tubulin polymerization. As an inhibitor of cell replication, RP 56976 is 2.5-fold more potent than taxol in J774.2 and P388 cells and at least 5-fold more potent in taxol-resistant cells.

Direct photoaffinity labeling of tubulin with taxol.

BACKGROUND: Taxol is a potent inhibitor of the replication of eukaryotic cells and has significant antitumor activity in human malignancies. The drug induces the formation of bundles of stable microtubules and blocks cells in the mitotic phase of the cell cycle. In vitro, taxol enhances the polymerization of tubulin to microtubules that are resistant to depolymerization. Although it is evident that taxol interacts with the tubulin-microtubule system, no information has been available on the binding site for the drug on the microtubule. PURPOSE: Our purpose was to determine if taxol binds to one or both of the tubulin subunits. METHODS: In the absence of a photoaffinity-labeled analogue of taxol, [3H]taxol was used directly to photolabel tubulin. A complex of microtubule protein and [3H]taxol was irradiated by ultraviolet light and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS AND CONCLUSIONS: The radiolabeled drug preferentially binds covalently to the beta-subunit of tubulin, and the binding can be competed with unlabeled taxol. IMPLICATIONS: This observation is the first step in a study to determine the binding site for taxol on the microtubule.

Electrophoretic analysis of P-glycoproteins produced by mouse J774.2 and Chinese hamster ovary multidrug-resistant cells.

P-glycoprotein (P-gp) plays a fundamental role in multidrug resistance. The quantity of P-gp relates to the degree of drug resistance. A comparison was made between P-gps in mouse and hamster cell lines in both Laemmli and modified Fairbanks gel systems. Both proteins are derived from precursors of similar size that undergo differential N-linked glycosylation. The electrophoretic mobility and the amount of P-gp are remarkably dependent on the conditions of analysis. Notably, boiling P-gp before Laemmli gel electrophoresis decreases its mobility by an amount that is equivalent to approximately equal to 15 kDa and results in an apparent diminution in the amount of protein. The latter effect can give a false impression concerning the quantity of P-gp in cells.

Characterization of the association of radiolabeled bleomycin A2 with HeLa cells.

The association of [3H]bleomycin A2 and Cu(II):[3H]bleomycin A2 with HeLa cells has been characterized. Under the conditions of our experiments, approximately 0.1% of the total drug in the medium associates with HeLa cells. Both forms of the drug bind to HeLa cells in a specific and saturable manner, with a Km of 20 microM and a Vmax of 2.5 pmol/min/10(6) cells. Scatchard analysis of the specific binding data demonstrates a single set of high-affinity binding sites. Cytotoxic activities of both forms of the drug are similar, with a 50% lethal dose of 0.5 microM at 48 hr. The specific binding in HeLa cells of either the labeled metal-free drug or its copper complex is reversible by a 100-fold excess of either unlabeled drug. Interaction of the drug with cells is temperature sensitive but is unaffected by metabolic poisons, suggesting that this process is not energy dependent. Isolation of DNA from HeLa cells incubated with the drug indicates that 1 mol of either [3H]bleomycin A2 or Cu(II):[3H]bleomycin A2 binds per 10(8) nucleotides. Further studies with the radiolabeled drug are required to define precisely the mechanisms involved in bleomycin uptake and compartmentalization within the cell.

A phosphoglycoprotein associated with taxol resistance in J774.2 cells.

Taxol is an inhibitor of cell replication that promotes the assembly of microtubules in vitro and in cells. In the murine macrophage-like cell line J774.2, a taxol-resistant subline (J7/TAX-50) has been developed in vitro by growing the cells in increasing drug concentrations. These cells, which are approximately 800-fold resistant to taxol, display some cross-resistance to colchicine, vinblastine, puromycin, doxorubicin, and actinomycin D but remain sensitive to bleomycin. Binding of radiolabeled drug to the resistant cells is reduced by approximately 90%. Resistant cells grown in the absence of drug for 10 days (J7/TAX-0D10), 1 month (J7/TAX-0D30), and 8 months (J7/TAX-0D240) regain a major portion (27, 92, and 99%, respectively) of their sensitivity to the drug. However, binding of the drug to the J7/TAX-0D30 and J7/TAX-0D240 cells is increased to only 20 and 70%, respectively, of that measured with sensitive cells. Analysis of plasma membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining reveals the presence of a prominent protein with an approximate molecular weight of 135,000 in the resistant line that is essentially absent from the parental line and from both of the J7/TAX-0D30 and J7/TAX-0D240 lines. Although this protein can be seen in J7/TAX-0D10, its quantity is diminished. The Mr 135,000 protein is also observed in the resistant cells when they are labeled with [3H]leucine, [35S]methionine, [3H]glucosamine, or [32P]orthophosphate. Plasma membranes from colchicine- or vinblastine-resistant J774.2 cells also contain prominent phosphoglycoproteins, with approximate molecular weights of 145,000 and 150,000, respectively. Partial purification of the Mr 135,000 glycoprotein by agarose-bound ricinus communis agglutinin I-agarose affinity chromatography indicates that it accounts for approximately 4 to 5% of total membrane protein. A Mr 100,000 phosphoglycoprotein, present in the membranes of J774.2 cells is essentially absent in J7/TAX-50 cells after labeling with [3H]glucosamine or [32P]orthophosphate. Phosphoamino acid analysis of the Mr 135,000 and 100,000 phosphoglycoproteins reveals that the phosphorylation sites are serine and threonine residues. There appears to be a good correlation between the presence of the Mr 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.

Taxol mediates serine phosphorylation of the 66-kDa Shc isoform.

In the human lung carcinoma cell line A549, Taxol (20 nM) causes a decreased electrophoretic mobility of the 66-kDa Shc isoform (p66shc), beginning 4 h after drug exposure, and reaching a maximum at 9-18 h. No shift was observed for the 52- and 46-kDa isoforms of Shc. The electrophoretic mobility shift of p66shc caused by Taxol is not the result of tyrosine phosphorylation, and there is no indication of a Shc/Grb2 complex in Taxol-treated A549 cells. This modification is blocked by the serine/threonine protein phosphatase 2A. In vivo 32P-labeling and subsequent phosphoamino acid analysis of p66shc indicated that both the original and the shifted p66shc were predominantly serine phosphorylated. Cyanogen bromide digestion of p66shc produced a phosphorylated fragment with an apparent molecular weight of approximately 7.9 kDa from the untreated cells and two phosphorylated fragments, of approximately 7.9 and approximately 9.6 kDa, from the Taxol-treated cells. The domain of Taxol-induced serine phosphorylation is thought to be in the cyanogen bromide fragment containing residues 2-65. The Taxol-induced electrophoretic mobility shift of p66shc was inhibited by the protein synthesis inhibitor, cycloheximide, but not by the mitogen-activated and extracellular signal-regulated protein kinase kinase (MEK) inhibitor, PD98059. This mobility shift did not occur in Taxol-resistant A549-T12 cells treated with 20 nM Taxol. In addition to Taxol, other microtubule-interacting drugs caused a decreased electrophoretic mobility of p66shc. This Taxol-mediated serine phosphorylation seen in p66shc may result from a MEK-independent signaling pathway that is activated in cells that have a prolonged or abnormal mitotic phase of the cell cycle and may play a role in signaling events that lead to cell death.

Mechanisms of Taxol-induced cell death are concentration dependent.

Although the ability of Taxol to stabilize cellular microtubules is well accepted, the mechanisms by which Taxol induces growth arrest and cell death remain unclear. Recent evidence indicates that Taxol alters specific intracellular signal transduction events, such as the activation of Raf-1 kinase, that may be essential for drug-induced apoptosis. To determine whether Raf-1 kinase activation occurs at different concentrations of Taxol and in response to disruption of the normal microtubule cytoskeleton, A549 cells were treated with different concentrations of Taxol after which Raf-1 activation and the microtubule cytoskeleton were analyzed. Raf-1 activation was observed at Taxol concentrations of 9 nM and greater. However, disruption of the normal microtubule cytoskeleton was seen at lower Taxol concentrations (1-7 nM), indicating that this process begins in the absence of Raf-1 activation. Raf-1 activation correlated with the induction of a G2-M block. Depletion of Raf-1 resulted in the accumulation of cells in the G2-M phase of the cell cycle, suggesting that Raf-1 may play an important role in the passage through mitosis. Supporting this idea, Raf-1 was activated in mitotic cells. Low concentrations of Taxol induced cell death in the absence of Raf-1 activation, indicating that Taxol-induced cell death is not dependent on Raf-1 activation. At concentrations of drug lower than the critical concentration required for Raf-1 activation, p53 and p21(WAF-1) were induced independently of Raf-1. These studies suggest that Taxol-mediated cell death may result from two different mechanisms. At low Taxol concentrations (< 9 nM), cell death may occur after an aberrant mitosis by a Raf-1 independent pathway, whereas at higher Taxol concentrations (> or = 9 nM) cell death may be the result of a terminal mitotic arrest occurring by a Raf-1-dependent pathway.

Relationship between the structure of taxol and other taxanes on induction of tumor necrosis factor-alpha gene expression and cytotoxicity.

Taxol is an antitumor drug with cytotoxic properties that correlate with its microtubule-stabilizing activities. It has been reported that taxol parallels lipopolysaccharide in its effects on the induction of tumor necrosis factor-alpha (TNF-alpha) gene expression in macrophages (C. Bogdan and A. Ding, J. Leukocyte Biol., 52: 119-121, 1992; C. L. Manthey, M. E. Brandes, P. Y. Perera, and S. Vogel, J. Immunol., 149: 2459-2465, 1992; J. M. Carboni, C. Singh, and M. A. Tepper, Natl. Cancer Inst. Monogr., 15: 95-101, 1993). Structure-activity studies using taxol and related taxanes have been done to determine the relationship between the effects of taxol on TNF-alpha gene expression and its cytotoxic and microtubule-stabilizing activities. Using Northern blot analysis, it was found that changes in the structure of taxol that did not alter cytotoxicity did prevent the induction of TNF-alpha gene expression. The data presented in this paper demonstrate that the effects of taxol on TNF-alpha gene expression are distinct from its known cytotoxic properties.

Chemical synthesis of radiolabeled bleomycin A2 and its binding to DNA.

A method for the preparation of biologically active [3H]- and [13C]bleomycin A2 is described. Demethyl Cu(II):bleomycin A2, isolated after pyrolysis of Cu(II):bleomycin A2, was methylated with either [3H]-or [13C]methyl iodide, which resulted in Cu(II):bleomycin A2 labeled in the dimethylsulfonium moiety. Copper was removed by treatment with dithizone in chloroform, and structures were verified by thin-layer chromatography and 1H and 13C nuclear magnetic resonance spectroscopy. Copper-free [3H]-and [13C]bleomycin A2 are active in the degradation of DNA in vitro. Gel exclusion chromatograhy and equilibrium dialysis were used to determine the apparent equilibrium constants for binding of [3H]bleomycin A2 and Cu(II):[3H]bleomycin A2 to calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate. In 2.5 mM sodium phosphate buffer, pH 7.0, binding data obtained by gel filtration with calf thymus DNA reveal an apparent equilibrium constant for [3H]bleomycin A2 of 5.7 X 10(5)/mol and for Cu(II):[3H]bleomycin A2 of 3.9 X 10(5)/mol. One molecule of [3H]bleomycin A2 binds for every 3.7 base pairs in DNA, and one molecule of Cu(II):[3H]bleomycin A2 binds for every 2.8 base pairs in DNA. Analysis of binding data with calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate obtained by equilibrium dialysis reveals, in each instance, 2 types of binding sites for both the copper and metal-free form of the antibiotic. For those sites in calf thymus DNA with tighter binding affinity, the apparent equilibrium constant for [3H]bleomycin A2 was 6.8 X 10(5)/mol and for the Cu(II):[3H]bleomycin A2 complex, 4.4 X 10(5)/mol. As seen with calf thymus DNA, the affinity of [3H]bleomycin A2 is slightly greater than that of Cu(II):[3H]bleomycin A2 for the synthetic DNAs, although more of the copper form of the drug binds to these polymers.

Structure-activity study of the inhibition of microtubule assembly in vitro by podophyllotoxin and its congeners.

This study investigates the inhibition of microtubule assembly in vitro by podophyllotoxin and its derivatives, which include in part the antitumor compounds 4'-demethylepipodophyllotoxin ethylidene beta-D-glucoside (VP-16-213) and 4'-demethylepipodophyllotoxin thenylidene beta-D-glucoside (VM-26); the cyclic ethers, cyclic sulfides, and cyclic sulfones of podophyllotoxin and deoxypodophyllotoxin; epipodophyllotoxin; picropodophyllotoxin; and several 4'-demethyl compounds. The inhibitory activity of these derivatives is sensitive to the configuration and size of substituents at position 4 in ring C and to steric features of substituents at position 12 in ring D. Decreasing activity correlates with the increasing size of the substituent at position 12, as indexed by their van der Waals radii. These results suggest that rings C and D of these drugs are involved in their interaction with the podophyllotoxin-binding site in tubulin.

Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells.

Vinca alkaloids are used extensively in the treatment of childhood acute lymphoblastic leukemia (ALL) and despite their usefulness, drug resistance remains a serious clinical problem. Vinca alkaloids bind to the beta-tubulin subunit of the alpha/beta-tubulin heterodimer and inhibit polymerization of microtubules. Recent studies have implicated altered beta-tubulin isotype expression and mutations in resistance to microtubule-stabilizing agents. Microtubule-associated protein (MAP) MAP4 binds to and stabilizes microtubules, and increased expression is associated with decreased sensitivity to microtubule-depolymerizing agents. To address the significance of beta-tubulin and MAP4 alterations in childhood ALL, two CCRF-CEM-derived Vinca alkaloid resistant cell lines, VCR R (vincristine) and VLB100 (vinblastine), were examined. Decreased expression of class III beta-tubulin was detected in both VCR R and VLB100 cells. VCR R cells and to a lesser extent VLB100 cells expressed increased levels of MAP4 protein. Increased microtubule stability was observed in these VCR R cells as identified by the high levels of polymerized tubulin (45.6 +/- 2.6%; P < 0.005) compared with CEM and VLB100 cells (24.7 +/- 3.3% and 24.7 +/- 2.5%, respectively). Expression was associated with a single MAP4 isoform in the polymerized microtubule fraction in CEM and VCR cells. In contrast, VLB100 cells expressed a lower molecular weight isoform in the polymerized fraction. Two-dimensional-PAGE and immunoblotting revealed marked posttranslational changes in class I beta-tubulin in VCR R cells not evident in CEM cells. Sequencing of the beta-tubulin (HM40) gene identified a point mutation in VCR R cells in nucleotide 843 (CTC-->ATC; Leu(240)-->Ile) that was not present in CEM or VLB100 cells. This mutation resides in a region of beta-tubulin that lies in close proximity to the alpha/beta tubulin interface. Multiple alterations related to normal microtubule function were identified in ALL cells selected for resistance to Vinca alkaloids, and these alterations may provide important insight into mechanisms mediating resistance to Vinca alkaloids.

A homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the intestine of Xenopus laevis.

P-glycoprotein is an integral membrane protein that functions in multidrug resistance (MDR) cells as a drug efflux pump to maintain intracellular concentrations of antitumor drugs below cytotoxic levels. A homologue of the mammalian mdr gene has been isolated and characterized from Xenopus laevis (Xe-mdr). The cDNA was isolated from a tadpole cDNA library using the full length mouse mdrlb cDNA as a probe. The Xe-mdr encodes a protein that is 66% identical to the mouse mdrlb and 68% identical to the human mdrl. The predicted structure of the Xe-mdr gene product identifies twelve membrane spanning domains and two ATP binding sites both of which are the hallmark of the ABC (ATP binding cassette) transporters. Xe-mdr mRNA is expressed as a single message of 4.5 kb and is found predominantly in the intestine. Xe-mdr message is increased 3- to 4-fold in the ileum compared to the rest of the small intestine. In situ hybridization of sequential sections from the small intestine localized the expression of the Xe-mdr to the cells lining the lumenal epithelium. Brush border membrane vesicles prepared from the small intestine of Xenopus laevis effluxed vinblastine in an ATP-dependent manner. Efflux was decreased by verapamil, a known inhibitor of P-glycoprotein function. These studies indicate that the structure of Xe-mdr has been conserved and suggest that the protein has a role in maintaining the function of the normal intestine in Xenopus.

Mechanism of action of the 12,13-epoxytrichothecene, anguidine, an inhibitor of protein synthesis.

Anguidine, muconomycin A, T-2 toxin, crotocin and trichodermin, a group of 12,13-epoxytrichothecenes, inhibit protein synthesis in HeLa cells and in rabbit reticulocyte lysates. These five mycotoxins can be divided into two groups on the basis of the reversibility of their effects in HeLa cells, and kinetics of inhibition and effects on polyribosome structure in rabbit reticulocyte lysates. Anguidine, muconomycin A and T-2 toxin are irreversible inhibitors of protein synthesis; crotocin and trichodermin are reversible inhibitors of protein synthesis. After addition of low concentrations (1 muM) of anguidine, muconomycin A or T-2 toxin to rabbit reticulocyte lysates, polyribosomes are broken down to monosomes. At higher concentrations, 1 mM, these drugs begin to freeze the polyribosomes. Crotocin and trichodermin freeze the polyribosomes at a concentration of 10 muM. We conclude that anguidine, muconomycin A and T-2 toxin act primarily as inhibitors of initiation of protein synthesis, whereas crotocin and trichodermin inhibit the process of chain elongation.

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 beta-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 delta-lactone ring of (+)-discodermolide overlays with the C13 side chain of Taxol, concurred with the results of the SAR analysis.